Gaël Chételat
email: chetelat@cyceron.fr

MD Javier Arbizu

Henryk Barthel

Valentina Garibotto

Ian Law

Prof Silvia Morbelli

Elsmarieke Van De Giessen

Federica Agosta

Frederik Barkhof

David J Brooks

Maria C Carrillo

Bruno Dubois

Anders M Fjell

Giovanni B Frisoni

Oskar Hansson

Karl Herholz

Brian F Hutton

Clifford R Jack

Adriaan A Lammertsma

Susan M Landau

Prof Satoshi Minoshima

Flavio Nobili

Agneta Nordberg

Rik Ossenkoppele

Wim Jg Oyen

Prof Daniela Perani

Gil D Rabinovici

Prof Philip Scheltens

Victor L Villemagne

Henrik Zetterberg

Alexander Drzezga

Prof. H Barthel Md

Prof W J G Oyen

Amyloid-PET and 18F-FDG-PET in the diagnostic investigation of Alzheimer's disease and other dementias

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

Summary

Various biomarkers are available to support diagnosis of neurodegenerative diseases in clinical and research settings. Among those are molecular imaging biomarkers, such as amyloid-PET and [ 18 F]fluorodeoxyglucose (FDG)-PET that assess brain amyloid deposition and glucose metabolism, respectively. However, because evidence is not conclusive yet, there is confusion in the field regarding the optimal timepoint, combination, and order of these PET-biomarkers in the diagnostic work-up. After an agreement achieved among interdisciplinary experts in the field, weighting the specific utility of the individual biomarkers based on available evidence and clinical expertise, we propose a diagnostic algorithm suggesting the optimal timepoint for these PET-biomarkers, taking into account the other available established biomarkers, for early and differential diagnosis of neurodegenerative diseases that can lead to dementia. We propose three main diagnostic pathways with distinct biomarker sequences, in which amyloid and FDG-PET are placed at different positions in the diagnostic work-up, depending on the clinical presentation. Our proposal could maximize the information provided by each PET biomarker while considering their complementary strengths, i.e. proof of amyloidopathy for the former and assessment of the pattern and extent of neurodegeneration for the later. This proposed algorithm could support diagnostic decision making in specialist clinical settings with access to these biomarkers and should stimulate further research towards optimal diagnosis.

I. Introduction

The early and differential diagnosis of neurodegenerative diseases leading to dementia is still challenging. 1 The field is moving toward a biological definition of dementia, so that the role of biomarkers in diagnosis is becoming predominant. [START_REF] Jack | NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease[END_REF][START_REF] Mckhann | The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease[END_REF][START_REF] Dubois | Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria[END_REF][START_REF] Albert | The diagnosis of mild cognitive impairment due to Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease[END_REF] Considering other biomarkers, amyloid-PET and FDG-PET imaging provide valuable and complementary information. [START_REF] Teipel | Multimodal imaging in Alzheimer's disease: validity and usefulness for early detection[END_REF][START_REF] Zhang | Diagnostic accuracy of 18F-FDG and 11C-PIB-PET for prediction of short-term conversion to Alzheimer's disease in subjects with mild cognitive impairment[END_REF][START_REF] Laforce | Molecular imaging in dementia: Past, present, and future[END_REF][START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF][START_REF] Rascovsky | Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia[END_REF][11][START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies[END_REF][START_REF] Höglinger | Clinical diagnosis of progressive supranuclear palsy: The movement disorder society criteria[END_REF][START_REF] Prestia | Prediction of AD dementia by biomarkers following the NIA-AA and IWG diagnostic criteria in MCI patients from three European memory clinics[END_REF] Therefore, these biomarkers are being used extensively for diagnosis of neurodegenerative diseases in research studies and are recommended by some guidelines for the diagnosis of AD and several non-AD neurodegenerative diseases. [START_REF] Jack | NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease[END_REF][START_REF] Dubois | Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria[END_REF][START_REF] Mckhann | The diagnosis of dementia due to Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease[END_REF][START_REF] Rascovsky | Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia[END_REF]11 For various reasons, including cost, availability, and reimbursement (see below section IV on practical issues), the generalization of this use in all clinical settings is not feasible yet. However, many academic memory clinics already use these biomarkers to support assessment and management of patients. [START_REF] Teipel | Multimodal imaging in Alzheimer's disease: validity and usefulness for early detection[END_REF][START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF] There is widespread evidence that combining biomarkers improves diagnostic accuracy. [START_REF] Laforce | Molecular imaging in dementia: Past, present, and future[END_REF][START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF] However, faced with an arsenal of biomarkers (Figure 1), uncertainty has arisen regarding appropriate combination and/or order of application of PET imaging for differential diagnosis of dementias, as there is no commonly accepted consensus or theoretical framework concerning how they should be combined.

Recommendations for the ordering of these biomarkers into a meaningful sequence or combination are thus needed, since they are already in use.

This Personal View summarises recommendations and conclusions from an interdisciplinary group of experts comprised of nuclear medicine physicians, radiologists, neurologists, geriatricians, psychiatrists, clinical and basic neuroscientists and patient-advocates. These experts met at the European Association of Nuclear Medicine (EANM) Focus Meeting 2 (January 2019, Cannes, France) to discuss the role of PET imaging for early and differential diagnosis of neurodegenerative diseases leading to dementia (referred to as neurodegenerative diseases in what follows) and to converge on a diagnostic algorithm indicating the recommended order of tests depending on the clinical presentation. Our aim is not to provide an exhaustive review of the available evidence on the utility of PET imaging in dementia. Instead, we provide a consensual assessment of the optimal use of PET in the diagnostic process from the perspective of a group of experts, including clinicians involved in the care of patients with dementia. We judged biomarkers exclusively on the basis of their diagnostic qualities, while other factors, such as regional differences in their availability or national differences in their reimbursement, are discussed (section IV) but not taken into account. As such, our recommendations are not intended to replace national or other guidelines. Rather, they aim at providing a decision aid for situations in which thorough etiological and biomarker-supported diagnostic assessment is desired and considered meaningful for individual clinical reasons. More specifically, these recommendations are designed to facilitate the decisions on when in the diagnostic workup each of the biomarkers can be used and combined in a meaningful way. Also, as this algorithm focusses on the most validated PET imaging biomarkers for dementia, it remains to be seen how the scenario will change in the future, once new PET markers such as tau-PET, new MRI techniques, or fluid biomarkers advance.

Consequently, we first outline the current state of evidence on the complementary role of PET imaging in the diagnostic workup of neurodegenerative diseases and then propose a diagnostic algorithm. We also highlight the main challenges towards the clinical implementation of this algorithm, and indicate the main future research directions.

II. PET biomarkers in the differential diagnosis of mild cognitive impairment and dementia

Diagnosis of dementia is particularly difficult in the prodromal stages (when neuropathological changes may already be present but symptoms still mild), and also in atypical clinical presentations. In particular, the differential diagnosis between AD and either frontotemporal lobar degeneration (FTLD) or neurodegenerative parkinsonian syndromes (PS) may be very challenging, due to overlapping symptoms. Several lines of evidence support the use of PET neuroimaging within the framework of available biomarkers. [START_REF] Albert | The diagnosis of mild cognitive impairment due to Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease[END_REF][START_REF] Rascovsky | Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia[END_REF][11][START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies[END_REF][START_REF] Höglinger | Clinical diagnosis of progressive supranuclear palsy: The movement disorder society criteria[END_REF] In the following sections, we assess the advantages and disadvantages of the PET biomarkers -which provide the rationale for the diagnostic algorithm.

II.1. CSF biomarkers

The utility of PET imaging, and imaging biomarkers in general, has to face comparison with that of available and established cerebrospinal fluid (CSF) biomarkers. The diagnostic utility of core CSF biomarkers for AD pathology (Aβ42/Aβ40 ratio, total, and phosphorylated tau) is recognised by research guidelines, [START_REF] Molinuevo | Current state of Alzheimer's fluid biomarkers[END_REF] and these biomarkers are already in clinical use in many European countries in accordance with country-specific regulations. Reference methods and materials for CSF Aβ42 assay standardization, as well as high precision clinical chemistry tests on fully automated instruments, are in place, which bodes well for full implementation with uniform reference limits in clinical practice. [START_REF] Blennow | Biomarkers for Alzheimer's disease: current status and prospects for the future[END_REF] As compared to PET imaging biomarkers, the diagnostic information derived from CSF analysis is in part overlapping, in part complementary. Both methods provide insights on neurodegeneration, tau, and amyloid pathology. With regard to amyloid detection, both approaches are similarly validated and agreement between CSF and PET amyloid results is usually good, but not perfect. [START_REF] Leuzy | Concordance and Diagnostic Accuracy of [11C]PIB PET and Cerebrospinal Fluid Biomarkers in a Sample of Patients with Mild Cognitive Impairment and Alzheimer's Disease[END_REF] While CSF assessment is less expensive, PET allows better staging and monitoring of the extent and location of pathology. A list of advantages and disadvantages of CSF versus PET amyloid measures and recommendations for their use are summarised in Table 1. In greater detail, this question has been previously discussed elsewhere. [START_REF] Zetterberg | Biomarkers for Alzheimer's disease-preparing for a new era of diseasemodifying therapies[END_REF]

II.2 FDG-PET imaging

In dementia, brain hypometabolism detected with FDG-PET is a marker of neurodegeneration (Table 1).

