The Carotid Artery Multi-modality Imaging Prognostic (CAMP) Study
The Carotid Artery Multi-modality Imaging Prognostic (CAMP) Study
Carotid artery disease is a main cause of ischemic stroke and vascular dementia, and a highly
 prevalent disease. There is uncertainty about the optimal management of patients with
 serendipitously or systematically detected asymptomatic carotid artery disease, due to the
 paucity of information on the predictive features of serious vascular events. While percent
 diameter stenosis is currently the accepted standard to decide about local interventions
 (carotid artery stenting or endarterectomy), international guidelines also recommend the
 evaluation of qualitative features of carotid artery disease as a guide to treatment. There
 is, however, no agreement on which qualitative features are best predictors of events.
 Furthermore, a role for metabolic plaque profile, local mechanical and hemorheologic factors
 in triggering microembolization and silent ischemic events has been proposed from
 experimental studies. This inadequate knowledge leads to a poor ability to identify patients
 at higher risk and to an unwarranted dispersion of medical resources, lack of standardization
 in diagnostic methods, and the use of expensive and resource-consuming techniques.

 Against this background, the investigators aim at:

 1. Prospectively identifying the best predictors of (silent and overt) ischemic stroke and
 vascular dementia in patients with asymptomatic subcritical carotid artery disease, by
 identifying the non-invasive diagnostic features of the "vulnerable carotid plaque" as a
 possible guide for optimal - local and systemic - treatment.

 2. Transferring new ultrasound techniques possibly improving risk prediction to the
 clinical field

 3. Assess whether "smart", low-cost diagnostic methods, such as ultrasound-based
 evaluations integrating established and advanced techniques, may yield at least the same
 level of prospective information as more expensive and less cost-effective techniques.
BACKGROUND AND RATIONALE

 State of the art and preliminary data Stroke is a most relevant health burden, the second
 most common cause of death and the third cause of disability worldwide. Stroke is a
 heterogeneous disorder with several disease mechanisms implicated in its pathogenesis.
 Ischemic stroke accounts for 87% of all stroke cases. The TOAST (Trial of Org 10172 in Acute
 Stroke Treatment) and the more recent SSS-TOAST (Stop Stroke Study TOAST) classifications
 currently provide the most widely used schemes of stroke classification for clinicians.
 Accordingly, pathomechanisms comprise: (a) large artery atherothrombosis (i.e., a major brain
 artery or branch cortical artery); (b) cardioembolism; (c) small vessel occlusion (lacunar
 infarcts); (d) other determined etiology (i.e., hypercoagulable states, hematologic
 disorders); (e) undetermined etiology (cryptogenic embolism; incomplete patient evaluation).

 Silent stroke, usually defined as focal T2 hyperintensities >3 mm with correlative T1
 hypointensities at magnetic resonance imaging (MRI), has a reported incidence of 10-15% and a
 relevant significance in terms of cognitive impairment and correlations with clinically overt
 stroke. Silent stroke is a cerebrovascular ischemic (less frequently haemorrhagic) lesion
 detected by neuroimaging such as magnetic resonance imaging (MRI) that does not determine
 acute focal neurological deficits but causes cerebral damage in an unaware way. Typically,
 silent stroke (SS) affects regions associated with various mental processes, mood regulation
 and cognitive functions, and therefore is a leading cause of vascular cognitive impairment
 that impacts about one-third of individuals over the age of 70. Cerebral small vessel disease
 related to aging (and possibly to age-related arterial stiffness) leading to leucoaraiosis
 and lacunar infarctions is a main cause of SS, but also microembolic lesions due to atrial
 fibrillation and carotid stenosis may be involved.

 The diagnosis of SS stroke may be challenging. In many cases, subjects are not diagnosed
 until long after a stroke occurs or in case of progressive cognitive impairment.
 Occasionally, strategic lesions in cerebral areas related to cognitive function, the
 hippocampus or the thalamus, and, more frequently, incremental lesion load over time lead to
 vascular dementia (VD). VD is is the second most common form of dementia after Alzheimer's
 disease. The prevalence of the illness is about 5% in Western countries. Current
 epidemiologic research suggests that AF contributes to cognitive decline and dementia
 independent of a history of stroke. Further work is warranted to elucidate the potential
 mechanisms underlying this association, and better designed studies are needed to explore the
 possible cognitive benefits of different therapeutic options in patients with carotid
 disease. Potential pathophysiological pathways linking carotid disease with cognitive decline
 are microembolism, systemic inflammation, and cerebral hypoperfusion.