FDG-PET is particularly useful for early diagnosis, since it can show characteristic patterns of AD neurodegeneration earlier than MRI in individuals with MCI who will convert to the Alzheimer's dementia. [START_REF] Laforce | Molecular imaging in dementia: Past, present, and future[END_REF][START_REF] Yuan | Fluorodeoxyglucose-Positron-Emission Tomography, Single-Photon Emission Tomography, and Structural MR Imaging for Prediction of Rapid Conversion to Alzheimer Disease in Patients with Mild Cognitive Impairment: A Meta-Analysis[END_REF][START_REF] Caminiti | FDG-PET and CSF biomarker accuracy in prediction of conversion to different dementias in a large multicentre MCI cohort[END_REF] Previous studies have shown added value of FDG-PET over routine CSF or MRI tests to predict conversion to AD dementia in people with MCI, especially short term progression, [START_REF] Laforce | Molecular imaging in dementia: Past, present, and future[END_REF][START_REF] Shaffer | Predicting cognitive decline in subjects at risk for Alzheimer disease by using combined cerebrospinal fluid, MR imaging, and PET biomarkers[END_REF][START_REF] Blazhenets | Predictive Value of 18F-Florbetapir and 18F-FDG PET for Conversion from Mild Cognitive Impairment to Alzheimer Dementia[END_REF] with a drop in the misclassification rate from 32% (for CSF or MRI alone) to 20% for FDG-PET alone, and from 27% for CSF and MRI to 9% when adding FDG-PET. [START_REF] Shaffer | Predicting cognitive decline in subjects at risk for Alzheimer disease by using combined cerebrospinal fluid, MR imaging, and PET biomarkers[END_REF] Insight on short-term (2-3 years) clinical progression is crucial for patients and families to plan the future and for the clinician to adapt the clinical monitoring, and to target those patients more suitable to undergo therapeutic interventions. Based on the typical temporoparietal pattern of hypometabolism, its negative predictive value, ranging from 77% (CI: 64-87%) to 95% (CI: 75-100%), [START_REF] Arbizu | Clinical utility of FDG-PET for the clinical diagnosis in MCI[END_REF][START_REF] Jagust | What does fluorodeoxyglucose PET imaging add to a clinical diagnosis of dementia?[END_REF] and evidence supporting its validity for clinical use, [START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF][START_REF] Arbizu | Clinical utility of FDG-PET for the clinical diagnosis in MCI[END_REF][START_REF] Nobili | European Association of Nuclear Medicine and European Academy of Neurology recommendations for the use of brain 18 F-fluorodeoxyglucose positron emission tomography in neurodegenerative cognitive impairment and dementia: Delphi consensus[END_REF][START_REF] Smailagic | 18F-FDG PET for Prediction of Conversion to Alzheimer's Disease Dementia in People with Mild Cognitive Impairment: An Updated Systematic Review of Test Accuracy[END_REF] FDG-PET is recommended for evaluating people with MCI suspected of having underlying AD. FDG-PET has also proven to be useful to predict clinical outcome, at the individual level, in people with MCI who already had amyloid-PET. Thus, a normal FDG-PET scan would predict clinical stability during followup of several years (even in amyloid-positive cases) [START_REF] Iaccarino | Alzheimer's Disease Neuroimaging Initiative. Predicting long-term clinical stability in amyloid-positive subjects by FDG-PET[END_REF] while abnormal FDG-PET would be associated with increased risk of progressive cognitive deterioration (including in amyloid-negative cases). [START_REF] Caroli | Mild cognitive impairment with suspected nonamyloid pathology (SNAP): Prediction of progression[END_REF] FDG-PET is also useful for staging of disease and for differential diagnosis, because the patterns of brain hypometabolism are closely associated with type and severity of cognitive deficits, and relatively distinct in different neurodegenerative diseases and even among their variants. [START_REF] Laforce | Molecular imaging in dementia: Past, present, and future[END_REF] Characteristic patterns include posterior cingulate and temporal-parietal involvement in Alzheimer's disease (AD), and predominant frontal, peri-sylvian, and anterior temporal hypometabolism in the behavioural, non-fluent, and semantic variants of frontotemporal lobar degeneration (FTLD), respectively. [START_REF] Laforce | Molecular imaging in dementia: Past, present, and future[END_REF][START_REF] Shaffer | Predicting cognitive decline in subjects at risk for Alzheimer disease by using combined cerebrospinal fluid, MR imaging, and PET biomarkers[END_REF][START_REF] Silverman | Brain 18F-FDG PET in the diagnosis of neurodegenerative dementias: comparison with perfusion SPECT and with clinical evaluations lacking nuclear imaging[END_REF][START_REF] Bergeron | Prevalence of amyloid-β pathology in distinct variants of primary progressive aphasia[END_REF] Specific FDG-PET hypometabolism patterns also include atypical AD variants, primary progressive aphasias, and atypical parkinsonisms. FDG-PET is, therefore, included in the diagnostic criteria of several neurodegenerative diseases: 1) behavioural variant of FTLD; [START_REF] Rascovsky | Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia[END_REF] 2) primary progressive aphasias; 11 3) dementia with Lewy bodies (DLB); 12 4) progressive supranuclear palsy (PSP). [START_REF] Höglinger | Longitudinal magnetic resonance imaging in progressive supranuclear palsy: A new combined score for clinical trials[END_REF] The use of FDG-PET remains limited, however, with regard to definite validation of the neuropathology underlying the detected patterns of hypometabolism.

According to the 5-phase strategic roadmap defined to foster clinical validation of biomarkers, FDG-PET is the PET biomarker that has reached the most advanced phase of validation. [START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF] Analytical validity (phase 1) is completed, clinical validity (phases 2 and 3) almost achieved, and preliminary evidence for clinical utility (phase 4 and 5) is available. 9

II.3. Amyloid-PET imaging

Amyloid-PET is an established neuroimaging technique with standardised tracer-specific visual reading procedures, and documented high reproducibility across PET centres. It allows non-invasive in vivo detection of amyloid plaques, one of the main neuropathological landmarks of Alzheimer's disease with very high sensitivity and specificity in patients with confirmed AD (96%, CI 80-100% and 100%, CI 78-100 respectively in people who had autopsy within 1 year of PET imaging). [START_REF] Clark | Cerebral PET with florbetapir compared with neuropathology at autopsy for detection of neuritic amyloid-β plaques: a prospective cohort study[END_REF] Amyloid-PET also allows detection of amyloid-pathology in the clinically atypical variants of AD such as posterior cortical atrophy, the frontal-executive variant or in the logopenic variant. [START_REF] Bergeron | Prevalence of amyloid-β pathology in distinct variants of primary progressive aphasia[END_REF][START_REF] Mcginnis | Approach to atypical Alzheimer's disease and case studies of the major subtypes[END_REF] It does however not allow to differentiate between distinct amyloid-positive disorders which may show similar amyloid-deposition patterns.

Many amyloid tracers have been developed, [START_REF] Villemagne | Aβ-amyloid and Tau Imaging in Dementia[END_REF] three of which have reached clinical approval and commercial availability: [START_REF] Leuzy | Concordance and Diagnostic Accuracy of [11C]PIB PET and Cerebrospinal Fluid Biomarkers in a Sample of Patients with Mild Cognitive Impairment and Alzheimer's Disease[END_REF] F-florbetapir (Amyvid™), [START_REF] Leuzy | Concordance and Diagnostic Accuracy of [11C]PIB PET and Cerebrospinal Fluid Biomarkers in a Sample of Patients with Mild Cognitive Impairment and Alzheimer's Disease[END_REF] F-florbetaben (NeuraCeq®), and [START_REF] Leuzy | Concordance and Diagnostic Accuracy of [11C]PIB PET and Cerebrospinal Fluid Biomarkers in a Sample of Patients with Mild Cognitive Impairment and Alzheimer's Disease[END_REF] F-flutemetamol (Vizamyl TM ). They are essentially equivalent in clinical practice, [START_REF] Laforce | Molecular imaging in dementia: Past, present, and future[END_REF][START_REF] Villemagne | Aβ-amyloid and Tau Imaging in Dementia[END_REF] and standardisation approaches have been developed to allow for direct quantitative comparison between the different tracers. [START_REF] Klunk | The Centiloid Project: standardizing quantitative amyloid plaque estimation by PET[END_REF] A meta-analysis of studies evaluating amyloid-PET's ability to predict MCI conversion to the Alzheimer's clinical syndrome of dementia demonstrated a sensitivity of 93% (CI: 71.3-99.9%) and a specificity of 56% (CI: 47.2-64.8%). [START_REF] Zhang | Diagnostic accuracy of 18F-FDG and 11C-PIB-PET for prediction of short-term conversion to Alzheimer's disease in subjects with mild cognitive impairment[END_REF] When comparing the ability to predict progression to Alzheimer's clinical syndrome of dementia, a slightly higher sensitivity has been reported

for amyloid-PET over FDG-PET, although FDG-PET has a higher specificity and a better accuracy for predicting short-term progression. [START_REF] Blazhenets | Predictive Value of 18F-Florbetapir and 18F-FDG PET for Conversion from Mild Cognitive Impairment to Alzheimer Dementia[END_REF][START_REF] Mallik | Clinical Amyloid Imaging[END_REF] A recent report shows that in amyloid positive cognitively unimpaired and MCI individuals, a negative FDG-PET was associated with clinical stability for several years. [START_REF] Iaccarino | Alzheimer's Disease Neuroimaging Initiative. Predicting long-term clinical stability in amyloid-positive subjects by FDG-PET[END_REF] This may be due to the fact that amyloid-pathology may appear long before onset of clinical symptoms. Indeed, positive amyloid scans are found in 10% to 44% of cognitively unimpaired elderly aged 50 to 90 years old, with yet unknown clinical relevance. [START_REF] Jansen | Prevalence of cerebral amyloid pathology in persons without dementia: a meta-analysis[END_REF] Currently, amyloid imaging alone is considered insufficient to predict time to clinical conversion in prodromal and asymptomatic stages. Also, the value of amyloid-imaging for disease staging may be limited, as it does not show close correlation with symptom severity and develops a plateau in later disease stages. [START_REF] Mallik | Clinical Amyloid Imaging[END_REF][START_REF] Villain | Regional dynamics of amyloid-β deposition in healthy elderly, mild cognitive impairment and Alzheimer's disease: a voxelwise PiB-PET longitudinal study[END_REF] Amyloid imaging has almost achieved analytical validity (phase 1) and clinical validity (phases 2 and 3). [START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF] Yet, more data is needed using the harmonised procedure to improve reliability of results across tracers and on the effects of covariates (e.g. age, sex, APOE genotype, disease duration, comorbidities) on controls and patients.

Regarding the clinical utility of amyloid-PET, meta-analyses have consistently shown that amyloid imaging is associated with changes in aetiological diagnosis, increases in diagnostic confidence, and changes in patient management in up to 60% of subjects. [START_REF] Barthel | Clinical Use and Utility of Amyloid Imaging[END_REF][START_REF] Fantoni | A Systematic Review and Aggregated Analysis on the Impact of Amyloid PET Brain Imaging on the Diagnosis, Diagnostic Confidence, and Management of Patients being Evaluated for Alzheimer's Disease[END_REF] These changes are more often due to a negative amyloid-PET result and more frequently in older (>65 years) patients. Such a case study is illustrated in Panel 3. [START_REF] De Wilde | Association of Amyloid Positron Emission Tomography With Changes in Diagnosis and Patient Treatment in an Unselected Memory Clinic Cohort[END_REF] Appropriate use criteria for amyloid-PET have been proposed, identifying the following patient populations as the most likely to benefit from the procedure: [START_REF] Johnson | Appropriate use criteria for amyloid PET: A report of the Amyloid Imaging Task Force, the Society of Nuclear Medicine and Molecular Imaging, and the Alzheimer's Association[END_REF] (1) people with MCI in which clinical uncertainty exists (2) patients with a dementia syndrome suggestive of AD, but with an atypical presentation or suspected mixed etiology; and (3) patients with early-onset progressive cognitive decline. A very large prospective multicentre trial (IDEAS), including already more than 11 000 participants, recently demonstrated that amyloid-PET performed in patients selected according to the AUC-criteria resulted in a change in management in a relevant proportion (about 60% of patients) [START_REF] Rabinovici | Association of Amyloid Positron Emission Tomography With Subsequent Change in Clinical Management Among Medicare Beneficiaries With Mild Cognitive Impairment or Dementia[END_REF] -which strongly justifies the application of this method in the corresponding population. Other studies have shown that amyloid imaging can also have clinical utility in individuals not fulfilling the AUC criteria. [START_REF] De Wilde | Association of Amyloid Positron Emission Tomography With Changes in Diagnosis and Patient Treatment in an Unselected Memory Clinic Cohort[END_REF][START_REF] Altomare | Quantitative appraisal of the Amyloid Imaging Taskforce appropriate use criteria for amyloid-PET[END_REF][START_REF] Apostolova | Critical review of the Appropriate Use Criteria for amyloid imaging: Effect on diagnosis and patient care[END_REF] This includes subjects with clinical probable AD (who may be amyloid-negative), patients with atypical non-amnestic phenotypes of cognitive impairment who do not fulfil criteria for "possible" AD and subjects with subjective cognitive decline. [START_REF] Mcginnis | Approach to atypical Alzheimer's disease and case studies of the major subtypes[END_REF][START_REF] Salloway | Two Phase 3 Trials of Bapineuzumab in Mild-to-Moderate Alzheimer's Disease[END_REF][START_REF] Slot | Subjective cognitive decline and rates of incident Alzheimer's disease and non-Alzheimer's disease dementia. Alzheimer's & Dementia[END_REF] Another large multicentre study (AMYPAD-DPMS; registration number EudraCT: 2017-002527-21) [START_REF] Frisoni | AMYPAD Diagnostic and Patient Management Study: Rationale and design. Alzheimer's & Dementia[END_REF] is ongoing including patients beyond AUC criteria, e.g. fulfilling criteria for probable AD or for subjective cognitive decline, that would allow provide answers regarding the clinical impact and utility of amyloid imaging in these populations (see supplementary materials).

II.4. PET biomarkers in the FTLD spectrum

The complexity of dementia clinical diagnosis is well illustrated within the FTLD spectrum, the family of clinically and neuropathologically heterogeneous disorders characterised by progressive degeneration of the frontal or anterior temporal lobes. [START_REF] Slot | Subjective cognitive decline and rates of incident Alzheimer's disease and non-Alzheimer's disease dementia. Alzheimer's & Dementia[END_REF] Symptoms of FTLD may notably resemble those of AD particularly of its atypical forms. FDG-PET has a long-standing role in the diagnosis of FTLD based on signature patterns of hypometabolism associated with specific clinical syndromes. [START_REF] Frisoni | AMYPAD Diagnostic and Patient Management Study: Rationale and design. Alzheimer's & Dementia[END_REF][START_REF] Bang | Frontotemporal dementia[END_REF][START_REF] Rabinovici | Amyloid vs FDG-PET in the differential diagnosis of AD and FTLD[END_REF] Recently, amyloid-PET has proven useful in discriminating AD from FTLD aetiologies, since amyloid plaques are a core feature of AD neuropathology but typically not part of the FTLD neuropathological spectrum. [START_REF] Bang | Frontotemporal dementia[END_REF][START_REF] Ma | Rates of Amyloid Imaging Positivity in Patients With Primary Progressive Aphasia[END_REF] The major advantage of amyloid-PET over FDG-PET is that it can highlight a specific pathology when FDG-PET cannot inform on the pathological aetiology underlying a metabolic abnormality. [START_REF] Foster | FDG-PET improves accuracy in distinguishing frontotemporal dementia and Alzheimer's disease[END_REF] Because of its high sensitivity to detect fibrillar amyloid pathology, a negative amyloid-PET scan can also be used to reliably rule out AD as the underlying aetiology in patients with complex presentations such as primary progressive aphasia variants (or corticobasal syndrome -see below). [START_REF] Bouwman | Diagnostic utility of FDG-PET in the differential diagnosis between different forms of primary progressive aphasia[END_REF][START_REF] Dubois | Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria[END_REF] In general, amyloid-PET is useful when a differential diagnosis between AD and non-AD causes of dementia is needed. FDG-PET can further address differential diagnosis within the FTLD spectrum in amyloid negative Page 11 of 37 patients, or when the final diagnosis is still unclear after amyloid-PET and/or CSF analyses, (e.g. in patients with mixed pathological features).

II.5. Neuroimaging biomarkers in parkinsonian syndromes with cognitive decline

Parkinsonian syndromes causing dementia include Parkinson's disease (PD), DLB, PSP, and corticobasal degeneration. The assessment of integrity of the nigrostriatal dopaminergic pathway with DaT-SPECT has been approved by the United States Food & Drug Administration and the European Medicines Agency to support the differential diagnosis between DLB and AD. The availability of this tool has increased the diagnostic accuracy for DLB, which is usually difficult due to overlapping symptoms with AD, vascular cognitive impairment, and even FTLD. [START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies[END_REF] Some DLB patients may not show all of the core clinical symptoms (e.g. they may present with minor or even no apparent PS). The use of DaT-SPECT should also be considered in these cases, e.g. if other core clinical features of DLB such as fluctuation, visual hallucinations or REM sleep behaviour disorder are observed, especially given the side effects observed in DLB patients to neuroleptics sometimes prescribed in patients with dementia. [START_REF] Mckeith | Neuroleptic sensitivity in patients with senile dementia of Lewy body type[END_REF] Since dopaminergic deficits can be present in all neurodegenerative PS, DaT-SPECT cannot be used for the differential diagnosis between these diseases. In contrast, FDG-PET can distinguish between specific patterns of hypometabolism of PD/DLB and all other neurodegenerative PS. FDG-PET has thus been recommended for differential diagnosis within neurodegenerative PS by the EANM-EAN taskforce. [START_REF] Hellwig | 18F]FDG-PET is superior to [123I]IBZM-SPECT for the differential diagnosis of parkinsonism[END_REF][START_REF] Walker | Clinical utility of FDG PET in Parkinson's disease and atypical parkinsonism associated with dementia[END_REF] PS are also systemic disorders, and in particular, [ 123 I]meta-iodobenzylguanidine (mIBG) myocardial scintigraphy assessing cardiac sympathetic nerve endings has been included in the clinical flow chart for the differential diagnosis between AD and DLB, as well as between PD and other neurodegenerative PS. [START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies[END_REF][START_REF] Tiraboschi | 123 I-2β-carbomethoxy-3β-(4-iodophenyl)-N -(3-fluoropropyl) nortropane single photon emission computed tomography and 123 I-metaiodobenzylguanidine myocardial scintigraphy in differentiating dementia with lewy bodies from oth[END_REF] When neurodegenerative PS is suspected, either mIBG scanning or FDG-PET could be considered depending on experience and availability (see Figure 2 for details). 59

III. Proposed algorithm for differential diagnosis

Based on the evidence summarised above on the relative strengths and limitations of each biomarker and their complementarity, but also relying on pragmatic considerations on how to reach a conclusion in a specific clinical situation, we propose a diagnostic algorithm reflecting the optimal combination of biomarkers according to different clinical situations (Figure 2). Further information concerning methodology used for reaching agreement among the co-authors of this Personal View are provided as supplementary materials. In addition to potentially supporting diagnostic decision making in specialized centers, this diagnostic algorithm is proposed as a theoretical framework to guide researchand to establish a standard for comparison with alternative algorithms (see validation section IV.1.)

III.1. Structural imaging as a first step

In situations when biomarker-based diagnostic work-up is clinically needed to establish a reliable etiological diagnosis, structural imaging is recommended as the very first step following clinical and neuropsychological evaluation, before other imaging biomarkers. Indeed, structural neuroimaging allows detecting other pathologies that might be responsible for cognitive decline (e.g. hydrocephalus, tumours, or vascular lesions, although the contribution of vascular lesions to the observed cognitive deficits cannot be determined with structural imaging). [START_REF] Frisoni | In vivo mapping of incremental cortical atrophy from incipient to overt Alzheimer's disease[END_REF] Furthermore, structural imaging can help assess the presence and degree of co-morbidities (e.g. vascular)

frequently found in neurodegenerative disorders, which can affect the clinical manifestation and may be treatable.

In addition, topographical patterns of atrophy assessed by structural imaging are associated with specific neurodegenerative conditions, [START_REF] Koikkalainen | Differential diagnosis of neurodegenerative diseases using structural MRI data[END_REF] whereas the overall degree of atrophy is associated with disease-specific clinical features and severity, and tracks the progression of neurodegeneration. [START_REF] Frisoni | In vivo mapping of incremental cortical atrophy from incipient to overt Alzheimer's disease[END_REF][START_REF] Gordon | Spatial patterns of neuroimaging biomarker change in individuals from families with autosomal dominant Alzheimer's disease: a longitudinal study[END_REF] Regarding the structural imaging modality, MRI has frequently been considered as a superior tool, as compared with computed tomography (CT) for dementia workup. [START_REF] Albert | The Use of MRI and PET for Clinical Diagnosis of Dementia and Investigation of Cognitive Impairment. Report of the neuroimaging work group of the Alzheimer's Association[END_REF] However to date there is no strong evidence towards recommending the one or the other.

III.2. A three-pathway algorithm

In general, after the clinical/neuropsychological evaluation and structural imaging, the added value of other diagnostic biomarkers may be especially high in cases with clinical diagnostic uncertainty (e.g. atypical appearance or mixed presentation such as combined cognitive, behavioural, and motor symptoms).

IV. Practical challenges and limitations of the algorithm

The suggested algorithm is based on existing evidence but also on several assumptions and expert opinions. Thus, several aspects will require further clarification and additional research in the future and some controversies also remained among the authors. These aspects are further discussed below.

IV.1. Validation

The algorithm is a theoretical proposal based on evidence and pragmatic considerations on how to reach a conclusion in a specific clinical situation, reflecting in depth discussions and converging agreement of experts from various disciplines. It is proposed as a timely model to support and potentially homogenize heterogeneous clinical practice, and to structure research and serve as a basis to future development and comparison to alternative models. There is extensive evidence of analytical and early clinical validity for individual biomarkers, but more evidence is needed on multimodal imaging approaches and, particularly, the order of tests. The systematic validation of each arm of the algorithm will be challenging given the high number of possible combinations. However, the added value for diagnosis of the specific combinations shown in the proposed algorithm will have to be demonstrated and compared to alternative algorithms that might arise based on new evidence and/or possible distinct pragmatic considerations.