 STUDY DESIGN This is a monocenter, prospective, longitudinal, observational study. This is a
 no-profit study, granted by Regione Toscana - Bando Salute 2018.

 Setting Eligible participants (see inclusion and exclusion criteria below) will be selected
 and recruited in the outpatient setting of the UO Cardiologia 1 - AOUP. Exclusion and
 inclusion criteria will be evaluated according the previous medical history and imaging tests
 of the participant. All participants identified as eligible will receive all the necessary
 information about the study and will sign the informed consent under the supervision of the
 Principal Investigator. In case of enrollment, a detailed plan will be prepared for each
 participant to schedule the study evaluations to be integrated with the routine examinations.

 STUDY OUTCOMES Variables

 Different variables will be assessed and analyzed, in particular:

 1. Baseline neurological, psychological and neurocognitive evaluation

 2. Baseline brain MRI evaluation

 3. Angio-MRI for carotid stenosis assessment and plaque tissue characterization

 4. Angio-CT evaluation for carotid stenosis assessment and plaque characterization

 5. Multigate Doppler (MGD) Investigation of Carotid Artery Stenosis

 1. Carotid stenosis assessment

 2. Wall shear rate and flow separation analysis

 6. Advanced ultrasound techniques for plaque vulnerability and local mechanical properties
 assessment a. Ultrasonographic markers of plaque composition b. Validation of 3D US for
 carotid plaque stenosis severity and morphology assessment c. Radiofrequency data for
 assessment of fibrous cap thickness, plaque deformation, and local arterial waveform and
 stiffness

 7. Follow-up (2-years) brain MRI evaluation

 8. Follow-up (2 years) neurological, psychological and neurocognitive evaluation

 1. Baseline neurological, psychological and neurocognitive evaluation

 Each participant will be evaluated at baseline and at the 2-year follow-up with a
 detailed medical history and physical examination. A detailed assessment of cognitive
 decline/dementia will be performed as below detailed:

 - Montreal Cognitive Assessment (MoCA), a brief screening tool wide used and sensitive
 for mild cognitive impairment. Moreover, performing a subtests analysis of MoCA results
 will allow to analyse performance visuospatial and executive functions, attention, and
 recall.

 -  Adopting a more focus on specific cognitive functions, and following results of two
 recent studies (The Tromso study - Mathiesen et al., 2004; Cheng et al., 2012) in
 asymptomatic carotid stenosis, the investigators will also assess:

 • Working memory: Backward Digit Span

 • Attention and psychomotor speed: Trail Making Test (Parts A and B)

 • Motor functioning: Grooved Pegboard Test

 • Memory: Verbal and Visual Paired Association (immediate and delayed recall -
 subtest of the Wechsler Memory Scale)

 -  Complex visuospatial perception: Modified Complex Figure Test (copy and
 recall)

 -  Finally, it is also important to evaluate subjective dizziness and changes in
 cognitive status, proved sensitive to carotid stenosis, using two brief self-report
 questionnaires:

 • The Dizziness Handicap Inventory

 • The Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE)

 -  The total assessment time will be around 30 minutes. The Clinical Psychology
 Unit will be responsible for preliminary tutorship of Centers about
 questionnaires administration, and subsequent centralized analysis.

 2. Baseline brain MRI evaluation Silent brain infarction will be defined as focal T2
 hyperintensity >3 mm with correlative T1 hypointensities at MRI. This evaluation will be
 matched with the similar 2-year follow-up examination, to assess the occurrence of
 silent strokes.

 3. Angio-MRI for carotid stenosis assessment and plaque tissue characterization Angio-MRI
 of carotid arteries will be performed at baseline and after a two-year follow-up for
 carotid plaque characterization.

 4. Angio-CT evaluation for carotid stenosis assessment and plaque characterization
 Estimation of lumen stenosis and vessel anatomy with contrast agents. Specific
 algorithms (quantitative evaluation of calcifications - lipid core - haemorrhage): the
 two main CT techniques for plaque characterization here used will be multi-detector-row
 CT and dual-source CT. Multi-detector-row CT will characterize plaque calcification,
 ulcerations, fibrous plaque thickness, intra- plaque haemorrhage and the presence of
 lipid-rich necrotic cores, based on voxel Hounsfield units density (HUs).