IV.2. Practical issues: availability, adverse effects, cost-effectiveness and sustainability

The widespread and consistent use of biomarkers not only relies on their performance for early and differential diagnoses, but also on more practical issues such as training status, availability of and distance to PET scanners and cyclotrons, and adverse effects (as listed in Table 2). These considerations have not been taken into account in this Personal View, because our focus has been purely on the diagnostic value of the individual biomarkers. Thus, these questions will require attention in future studies.

Because PET imaging can be relatively expensive (the cost of an amyloid-PET scan for instance is 2000-2500 € in Europe and 3000-4500 US$ in the United States), the issue of cost-effectiveness is particularly relevant in the context of lacking effective treatment. There is no sufficient evidence available yet on the impact of biomarkers on disease burden or cost-effectiveness (Phase 5 secondary aim). [START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF] Overall, an examination with high sensitivity and specificity enhances diagnostic confidence and might reduce costs and the number of patient visits and additional tests, as it has been demonstrated for amyloid PET imaging. [START_REF] Shea | Impact of Amyloid PET Imaging in the Memory Clinic: A Systematic Review and Meta-Analysis[END_REF][START_REF] Carswell | Clinical utility of amyloid PET imaging with (18)F-florbetapir: a retrospective study of 100 patients[END_REF] Objective evidence from health economic studies is needed to systematically assess cost-effectiveness and ideally considering various situations of treatment availability and efficacy. In addition to cost-effectiveness, medical diagnostic tools in the future may have to face discussion with regard to sustainability. Indeed, imaging instrumentations require considerable amount of energy with regard to operation, cooling and computer technology. 68

IV.3. Reimbursement

Reimbursement of imaging tests from payers varies considerably across the world. [START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF] The right-to-know of the patients may need to be balanced against the economic circumstances and possibilities of the respective health care systems. Reimbursement of FDG-PET is mainly provided for the differential diagnosis of AD versus FTD in the US and many European countries. Amyloid PET is available in most high-income countries but reimbursement is limited. [START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF] In 2013, the US Centers for Medicare & Medicaid Services issued a National Coverage

Decision concluding yet insufficient evidence of clinical utility to justify general reimbursement for amyloid-PET. [START_REF] Jacques | Decision Memo for Beta Amyloid Positron Emission Tomography in Dementia and Neurodegenerative Disease (CAG-00431N)[END_REF] However, they agreed to cover scans in clinical studies investigating whether amyloid-PET improves health outcomes. Understandably, health systems are extremely cautious to endorse any expensive test which might be indicated in a large segment of the population. This could change dramatically, once disease-modifying drugs become available.

IV.4. Ethical aspects of early diagnosis and disclosure of biomarker information

Disclosing information on early ongoing neurodegenerative disease or even prediction is linked to major ethical considerations. From the patient perspective, the right-to-know and the right not to know should be taken into account. On the one hand disclosing such serious information may cause anxiety and worry, potentially even long before symptomatic onset of disease and without access to therapy options. On the other hand, early and accurate diagnosis allows people with dementia diseases and their families to build a care team and seek education and support services. It provides an opportunity for creating advance directives and financial planning, enables earlier access to appropriate treatments, and opens opportunities for enrolment in clinical trials. A recent study concerning the potential benefits of the clinical use of amyloid-PET showed that over 80% of patients and caregivers would support use and reimbursement of the test to inform legal, financial, and long-term health care choices. [START_REF] Mustafa | Patient and Caregiver Assessment of the Benefits From the Clinical Use of Amyloid PET Imaging[END_REF] Further research is required in this area and in ongoing studies, the context for appropriately disclosing the results of the test to patients and families are being defined. 71

IV.5. Alternatives to the diagnostic algorithm

It needs to be emphasized that the in part cost-and energy-expensive biomarker tests discussed here will not in all cases be able to solve the diagnostic problem, even when applied in a systematic order or combination.

Furthermore, the potential benefit for the individual patient needs to be carefully reflected, when considering the available options for biomarker-supported diagnosis. Depending on the situation of the patient with regard to age, stage of disease, psychological constitution, comorbidities, etc., the diagnostic strategies may considerably deviate from a standardized pathway. It is essential to involve patients and their caregivers into this decision. Thus, in the clinical work-up of neurodegenerative disorders, the most important factor remains to be the discussion with the patients and their caregivers about the meaning of the symptoms, the prognosis and the therapeutic options as well as about the value and the consequences of further diagnostic steps. Particularly in view of the often still limited therapeutic options, other strategies such as watchful waiting or systematic clinical-neuropsychological follow-up examinations may represent preferable alternatives.

IV.6. Controversies with regard to disease models

There are many questions unresolved regarding the development of neurodegenerative disorders and the answers to these questions may in the future affect the way diagnostic questions are asked or how diagnostic algorithms will be drafted. The dominant hypothesis in Alzheimer's disease up to now is the amyloid hypothesis, whereby amyloid deposition is considered as an early causal event of the disease. [START_REF] Hardy | Alzheimer's disease: the amyloid cascade hypothesis[END_REF] This hypothesis has led to the amyloid/tau/(neurodegeneration) (A/T/(N) model (see supplementary materials), [START_REF] Jack | NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease[END_REF] which is well-accepted but also stimulated much debate and criticisms. [START_REF] Glymour | Will biomarker-based diagnosis of Alzheimer's disease maximize scientific progress? Evaluating proposed diagnostic criteria[END_REF][START_REF] Garrett | A Critique of the 2018 National Institute on Aging's. Research Framework: Toward a biological definition of Alzheimer's disease[END_REF][START_REF] Morris | Questions concerning the role of amyloid-β in the definition, aetiology and diagnosis of Alzheimer's disease[END_REF] Multimodal imaging may allow testing and possible revision of these models, and it has already stimulated consideration of alternative models, including the dual pathway hypothesis, amyloid-independent mechanisms, and the vascular hypothesis. [START_REF] Chételat | Aβ-independent processes-rethinking preclinical AD[END_REF][START_REF] Pimplikar | Amyloid-Independent Mechanisms in Alzheimer's Disease Pathogenesis[END_REF][START_REF] Small | Linking Aβ and Tau in Late-Onset Alzheimer's Disease: A Dual Pathway Hypothesis[END_REF][START_REF] Herrup | The case for rejecting the amyloid cascade hypothesis[END_REF][START_REF] De La | The Vascular Hypothesis of Alzheimer's Disease: A Key to Preclinical Prediction of Dementia Using Neuroimaging[END_REF] However, while drafting the diagnostic algorithm, we aimed at providing a model with diagnostic value largely on the basis of commonly agreed facts and independently from still controversial concepts and assumptions on disease causality.

V. Conclusion and future directions

The panellists at EANM Focus Meeting 2 agreed on recommendations regarding the use of PET imaging within the scope of available biomarkers for early and differential diagnosis of neurodegenerative diseases. After clinical/neuropsychological evaluation and structural imaging when needed, the decision on necessity and choice of the next biomarker depends on the specific clinical profile and the individual diagnostic question. Amyloid-PET allows to detect amyloidosis in vivo in a standardized fashion and has high negative predictive value toward Alzheimer's disease. Compared with amyloid CSF biomarkers, amyloid-PET is used to determine the location and extent of pathology, and detect brain areas of earliest amyloid accumulation [START_REF] Grothe | In vivo staging of regional amyloid deposition[END_REF] and changes over time. [START_REF] Villemagne | Aβ-amyloid and Tau Imaging in Dementia[END_REF][START_REF] Villain | Regional dynamics of amyloid-β deposition in healthy elderly, mild cognitive impairment and Alzheimer's disease: a voxelwise PiB-PET longitudinal study[END_REF] FDG-PET allows to detect neurodegeneration with greater sensitivity than structural MRI. Compared with CSF neurodegeneration biomarkers, topographical information on neurodegeneration obtained with FDG-PET (patterns of hypometabolism) is closely associated to type and severity of cognitive deficits, making this biomarker particularly useful for differential diagnosis, staging of disease extent and predicting short-term progression. [START_REF] Laforce | Molecular imaging in dementia: Past, present, and future[END_REF][START_REF] Caroli | Mild cognitive impairment with suspected nonamyloid pathology (SNAP): Prediction of progression[END_REF] Based on our current knowledge of the respective advantages and disadvantages of each

Declaration of interests

Panels, Tables, and Figures

Panel 1.

Search strategy and selection criteria

References for this Review were identified by searches of PubMed between November 1, 2018 and October 1, 2019 and from references to relevant articles. The search terms (alone or in combination) "Alzheimer's disease", "dementia", "neurodegenerative diseases", "MCI", "imaging", "biomarkers", "PET", "FDG", "amyloid imaging", "amyloid PET", "PIB", "flutemetamol", "florbetapir", "florbetaben", "amyloid markers", "magnetic resonance imaging (MRI)", "CSF biomarkers", "clinical validity", "clinical utility", "clinical acceptance", "management change", "diagnostic change" were used. Additional studies cited from the previously identified papers and known to the authors were also included. There were no language restrictions.

The final reference list was generated on the basis of relevance to the topics covered in this Review.

Panel 2. Glossary

Dementia

Dementia is a syndrome characterized by a progressive alteration of cognitive functions beyond that expected for age and education and severe enough to affect daily functioning. According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), dementia is categorised as a major neurocognitive disorder (major-NCD) and defined by a decline from a previous level of performance, and compared to appropriate norms, sufficient to interfere with independence, in at least one of the following cognitive functions: attention, planning, inhibition, learning, memory, language, visual perception, spatial skills, social skills or other cognitive functions. Cognitive impairment might be accompanied by deterioration in emotional control, social behaviour, or motivation.

Dementia is not a single disease; it is caused by a variety of brain diseases or injury, including by neurodegenerative diseases. The most frequent cause of dementia is Alzheimer's disease, which may represent 60-70% of cases according to the World Health Organization (WHO), with other major forms including vascular dementia, dementia with Lewy bodies and frontotemporal dementia. Dementia could also be caused by nonneurodegenerative diseases such as stroke, cancer or chronic alcoholism.

Page 29 of 37

Neurodegenerative diseases

Neurodegenerative disease is a generic term for all diseases characterized by a progressive loss of structure or function of neurons including neurodegenerative diseases leading to dementia, Parkinson's disease, motor neurone disease (or amyotrophic lateral sclerosis), cerebellar degenerations, Huntington's disease, fatal familial insomnia and prion diseases. Neurodegeneration causes cognitive deterioration which might progress to dementia. This paper focuses on neurodegenerative diseases leading to dementia. However, this is not the case for all neurodegenerative diseases. In Parkinson's disease for instance, motor symptoms might remain isolated or be accompanied by cognitive symptoms leading to dementia. The causes for neurodegenerative diseases are not fully understood but most likely include genetic and environmental factors. Neurodegenerative diseases are characterized by presence of neuropathologies -which reflect the alteration of specific proteins, and are most often used to define the disease and as the gold-standard for diagnosis. There is an overlap between diseases in the proteins involved. The tau protein for instance is involved in both Alzheimer's disease and some forms of frontotemporal lobar diseases.

Mild Cognitive Impairment (MCI)

MCI, called as mild neurocognitive disorder (mild-NCD) in the DSM-5, refers to a stage of cognitive impairment that is beyond that expected for age and education but, in opposition to dementia, not severe enough to affect daily functioning. Criteria for MCI include self or informant-reported memory/cognitive complaint. Several subtypes have been defined based on the predominant cognitive domain that is affected. It has been demonstrated

that MCI is associated with an increased risk of developing dementia, with the amnestic subtype showing high conversion to Alzheimer's type dementia.

Biomarkers

Biomarkers refer to biological measurements that could be obtained in vivo and allow to quantify biological and pathological conditions / processes or therapeutic effects. In the field of neurodegenerative diseases, several biomarkers are available from CSF or blood sampling, or from neuroimaging. In this paper we focus on neuroimaging and especially on PET biomarkers. The most widely used and validated PET biomarkers for Alzheimer's disease include Amyloid-PET and FDG-PET, while Tau-PET biomarkers are entering the field.

Diagnostic algorithm

A diagnostic algorithm is supposed to support the clinician in the decision on which diagnostic test to employ in which situation, depending on the clinical presentation of the patient, the probability of the underlying disease and the expected consequences of the diagnostic procedure. While several biomarkers are available for neurodegenerative diseases, uncertainty has arisen regarding appropriate combination and/or order of application of these biomarkers as there is no consensus or theoretical framework concerning how they should be combined.

In this paper we aimed to propose a diagnostic algorithm suggesting the optimal timepoint for available PET- The common side effects (>1/100 and < 1/10) reported with fluorinated tracers are: injection site irritation and pain, flushing, increased blood pressure, and headache.

The common side effect of CSF sampling is postlumbar puncture headache that occurs in 1 to 10% of the investigations in memory clinic settings; [START_REF] Zetterberg | Low incidence of post-lumbar puncture headache in 1,089 consecutive memory clinic patients[END_REF][START_REF] Duits | Performance and complications of lumbar puncture in memory clinics: Results of the multicenter lumbar puncture feasibility study[END_REF] more serious side effects of lumbar puncture, such as infection or brain herniation, might occur in the clinical setting, albeit extremely rarely.

Variability of the measure across centres and methods

Low [START_REF] Mallik | Clinical Amyloid Imaging[END_REF] Used to be considerable, [START_REF] Mattsson | CSF biomarker variability in the Alzheimer's Association quality control program. Alzheimer's & dementia : the journal of the Alzheimer[END_REF] but recently commercialised fully automated assays might solve the problem. 16

Individual variability of values in healthy subjects

Low [START_REF] Mallik | Clinical Amyloid Imaging[END_REF] Quite high but could be corrected for by measuring changes over time and/or employing the ratio-based approaches. [START_REF] Zetterberg | Intra-individual stability of CSF biomarkers for Alzheimer's disease over two years[END_REF]

Sensitivity to detect change over time / in the disease course

Yes, but low: possibly fast accumulation during the negative to positive transition, [START_REF] Jack | Tracking pathophysiological processes in Alzheimer's disease: an updated hypothetical model of dynamic biomarkers[END_REF] followed by protracted increase before reaching a plateau No: Stable (studies show that CSF Aβ42 changes 10-20 years prior to dementia and remains stable during the clinical phase of the disease). [START_REF] Zetterberg | Intra-individual stability of CSF biomarkers for Alzheimer's disease over two years[END_REF][START_REF] Blennow | Longitudinal stability of CSF biomarkers in Alzheimer's disease[END_REF] Sensitivity for amyloid pathology 91-98% [START_REF] Clark | Cerebral PET with florbetapir compared with neuropathology at autopsy for detection of neuritic amyloid-β plaques: a prospective cohort study[END_REF][START_REF] Sabri | Florbetaben PET imaging to detect amyloid beta plaques in Alzheimer's disease: Phase 3 study[END_REF][START_REF] Salloway | Performance of [18F]flutemetamol amyloid imaging against the neuritic plaque component of CERAD and the current (2012) NIA-AA recommendations for the neuropathologic diagnosis of Alzheimer's disease[END_REF] 80-96% [START_REF] Shaw | Cerebrospinal fluid biomarker signature in Alzheimer's disease neuroimaging initiative subjects[END_REF][START_REF] Tapiola | Cerebrospinal fluid {beta}-amyloid 42 and tau proteins as biomarkers of Alzheimer-type pathologic changes in the brain[END_REF] Specificity for amyloid pathology 87-100% [START_REF] Clark | Cerebral PET with florbetapir compared with neuropathology at autopsy for detection of neuritic amyloid-β plaques: a prospective cohort study[END_REF][START_REF] Sabri | Florbetaben PET imaging to detect amyloid beta plaques in Alzheimer's disease: Phase 3 study[END_REF][START_REF] Salloway | Performance of [18F]flutemetamol amyloid imaging against the neuritic plaque component of CERAD and the current (2012) NIA-AA recommendations for the neuropathologic diagnosis of Alzheimer's disease[END_REF] 77-82% [START_REF] Shaw | Cerebrospinal fluid biomarker signature in Alzheimer's disease neuroimaging initiative subjects[END_REF][START_REF] Tapiola | Cerebrospinal fluid {beta}-amyloid 42 and tau proteins as biomarkers of Alzheimer-type pathologic changes in the brain[END_REF]

Information about N status

Possible with "early phase imaging" [START_REF] Florek | Dual Time-Point [18F]Florbetaben PET Delivers Dual Biomarker Information in Mild Cognitive Impairment and Alzheimer's Disease[END_REF] Available with total tau (but currently being questioned), might be with neurofilament levels but still need validation/confirmation/standardisation. [START_REF] Khalil | Neurofilaments as biomarkers in neurological disorders[END_REF]

Information about T status

None

Potentially available with phospho-tau levels

Information on extent of amyloidpathology

Available; distribution of amyloid pathology might indicate the amyloidosis stage. [START_REF] Grothe | In vivo staging of regional amyloid deposition[END_REF] Not available

Information on location of amyloidpathology

Available Not available

Potential to measure antiamyloid therapy effects

Conceivable

Conceivable with APP and Aβ peptides other than Aβ42 (BACE inhibitors would influence Aβ38 and -40 levels, as well as sAPPβ in the CSF). [START_REF] May | Robust central reduction of amyloid-β in humans with an orally available, non-peptidic β-secretase inhibitor[END_REF] Despite sometimes reflecting similar pathological processes, CSF and imaging biomarkers may possess complementary strengths and disadvantages. Certainly, the level of experience, establishment and standardisation as well as availability at a given centre will have to be considered with regard to the choice of the corresponding biomarker. Compared with amyloid-PET, CSF amyloid assessment is less expensive, and a conservative approach could be to use CSF sampling whenever possible. Then, amyloid-PET would only be necessary in a proportion of patients i) who refused the lumbar puncture (up to 10%); [START_REF] Duits | Diagnostic impact of CSF biomarkers for Alzheimer's disease in a tertiary memory clinic[END_REF] ii) in whom CSF sampling is contraindicated (about 5%); [START_REF] Nath | Atraumatic versus traumatic lumbar puncture needles: A systematic review and meta-analysis protocol[END_REF]

  These considerations are taken into account in the proposed algorithm indicating three different pathways with distinct sequences of tests for situations in which additional biomarker assessment is required after clinical assessment and structural imaging, with amyloid biomarkers, FDG-PET, or dopamine transporter (DaT)-SPECT being the preferred subsequent step depending on the clinical presentation. These different pathways are detailed in Figure2.

  biomarker together with logical and pragmatic considerations, the authors converged towards a diagnostic algorithm for the optimal combination of biomarkers depending on the clinical condition. The implementation of this algorithm in clinical practice will have to face challenges related with practicality, cost-effectiveness, ethical considerations, validation, controversies surrounding the underlying pathophysiological model and integration of future biomarker developments. There are currently numerous technologies, other tracers and biomarkers under development (see supplementary materials), which may open new diagnostic avenues and have an impact on the proposed algorithm. Importantly, advanced approaches/instrumentation such as integrated PET/MR imaging in combination with dual-phase PET acquisition may allow to acquire multimodal imaging in a one-stop manner, while remaining cost-effective and logistically feasible, thus omitting the need to decide on sequences of tests.[START_REF] Florek | Dual Time-Point [18F]Florbetaben PET Delivers Dual Biomarker Information in Mild Cognitive Impairment and Alzheimer's Disease[END_REF] Other most interesting future options include tau-PET which has recently obtained FDA-approval for one tracer in the US, synaptic density imaging, inflammation imaging, and blood biomarkers, as well as improvement of PET quantification methods or scanner equipment.ContributorsGC, JA, HB, VG, IL, SMo, and EvdG are part of the EANM Neuroimaging committee and the EANM Focus 2 Meeting Scientific Committee. JA, HB, VG, IL, SMo, and EvdG contributed equally to this work. FA, FB, DJB, MC, BD, AMF, GBF, OH, KH, BH, CJ, AL, SL, SMi, FN, AN, RO, WO, DP, GR, PS, VV, HZ, and AD were all members of the EANM Focus 2 expert panel, contributed to the content of the study, and reviewed the paper and PS also provided the case study.

Figure 1 .