 5. Multigate Doppler (MGD) Investigation of Carotid Artery Stenosis The University of Pisa
 has a long-standing collaboration with the Microeletronic System Design Lab (MSD-Lab) at
 the Department of Informational Engineering (DINFO) of Florence University, based upon
 an ultrasound advanced open platform (UlAOP) providing a detailed representation and
 quantification of flow velocity profile and wall shear rate by means of multiple
 spectral Doppler sample volumes (128) aligned perpendicularly through the longitudinal
 arterial axis.

 a) Carotid stenosis assessment A customized configuration developed in Florence will be
 used to provide in real time a simplified flow velocity profile in common and internal
 carotid artery, using 7 Doppler vectors aligned through the vessel transverse axis,
 before, within and after the plaque. This approach allows to overcome the assumption of
 a fully parabolic flow velocity profile through the vessel, which is instead variably
 skewed in the presence of curvatures and stenosis. The correlation of central flow
 velocity, peak flow velocity and flow velocity distribution with severity and shape of
 carotid stenosis as assessed by angio-TC will be investigated. The association of flow
 velocity values and skewness with MES and silent brain ischemic lesions will be also
 assessed.

 b) Wall shear rate and flow separation analysis By post-processing of MGD datasets, wall
 shear rate, oscillatory flow velocity patterns and retrograde flows will be estimated
 using a computational fluid dynamics (CFD) approach. The association of these parameters
 with MES and baseline and incident silent brain ischemic lesions will be assessed. This
 evaluation will be performed in a subset of 100 participants recruited in Pisa, no more
 than 2 months after the angio-TC evaluation

 6. Advanced ultrasound techniques for plaque vulnerability and local mechanical properties
 assessment After the baseline investigation, in a subset of participants, the ultrasound
 investigation of carotid arteries and plaques will be repeated by US system providing
 radiofrequency raw data for post-processing analysis. Conventional 2D US scan as well as
 acquisition of the RF-data sets will be performed. Two-dimensional images will be
 exploited for tissue characterization analysis; RF- datasets will be used to determine
 fibrous cap thickness, local plaque deformation, arterial contour- wave analysis with
 central pulsatile pressure and excess pressure estimate. This evaluation will be
 performed at baseline no more than 2 months after the angio-TC evaluation, and then
 repeated at the two-year follow-up.

 1. Ultrasonographic markers of plaque composition Carotid plaque characterization will
 be performed on 2D images at Lund University, by means of a customized software
 recently validated against immunohistochemistry by Nilsson J and Goncalves I within
 the context of a FP7 collaborative study on diabetes (IMI-SUMMIT project), with the
 participation of the University of Pisa (Palombo C et al).

 2. Validation of 3D US for carotid plaque stenosis severity and morphology assessment
 Plaque volume and shape assessment (concentric/eccentric, regular, irregular) will
 be assessed with a dedicated 3D probe implemented on a Philips cardiovascular US
 platform in 50 participants. After acquisition of a 3D dataset (Fully Automated
 Mechanical Transducer), including total plaque volume and maximum area reduction.
 Total ulcer volume and size will be integrated. Off-line texture analysis will be
 performed on a dedicated subset of images. Beside conventional 2D ultrasonographic
 assessment of stenosis severity (including Doppler findings and 2D findings),
 advanced ultrasonographic examinations will be performed with the aim of
 quantitative data acquisition on tissue characterization, detection of
 neovascularity, detection of total plaque burden (volume 3D), plaque deformation
 and flow profiling. This subproject will be performed only at baseline.

 3. Radiofrequency data for assessment of fibrous cap thickness, plaque deformation,
 and local arterial waveform and stiffness The RF-dataset provided by the Esaote
 systems will allow estimate of common carotid distensibility and plaque fibrous cap
 thickness and deformation thanks to an array of 16 parallel lines crossing the
 vessel longitudinal axis through 14 mm, with high spatial (10 μm) and temporal (660
 Hz) resolution. A pressure- calibrated local arterial distension waveform is also
 obtained, from which indices of local stiffness, pulsatile pressure, including the
 so-called "excess pressure", and wave reflection can be assessed.

 7. Follow-up (2-years) brain MRI evaluation This examination will be performed at the
 2-year follow-up and matched with the baseline examination, to assess new incident
 events. Silent stroke will be similarly defined as focal T2 hyperintensity >3 mm with
 correlative T1 hypointensities at MRI. At this visit, MRI will be limited to the brain,
 without carotid reassessment

 8. Follow-up (2 years) neurological, psychological and neurocognitive evaluation This will
 be done with a complete physical examination and the use of dedicated scales to perform
 an evaluation of signs of silent stroke or cerebral involution, as per item 1.