 1 Figure 1. Examples of images obtained with structural MRI or PET using different radiotracers listed in order of the level of current clinical establishment versus primarily research-oriented application (from left to right). Top row shows healthy controls, while bottom row shows individuals with a clinical syndrome of late-onset Alzheimer's dementia. First column illustrates structural MRI-based z-score deviations of grey-matter volume in patients compared to controls displayed in blue on the right hemisphere of a template image. Second column shows [18F]FDG-PET glucose metabolism displayed as a 3D-surface projection of the right hemisphere with normal metabolism shown in yellow/red and reduced metabolism in green/blue. Third column shows amyloid-PET with [11C]PiB, displayed as a 3D-surface projection of the right hemisphere, with high amyloid burden indicated in yellow/red and no/low amyloid deposition in green/blue. Fourth column shows tau-PET with [18F]AV-1451, displayed as a 3D-surface projection of the right hemisphere, with high tau-tracer retention shown in yellow/red and no/low tau-tracer retention in green/blue (columns 1-4: images courtesy of University of Cologne, Germany). Fifth column shows TSPO-PET with [11C]PK11195, displayed as axial slices (caudal aspect, frontal to the top) with yellow/red showing elevated TSPO expression reflecting neuroinflammation (images courtesy of Prof. David Brooks, Aarhus University, Denmark). Sixth column shows SV2A-PET of synaptic density with [11C]UCB-J displayed as axial slices (caudal aspect, frontal to the top) with yellow/red showing normal synaptic density and green reflecting reduced synaptic density (images courtesy of Prof. Ming-Kai Chen, Yale School of Medicine, USA).

Figure 2 .

 2 Figure 2. Proposal for a diagnostic algorithm for early and differential diagnoses of dementia.

  [START_REF] Laforce | Molecular imaging in dementia: Past, present, and future[END_REF][START_REF] Frisoni | Strategic roadmap for an early diagnosis of Alzheimer's disease based on biomarkers[END_REF] Moreover, the choice of further diagnostic assessment should account for factors influencing pre-test probability (APOE genotype, family history, etc.,) as well as the potential consequences of the diagnosis. If consequences of the diagnosis are limited (e.g. other comorbidities dominating patient prognosis or lack of therapeutic alternatives in patients presenting with severe dementia) and once treatable conditions are excluded, there might not be a strong

reason to further strive to obtain an aetiological diagnosis with molecular imaging. Also, the diagnostic benefits of additional biomarker tests in the individual situation should be considered. For example, rates of amyloid positivity increase with age in APOE ε4 carriers, and the likelihood that the presence of amyloid is not responsible for cognitive decline or predictive of cognitive decline increases in very old APOE ε4 carriers.

[START_REF] Ossenkoppele | Prevalence of Amyloid PET Positivity in Dementia Syndromes[END_REF]

By contrast, a positive amyloid-PET scan is particularly helpful for ruling in AD in the differential diagnosis of early-onset dementia patients, since the a priori risk of incidental age-related amyloid pathology is low in young patients.

  financial support from Siemens Healthcare, outside the submitted work, and has a PSMA patent pending; BD reports personal fees from Biogen-Boehringer Ingelheim, grants from Merck Foundation for IM2A, outside the submitted work; KH reports grants from GlaxoSmithKline, outside the submitted work; BH reports other from GE Healthcare, other from Siemens, other from GSK, outside the submitted work; CJ consults for Lily and serves on an independent data monitoring board for Roche but he receives no personal compensation from any commercial entity. He receives research support from NIH and the Alexander Family Alzheimer's Disease Research Professorship of the Mayo Foundation, other from Lily, other from Roche, outside the submitted work; OH has acquired research support (for the institution) from Roche, GE Healthcare, Biogen, AVID Radiopharmaceuticals and Euroimmun. In the past 2 years, he has received consultancy/speaker fees (paid to the institution) from Biogen and Roche; SL reports personal fees from Cortexyme, Inc, personal fees from

	Research, Genzyme-Sanofi, IXICO Ltd, GeNeuro, and Apitope, grants for AMYPAD (IMI), EuroPOND
	(H2020), UK MS Society, Dutch MS Society, PICTURE (IMDI-NWO), NIHR UCLH Biomedical Research
	Centre (BRC), ECTRIMS-MAGNIMS, speakers' bureau for Biogen-IDEC and IXICO; DJB reports grants and
	personal fees from GE Healthcare, outside the submitted work; AD reports personal fees and non-financial

HB, MC, GC, AMF, VG, AL, IL, FN, AN, RO, WO, DP, and EvdG have nothing to declare. GC has received research support from the European Union's Horizon 2020 research and innovation program (grant agreement N °667696), Inserm, Fondation d'entreprise MMA des Entrepreneurs du Futur, Fondation Alzheimer, Programme Hospitalier de Recherche Clinique, Région Normandie, Association France Alzheimer et maladies apparentées and Fondation Vaincre Alzheimer (all to Inserm), and personal fees from Fondation d'entreprise MMA des Entrepreneurs du Futur. No stock options, patents, or royalties. GBF has received research funds and personal fees from GE Healthcare, Avid Lilly, and Life Molecular Imaging; FA reports personal fees from Elsevier INC, personal fees from Biogen Idec, personal fees from Novartis, outside the submitted work; JA reports grants from General Electric, grants from Piramal, grants from Lilly-Avid, personal fees from Biogen, personal fees and non-financial support from Araclon, personal fees and non-financial support from Advanced Accelerator Applications, personal fees from Bayer, outside the submitted work; FB reports consultancies for Bayer-Schering Pharma, Biogen-Idec, TEVA, Merck-Serono, Novartis, Roche, Jansen support from GE Healthcare, personal fees and non-financial support from Life Molecular Imaging (Piramal), personal fees and non-financial support from Lilly / Avid, during the conduct of the study, grants, personal fees and non-NeuroVision, outside the submitted work; SMi reports consultancy for Hamamatsu Photonics; SMo reports speaker's honoraria from GE Healthcare; GR reports grants from NIH, grants from Alzheimer's Association, grants from American College of Radiology, grants from Tau Consortium, grants from Michael J. Fox Foundation, grants from Association for Frontotemporal Degeneration, grants from Avid Radiopharmaceuticals, grants from Eli Lilly, grants from GE Healthcare, grants from Life Molecular Imaging, personal fees from Genentech, personal fees from Merck, personal fees from Eisai, from Lundbeck, outside the submitted work; PS reports other from Medavante, other from ProBiodrug AG, other from EIP Pharma, other from Novartis AG, other from Toyama, outside the submitted work; VV reports grants from Life Molecular Imaging, grants from Avid Radiopharmaceuticals, grants from GE Healthcare, outside the submitted work; HZ reports that he has served at scientific advisory boards of Roche Diagnostics, Samumed, Wave and CogRx and has given open lectures sponsored by Alzecure.

Table 1 . PET biomarkers for dementia.

 1 biomarkers, taking into account the other available established biomarkers, for early and differential diagnosis of neurodegenerative diseases that can lead to dementia. An interdisciplinary group of experts converge on a diagnostic algorithm indicating the recommended order of tests depending on the clinical presentation. Case vignette Mr. B is a 75-year-old man, who is presented to a neurologist by his wife because of progressive complaints of memory loss and occasional word finding difficulties. Despite his age, he still works as a tax-consultant and helps friends and family members with their finances. His "clients" notice that he has become slow in finishing the tax filings and recently some of them were returned because they were incomplete and full of mistakes. He was never very good with the computer, but lately his wife had to help him making wire transfers and writing emails. He has 2 children and 5 grandchildren, of which he sometimes mixes up the names. His wife reports that his gait has also become slower; he used to walk 3 meters ahead of her always during the weekly outings, but now he lags behind her.His MMSE score is 27/30 and the MOCA is 20/30, of which the latter is lower than expected, and indicative of executive dysfunction more than memory dysfunction. Because of a history of mild hypertension since he was 40 years and mild diabetes since 7 years, both well controlled, the neurologist orders an MRI, that shows bilateral hippocampal atrophy grade 2 on the Scheltens scale and grade 3 White Matter Hyperintensities on the Fazekas scale, with 5 lacunar infarcts in the deep white matter and striatum, indicative of severe small vessel disease. The neurologist doubts whether all the cognitive complaints and findings can be attributed to the vascular damage and Page 31 of 37 wants to rule out Alzheimer's as co-pathology, also in view of the worse prognosis of the combination of both pathologies. She orders an amyloid PET scan which comes back negative, ruling out AD co-pathology. She makes a diagnosis of pure vascular cognitive impairment and intensifies his vascular care and offers life style advice. Mr B and his wife are relieved that AD is ruled out and subscribe to a fitness program at their local gym and put themselves on a Mediterranean diet.

	Panel 3.

Table 2 : Comparison of amyloid-PET and amyloid CSF assessments.

 2

		PET imaging	CSF sampling
	Costs	Relatively high	Relatively low
	Contraindications	None	Treatment with anti-coagulants, spinal defects
	Side effects

  (about 20%).100,101 This statement is confirmed by the experience in a context in which both amyloid-PET and CSF procedures are accessible and reimbursed, such as in Sweden.102 However, other than imaging, CSF analysis may not answer a number of questions such as location and extent of pathology. Recent studies also show that amyloid-PET has incremental diagnostic value when performed after CSF evaluation.103,104 It may be discussed that performing amyloid-PET in the first line may spare patients multiple visits and unnecessary invasive interventions and that this may contribute to a more direct, more comprehensive and standardised workup, particularly in diagnostic reference centres such as memory clinics. Furthermore, disease follow-up and treatment specific therapy monitoring may only be possible by means of suitable neuroimaging biomarkers, as e.g. CSF markers would not provide conclusive information on change in extent of pathology or neurodegeneration over time. Finally, the topographic/regional information provided by PET offer valuable information about the earliest pathological stages of amyloid accumulation.[START_REF] Grothe | In vivo staging of regional amyloid deposition[END_REF]

or iii) for whom CSF results are inconclusive due e.g. to technical problems or values close to threshold Page 33 of 37

  The diagnostic work-up should start with a clinical/neuropsychological evaluation, followed by structural imaging when needed to establish reliable etiological diagnosis. Further workup could be halted, if clinical and structural imaging information both converge towards a specific diagnosis, e.g., in patients with memory predominant profile with typical hippocampal atrophy, possibly with positive family history and/or ε4-positive APOE genotype when available, and/or if consequences of the diagnosis are limited (e.g. other comorbidities dominating patient prognosis). However, if tailored therapy concepts are the aim and/or decisions depend on a conclusive diagnosis and prognosis, additional biomarker assessment is required (referred to in the figure as: "situations with need for further testing"; see also supplementary materials for further details). In pathway 1, preferred if the main suspicion is AD, analysis of amyloid pathology would be the subsequent next step. Depending on the result, FDG-PET may be additionally required to obtain further prognostic/diagnostic information, e.g. on the extent of neurodegeneration or on a specific pattern of hypometabolism or with regard to short-term prognosis in MCI.Pathway 2 is recommended if AD is not the single most probable/suspected diagnosis or for older (>75-80 years) individuals. If the result of the FDG-PET scan is conclusive, no further test might be requested (examples 1); in contrast, if the pattern of FDG-PET hypometabolism is not conclusive, or if the reliable clarification of neuropathology is clinically relevant, a further amyloid test might be necessary (examples 2). Finally, in Pathway 3, we would recommend a DaT-SPECT (or mIBG imaging) as the primary test for all situations in which a movement disorder/parkinsonian syndrome is clinically in question; in some cases (abnormal DaT-SPECT), no further test is required (example 3), but if further specification is needed (i.e. if the DaT-SPECT is normal, or, if it is abnormal, to differentiate between all neurodegenerative PS), then an additional FDG-PET is recommended, followed by amyloid-PET if AD remains a possibility. More details and examples are given in Supplementary materials. This algorithm is a theoretical proposal and further validation of the order of tests is needed (see text for details).

Acknowledgements

The authors acknowledge John Bean PhD (Bean Medical Writing, Halle, Belgium and funded by EANM) for providing medical writing services which included taking the minutes of the EANM Focus 2 meeting, amending

text of the article to include EANM Focus Meeting 2 content, editing drafts to improve the accuracy of language, flow, organization, structure, and overall readability, and checking for grammatical and spelling errors. The authors are also acknowledge Susanne Koebe, Andreas Felser, Janine Effey, Petra Neubauer, Hanna Krippl, and Henrik Silber (EANM) for project management. This research was funded by the European Association of Nuclear Medicine and supported by unrestricted grants from Siemens Healthineers, Biogen, Cerveau Technologies, Life Molecular Imaging, and Lilly. These sponsors had no direct or indirect influence on the programme and content of the EANM Focus 2 meeting and the writing or content of this article. FB and BH are supported by the NIHR University College London Hospitals Biomedical Research Centre. The AMYPAD project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 115952. The IDEAS study was funded by the Alzheimer's Association, the American College of Radiology (ACR), Avid Radiopharmaceuticals, Inc. (a wholly owned subsidiary of Eli Lilly and Company), General Electric Healthcare and Life Molecular Imaging (formerly Piramal Imaging). The U.S. Centers for Medicare and Medicaid Services provided coverage for amyloid-PET scans in DEAS under coverage with evidence development.

Panel 4. Take-home messages

• PET neuroimaging biomarkers have an important role in the diagnosis workup of neurodegenerative diseases leading to dementia.

• Amyloid PET is most useful to prove or rule out AD and provide specific information on the underlying pathology.

• FDG-PET has great value for differential diagnosis of neurodegenerative diseases, prediction of shortterm outcome, and staging of neurodegenerative processes.

• Depending on the clinical presentation, specific combinations of biomarkers are of significant added value for early and differential diagnosis.


Aurélien Louvrier
email: au.louvrier@gmail.com

N Sigaux

C Meyer

M Benassarou

J C Lutz

Customized three-dimensionally printed mandibular external fixator

2 Key words: external fixator; 3D printing; computer-aided design; computer-aided manufacturing.

Abstract. The advent of customized three-dimensional (3D) printing allows the affordable manufacturing of sophisticated medical devices, thereby providing swift and simple solutions to specific needs in modern healthcare. Meanwhile, certain devices such as industrial mandibular external fixators (EFs) have become less and less available from medical device companies because of decreased indications. What is more, their handling is often complex. The authors report, step by step, the original design and uneventful clinical use of a 3D-printed, customized mandibular EF. This device was designed together with a positioning and drilling guide for the fixation of a septic mandibular pseudarthrosis. It provided an adequate and satisfactory balance between lightness and rigidity. A simple, accurate and safe placement of the EF was achieved thanks to the skin-supported positioner and drilling guide, thereby making the procedure minimally invasive and time-efficient. To our knowledge, this is the first reported clinical use of a 3D-printed, customized mandibular EF to date. Because such 3D technology is becoming increasingly available to a large number of surgeons, the authors believe that the present innovation could become an alternative to reusable standard EFs.

Indications for mandibular external fixators (EFs) are nowadays limited to the stabilization of lesions occurring in a compromised bone environment: pathologic fracture, septic pseudarthrosis and comminuted fractures [START_REF] Cornelius | External pin fixation for stabilization of the mandiblecomeback of a method: historical review and first experiences with the 'mandible external fixator[END_REF]. Because of the low frequency of such indications in modern healthcare systems, EFs have become difficult to procure from medical device companies. Moreover, commercial mandibular EFs are often complex because of their many parts which require high maintenance in order to verify the system's integrity. Industrial devices are sometimes difficult to adapt to specific situations and require a somewhat wide surgical approach in order to ensure the proper positioning of pins.

Since the first reported use of three-dimensional (3D) printing in maxillofacial surgery [START_REF] Mankovich | The display of three-dimensional anatomy with stereolithographic models[END_REF], this technology has become increasingly available to a large number of surgical teams over the past 10 years [START_REF] Louvrier | How useful is 3D printing in maxillofacial surgery?[END_REF]. This is due to the rapid development of affordable printers and the availability of opensource, computer-aided design software allowing patients 'bedside' printing.

The authors report, step by step, the original design and clinical use of a customized mandibular EF and its positioning/drilling guide, 3D-printed in the department of maxillofacial surgery. This technical innovation was designed for the fixation of a septic mandibular pseudarthrosis in a patient who had previously undergone radiotherapy for a squamous cell carcinoma of the tongue 3 years previously.

Technical note

A computed tomography (CT) scan was performed systematically to confirm the diagnosis of mandibular septic pseudarthrosis (Fig. 1). The surgical approach was intended to be as limited as possible and therefore, a customized 3D-printed mandibular EF together with a positioning and drilling guide were designed and manufactured according to the following procedure. The DICOM files from the patient's mandibular CT scan were converted to STL files using InVesalius 3.0® software (CTI, Campinas, São Paulo, Brazil). Surgical planning was conducted using Meshmixer® software (Autodesk, Mill Valley, CA, USA) (Fig. 2). It consisted of the insertion of three standard 3-mm pins (Stryker, Kalamazoo, MI, USA) on each side of the pseudarthrosis site whilst preserving the inferior alveolar nerve and the teeth apices. These pins were then virtually inserted through holes into two lateral, square-section hollow bars. These two lateral bars were secured with a third anterior bar, thereby completing the body of the EF. A mandibular skin-supported, customized positioning and drilling guide was then designed in order to accurately set the fixator. It was mainly intended to maintain a safe distance between the fixator and the skin, to guide the placement of the fixator and the percutaneous placement of the pins. This skin-supported guide encompassed the entire base of the mandibular body and chin so as to ensure good accuracy when placed on the patient and good peroperative stability. It was engineered with three slots on each side, allowing pin insertion. The guide also featured three flattened sections designed to support the bars of the fixator, thereby temporarily securing the two devices during positioning (Fig. 3). Both resulting files were then transferred to an Ultimaker®2 printer, (Ultimaker, Geldermalsen, Netherlands) to manufacture acrylonitrile butadiene styrene plastic devices.

The surgical procedure was conducted under general anaesthesia. A minimally invasive endobuccal approach was realized in order to perform plate and screw removal, bone curettage and avulsion of the mobile teeth in the front of the pseudarthrosis site. The operating site was washed before a mucosal closure. Bone samples were systematically collected for histological and bacteriological analysis. After dental occlusion checking, the skin-supported positioner and the body of the mandibular EF was then used as a drilling guide in order to insert the six pins into the bone percutaneously, through the holes in the lateral bars and the slots in the positioner. Each pin with a 10-mm working length was inserted into the bone over a 10-mm depth and secured in the mandibular EF using both a 2.3-mm-diameter screw tightened through the bar, and surgical cement injected into the two lateral hollow bars. Finally, the appropriate positions of the bone fragments and of the mandibular EF were confirmed by clinical examination (Fig. 4) and control CT-scan (Fig. 5).

Figures

Fig. 1 .

 1 Fig. 1. Preoperative computed tomography scan (three-dimensional reconstruction) showing a right

Fig. 2 .

 2 Fig. 2. Computer-aided-design of the mandibular external fixator and the skin-supported guide using

Fig. 3 .

 3 Fig. 3. Mandibular skin-supported positioning/drilling guide (lateral view).

Fig. 4 .

 4 Fig. 4. Patient with a customized three-dimensionally printed mandibular external fixator in position.

Fig. 5 .

 5 Fig. 5. Control computed tomography scan (axial view) showing the position of the pins into the bone

Fig. 6 .

 6 Fig. 6. Control computed tomography scan (3D reconstruction) after a 1-year follow-up showing

Acknowledgments. The authors thank Dr Olivier Mauvais, Dr Elise Weber for their clinical involvement.

Funding

This work was not funded.

Standard wound care was initiated postoperatively along with pain medication, prophylactic antibiotic treatment, antiseptic mouthwash and an exclusive enteral nutrition by nasogastric tube was prescribed for 2 weeks. Antibiotic treatment was adapted after bacterial identification in the bone samples. After 14 weeks, once healing of the pseudarthrosis was confirmed clinically and radiologically, the 3D-printed mandibular EF was removed under local anaesthesia. No recurrence of the pseudarthrosis was observed after a 32-month follow-up (Fig. 6).

Discussion

Customized medical 3D-printing allows for greater freedom and versatility in the development of innovative devices. The reported 'bedside' 3D-printed, customized mandibular EF provided an adequate and satisfactory balance between lightness and rigidity which was optimized by the injection of surgical cement into the two lateral hollow bars during the surgical procedure. Among the many advantages, the authors emphasize the simple, accurate and safe placement of the EF thanks to the skin-supported positioner and drilling guide, thereby making the procedure minimally invasive and time-efficient. To ensure proper peroperative positioning and stability, it is important that the guide includes the entire base of the mandibular body and the chin; in this way, the positioning of the skin-supported positioner and drilling guide is undeniable. The percutaneous placement of the pins is also an important point; the introduction must be gentle and the operator must control the absence of the lower lip mobility to ensure that the marginal branch of the facial nerve is not on the way; this precaution remains the same with the use of a conventional mandibular EF. Because 3D technology has become increasingly available to a large number of surgeons, the authors believe that the present innovation could become an alternative to reusable, standard EFs.

Competing interests

Ethical approval

This work was approved by the Ethical Committee of the Faculties of Medicine and Dentistry of Strasbourg and of the University Hospital. References: FC/file 2019/37.

Patient consent

Written patient consent was obtained.


Pauline Henrot
email: pauline.henrot@u-bordeaux.fr

François Moisan

Julien Seneschal

Alain Taïeb

Muriel Cario

Marie-Elise Truchetet

M.-E Truchetet

Is Hyperpigmentation in Systemic Sclerosis a Perivascular Dermal Tattoo?