 Tools/equipment: questionnaires. Expected results: assessment of possible neuropsychologic
 and cognitive status changes after two years + correlations between changes in
 neuropsychologic and cognitive status after follow-up and MES, carotid plaque features, local
 arterial mechanics, and atherosclerotic risk profile at baseline (longitudinal study) +
 correlations between changes in neuropsychologic and cognitive status after follow-up and
 changes in silent brain ischemic lesions (MRI), and in carotid plaque features and local
 arterial mechanics by ultrasound, conventional and advanced (longitudinal study).

 DATA MANAGEMENT Data collection All variables listed above will be safely reported on the
 case report form, collected and stored, including some self-reported questionnaires, i.e. the
 Dizziness Handicap Inventory and the Informant Questionnaire on Cognitive Decline in the
 Elderly (IQCODE).

 Data management All data will be managed in compliance with the General Data Protection
 Regulation (GDPR). A dedicated platform (Data Platform) in full compliance with the GDPR
 rules and the Italian Legislative Decree no. 196 dated 30/06/2003 will be developed. Digital
 and paper-based case report forms will be generated to collect participants' data. Data will
 be then uploaded on the Data Platform for safe storing by using cloud technologies and
 secured access credentials and protocols (TLS / HTTPS). All necessary measures to ensure full
 protection of data will be applied.

 Pseudonymisation will be guaranteed by a secured alphanumeric code, whose key will be
 password-protected and available only to the study investigators.

 Data storage The principal investigator (Prof. Raffaele De Caterina) will be responsible for
 data collection, management, storage and pseudonymisation.

 Statistical analysis Correlation and regression analysis will be performed in uni- and
 multivariate manner, after correction for collinearity. Bland-Altman Analysis will assess the
 predictive power and concordance of each methods for specific plaque features. Logistic
 regression analysis, Cox regression analysis and survival analysis will be performed
 according to different predictors, after determination of cut-off values for single
 continuous variables. Incremental analysis (Chi-square; Net reclassification) will be
 performed in order to assess the integrated value of combined (multi-imaging; soluble
 biomarkers; clinical) methods. Finally, an inter- and intra-observer analysis will be
 performed. A mathematical models combining the features derived from all the techniques used
 to construct the best model predictive of stroke and the development of neurocognitive
 decline.

 ADMINSITRATIVE ASPECTS Study financial support The study will be financially supported by
 Regione Toscana, Bando Salute 2018. There will be no interference from Regione Toscana on the
 scientific management of the study. Three financial tranches will be bestowed as for formal
 agreements (at the beginning of the study, at 18 months, at 36 months or termination of the
 study).

 Ethical considerations The study will be conducted according to the norms of Good Clinical
 Practice (ICH/GCP) and to the ethical guidelines of the Declaration of Helsinki. Enrollment
 will start only after the protocol is approved by the local Ethical Committee. Written
 informed consent will be obtained from each participant before enrollment.

 Informed consent acquisition Each participant will be carefully and thoroughly informed by
 the investigators involved in the study at the time of enrollment. Written informed consent
 will be requested only after a comprehensive discussion with the participant.

 There are no financial conflict of interest for any of the investigators involved in the
 study.
Inclusion Criteria:

 -  Asymptomatic carotid artery lesions between 40 and 60%, at carotid artery ultrasound,
 for which lesions an interventional strategy - beside optimal medical therapy - is not
 clearly defined.

 -  Patients with asymptomatic carotid artery stenosis higher than 60% will be enrolled
 when revascularization by either carotid endarterectomy (CEA) or stenting will be
 considered not indicated or not feasible

 -  Both genders (with capping implemented in order to ensure a 50±5% balance of males to
 females),

 Exclusion Criteria:

 -  Severe comorbidities a priori precluding the 2-year follow-up (cachexia; end-stage
 cancer; severe renal/respiratory insufficiency; advanced (class IV) heart failure;
 atrial fibrillation and other significant arrhythmias).

 -  Severe allergic diathesis; contraindications to contrast agents for angio-TC or MRI;
 claustrophobia; presence of prostheses/implanted electronic devices not suitable for
 MRI; lack of consent for any reason.