Keywords: dermal tattoo? 15, Figure: 1, Table: 1 Systemic Sclerosis

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

TO THE EDITOR

Sir, Systemic Sclerosis (SSc) is a rare but severe disease whose pathophysiology remains partly unknown, combining autoimmune features, vasculopathy, and systemic fibrosis [START_REF] Gabrielli | Scleroderma[END_REF]). Vascular involvement is a major issue: digital ulcers (DUs) are a biomarker of disease severity and higher mortality [START_REF] Hughes | Digital ulcers in systemic sclerosis[END_REF][START_REF] Meunier | Increased risk of mortality in systemic sclerosis-associated digital ulcers: a systematic review and meta-analysis[END_REF]; pulmonary hypertension and renal crisis are potentially lethal and silent at an early stage. Thus, better characterizing patients at risk is a crucial matter. In this context, we focused on pigmentary disorders in SSc, which affect about half of the patients and can impact their quality of life [START_REF] Jewett | Sociodemographic and Disease Correlates of Body Image Distress among Patients with Systemic Sclerosis[END_REF]. Recently, our group observed a link between the presence of diffuse hyperpigmentation and vascular involvement in SSc, especially DUs [START_REF] Leroy | Association of skin hyperpigmentation disorders with digital ulcers in systemic sclerosis: Analysis of a cohort of 239 patients[END_REF]). Here, we reveal microscopic distribution of melanin in SSc skin, among patients presenting or not hyperpigmentation.

In this observational cross-sectional study, we characterized arm skin melanin distribution in 26 SSc patients in a blinded manner. Written informed consent was obtained from all participants and the study was approved by the institutional ethical committee (CPP, 2012 A00081-42, Aquitaine). SSc patients were recruited in a case-control manner in order to compare melanin distribution between hyperpigmented or not SSc patients. Demographic and disease characteristics of SSc patients are presented Table 1. 14 (54%) of SSc patients presented diffuse hyperpigmentation (illustration: Figure 1a), accompanied with hypopigmented macules for 7 patients. The hyperpigmented group included a significantly higher proportion of diffuse cutaneous SSc (DcSSc) forms, as already reported [START_REF] Leroy | Association of skin hyperpigmentation disorders with digital ulcers in systemic sclerosis: Analysis of a cohort of 239 patients[END_REF], possibly due to the association between DcSSc and dark phototypes [START_REF] Gelber | Race and association with disease manifestations and mortality in scleroderma: a 20-year experience at the Johns Hopkins Scleroderma Center and review of the literature[END_REF]; indeed, this group tended to include darker phototypes than the "no pigmentary disorders" group, without reaching statistical significance (p=0.0809), which is consistent with the literature [START_REF] Reveille | Systemic sclerosis in 3 US ethnic groups: A comparison of clinical, sociodemographic, serologic, and immunogenetic determinants[END_REF].

Importantly, the proportion of patients with clinical vascular involvement was not statistically different between the two groups, which allows to avoid a potential bias related to the previously reported association between hyperpigmentation and vascular involvement [START_REF] Leroy | Association of skin hyperpigmentation disorders with digital ulcers in systemic sclerosis: Analysis of a cohort of 239 patients[END_REF].

In brightfield images, we observed a striking pattern of dark brown pigment deposit in the dermis of 8 SSc patients, particularly lining dermal vessels. We could not easily evidence any pigmented deposit in the superficial dermis as its thickness was greatly reduced [START_REF] Henrot | Decreased CCN3 in Systemic Sclerosis endothelial cells contributes to impaired angiogenesis[END_REF]). CD31 staining allowing the identification of endothelial cells confirmed that pigment was specifically located near the outside layer of dermal vessels (Figure 1b). We did not find any evidence of perivascular pigment around lymphatic vessels (Figure S1a). Fontana-Masson stain showing black pigment pointed out that the dermal pigment was at least partly constituted by melanin (Figure 1c), as supported by high-power view showing melanin granules (Figure 1c and Figure S1b). Perls and Sudan black B stains were negative, ruling out respectively haemosiderin and lipofuscin presence in the perivascular pigmented deposit (Figure S1c,d). Finally, bleaching with hydrogen peroxide completely washed out the perivascular pigment, confirming the melanin content (Figure S1e). CD68 immunostaining identifying macrophages showed that in some cases, the pigment was inside the cytoplasm of dermal melanophages, but not always (Figure 1d). Among SSc patients, those that exhibited such a pattern of perivascular pigmentation all belonged to a subgroup associating hyperpigmentation and vascular involvement (defined as presence of DUs and/or pulmonary hypertension and/or renal crisis) (Figure 1e). Among hyperpigmented SSc patients, presence of this histological pattern was significantly associated with vascular involvement (p=0.0063, chi2). Overall, the specificity of this histological pattern to indicate vascular involvement was 100% in our cohort; moreover, among SSc patients with clinical hyperpigmentation, sensitivity of the pattern to indicate vascular involvement was 80%.

Demographic and disease characteristics of the 8 SSc patients that presented this pattern are presented in Supplementary Table 1. This finding raises several points. First, such dermal pigmentation could account for the persistent tanning that some patients report, maybe due to the absence of pigment clearance once in the dermis (dermal tattoo) -it could also explain the peculiar skin color ("copper-like") observed in some patients. In this case, clinical hyperpigmentation would be explained by dermal pigmentation rather than increased epidermal pigmentation, and some samples even exhibited a marked decrease in epidermal pigmentation (Figure S1f).

Secondly, this histological pattern is probably not restricted to SSc skin, and dermal pigment is a common phenomenon among dark phototypes [START_REF] Ortonne | Latest Insights into Skin Hyperpigmentation[END_REF], including in the case of post-inflammatory hyperpigmentation, although the perivascular location has not been commonly described. However in our case, dermal pigmentation was also observed in fair phototypes; moreover, such pigment deposit was only incidentally seen in a cohort of 26 healthy control skin samples matched on sample location and phototype; overall, intensity of pigment deposit around dermal vessels was significantly higher in SSc patients compared to healthy controls (Figure S1g; p=0.0337).

Thirdly, the specific perivascular localization of pigment deposit suggests, in an original way, a pathophysiological relationship between vasculature and pigmentation, as seen in other diseases such as melasma [START_REF] Passeron | Long-lasting effect of vascular targeted therapy of melasma[END_REF]) and solar lentigo [START_REF] Hasegawa | Increased blood flow and vasculature in solar lentigo[END_REF], and recently highlighted by our group in SSc [START_REF] Leroy | Association of skin hyperpigmentation disorders with digital ulcers in systemic sclerosis: Analysis of a cohort of 239 patients[END_REF]. This also raises the question of the origin of the pigment deposit. It is unlikely that the observed deposits are the result of drugs inducing dermal pigmentation, such as hydroxychloroquine; indeed, pigmentary changes usually differ clinically (bluish-green macules) and microscopically (pigmented deposit Perls-positive and in the superficial dermis) [START_REF] Jallouli | Hydroxychloroquineinduced pigmentation in patients with systemic lupus erythematosus: a case-control study[END_REF], although a Perls-negative perivascular deposit has already been reported [START_REF] Puri | Hydroxychloroquine-induced hyperpigmentation: the staining pattern[END_REF]. However and most importantly, of the 8 patients that presented this pattern, 3 had no history of current or past drug uptake known to modify skin pigmentation, including hydroxychloroquine. In any case, the similar distribution suggests a shared mechanism, possibly triggered by a circulating factor. Thus, one could imagine an enhanced secretion of melanogenic factors by SSc endothelial cells, such as endothelin-1 [START_REF] Regazzetti | Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation[END_REF], combined to the loss of epidermal melanocytes due to an adhesion defect, subsequently falling into the dermis, similarly to vitiligo [START_REF] Picardo | A New View of Vitiligo: Looking at Normal-Appearing Skin[END_REF]. Dermal melanin would then be phagocyted by macrophages (Figure 1d) migrating towards the nearest vessel. However, this hypothesis does not fully explain the presence of melanin inside the cytoplasm of CD68-negative perivascular cells. The nature of these cells remains to be elucidated; notably, one should consider the possibility of a local melanin production by transdifferentiation of perivascular cells.

Altogether, perivascular melanin could help to detect vascular involvement specifically among SSc patients presenting clinical hyperpigmentation. However, this finding needs to be assessed in larger cohorts of hyperpigmented SSc patients in order to validate the association with clinical vascular involvement. Moreover, the cross-sectional design of the study does not allow determining if the perivascular melanin deposit occurred before or after the development of the vascular injury. Prospective studies are warranted in order to address the possibility of this histological pattern to be used as a predictive marker. Of note, despite various duration of pigmentary changes in SSc patients, we did not see any correlation between the intensity of the pigmented deposit and disease duration (data not shown). Nevertheless, this does not refute the hypothesis that the pigmented deposit occurs early in the development of the disease. Future research should also focus on the mechanism of melanin deposit in the dermis, particularly this peculiar localisation around dermal vessels, to further establish the link between hyperpigmentation and vasculopathy.

TABLES Table 1: characteristics of included SSc patients.

Phototype: according to Fitzpatrick's classification. Systemic therapies: current or past (whatever the duration). NSAID: Non-steroidal anti-inflammatory drugs (if long-term treatment). (e): Flow-chart of SSc patients repartition in our cohort. NVasc = no vascular involvement (defined as presence of DUs and/or pulmonary hypertension and/or renal crisis), Vasc = vascular involvement. Among hyperpigmented SSc patients, presence of this histological pattern is significantly associated with vascular involvement (p=0.0063, chi2).

FIGURE LEGEND

Figure 1 :

 1 Figure 1: SSc patients presenting hyperpigmentation and vascular involvement specifically

Acknowledgements

This work was funded by Association des Sclérodermiques de France and Société Française de Dermatologie. The authors wish to thank Dr Hugues Begueret, from the Pathology Department of Bordeaux University Hospital, for providing the Perls stain, as well as Catherine Pain for technical assistance, and all the clinicians and patients who took part in the SSc-VISS cohort.

CRediT statement

Conceptualization and writing -original draft: PH, MET and MC; investigation: PH, MC and FM; formal analysis: PH; resources: MET and JS; supervision: MET, AT and MC; visualization: PH and FM; writing-review and editing: PH, FM, JS, AT, MC and MET. All authors read and approved the final manuscript.

Funding: This work was conducted thanks to grants from Association des Sclérodermiques de France and Société Française de Dermatologie.

Data Availability statement

No datasets were generated or analyzed during the current study.

Conflict of interest: None.

Keywords: scleroderma, pigmentary disorders, melanin, dermal vessels

Conflict of Interest

The authors state no conflict of interest.