The European Unstable Carotid Artery Plaque Study (EUCAPS)

Project protocol by David Russell, MD, PhD, FESO, FRCPE

Department of Neurology, Oslo University Hospital and Institute of Clinical Medicine, University of Oslo

I. Background

Each year, there are approximately 1million strokes in the European Union, making it by far the most common neurological disorder. Approximately 25% of men and 20% of women can expect to experience a stroke if they live to be 85 years old, and stroke is the second most common cause of death worldwide (1). However, mortality data underestimate the true burden of stroke because in contrast to coronary heart disease (CHD) and cancer, the major burden of stroke is chronic disability rather than death. Stroke patients frequently require long hospital stays followed by on-going support in the community, or nursing home care. Stroke is the number one cause of disability in the European Union and consequently a major drain on health care funding. The costs of stroke are estimated to be nearly twice those of CHD, accounting for 6% of total national health service and social services expenditures (2).The total incidence of stroke is projected to increase by 50% the next 20 years because of the rapid increase in the elderly population (3), and it is predicted that stroke will account for 6.2% of the total burden of illness in 2020 (3).Thus, without more effective strategies for the prevention, treatment, and rehabilitation of stroke, the cost of this disease will increase dramatically.
Although individual European governments and the World Health Organization (WHO) have highlighted the importance of the prevention and treatment of stroke (4); stroke research is grossly underfunded, and lags a long way behind heart disease and cancer (5-6).
A significant proportion of strokes are thromboembolic in nature, arising from an atherosclerotic plaque at the carotid bifurcation. Such strokes have been shown to be effectively preventable by carotid endarterectomy or by emerging therapeutic strategies such as carotid angioplasty and stenting. In current clinical practice patient selection for revascularization primarily involves identification of the severity of luminal stenosis. This is usually measured through conventional imaging modalities such as
ultrasound, intra-arterial digital subtraction angiography (DSA) or, more recently, MR or CT angiography.

It is, however, increasingly clear that the degree of luminal stenosis alone may not be the best predictor of risk. Owing to the process of arterial remodeling, the lumen of an artery may not be compromised despite the presence of significant atherosclerotic burden. Strokes may occur as a result of non-stenotic carotid disease, and conversely, a non-negligible proportion of patients with significant carotid stenosis may remain completely asymptomatic throughout their life-time (7).

There is thus growing research interest in developing imaging modalities which may identify in vivo certain key morphological features such as the thin or ruptured fibrous cap, the large underlying necrotic lipid core, the presence of intraplaque haemorrhage or thrombus, or plaque neovascularisation which are thought to be associated with increased risk.
In parallel with imaging approaches, which aim to primarily assess the structural components of the carotid plaque, molecular imaging attempts to visualize specific biological processes occurring within the plaque in vivo at a cellular level. Such processes include inflammation, angiogenesis and apoptosis are now known to play a key role at all stages of atherosclerosis including initiation, progression or plaque rupture. The ability to image such biological processes has crucial advantages over conventional structural imaging. Molecular imaging holds the premise of earlier detecting potentially 'unstable' plaques before they actually become symptomatic.

Recent research has shown that inflammation plays a key role in atherogenesis and plaque
destabilization. However, although the participation of inflammatory mediators in the atherosclerotic process has become widely recognized, the identification and characterization of the different actors, as well as their relative importance, have not been clarified. Further studies on these issues could identify new risk markers for atherosclerosis and plaque instability as well as new targets for therapy.

II. Approaches, hypotheses and choice of methods

Risk markers for atherosclerosis
Rikshospital´s Inter-departmental Cerebrovascular Research Group (RCRG) has recently reported on five possible markers and mediators of carotid plaque instability (8-12).
1. That the plasma levels of the protease Granzyme B (8), a potent inductor of apoptosis, were markedly increased in symptomatic patients with echolucent carotid plaques. Echolucent carotid plaques have been found to be associated with symptomatic carotid artery disease and they present a higher risk for future ischemic cerebrovascular events compared with echogenic plaques.
2. That the adipokine, visfatin, should be regarded as an inflammatory mediator, localized to foam cell macrophages within unstable atherosclerotic lesions that potentially plays a role in plaque destabilization (9).
3. That the CXCR2 ligand growth-related oncogene (GROa) may be involved in atherosclerosis and plaque destabilization (10).
4. That CD137-CD137L interactions in the vasculature may contribute to the progression and vulnerability of atherosclerotic lesions (11).
5. That the transmembrane glycoprotein CD36 may be a marker of plaque instability and symptomatic carotid atherosclerosis, possibly at least partly as a result of CD36 release to the circulation from foam cells within the atherosclerotic lesion (12).
However, these studies were carried out in small numbers of patients and require verification in a large study population. Moreover, further studies which will try to identify other and perhaps even more important markers and mediators of plaque destabilization in symptomatic carotid artery disease are clearly needed.
Imaging of Metabolic Activity

Positron Emission Tomography (PET) may be used to image inflammation and metabolic activity within the atherosclerotic plaque. Fluorine-18-labelled fluorodeoxyglucose (FDG) is a PET radiopharmaceutical that enters metabolically active cells through the same mechanisms as glucose. FDG is however not fully broken down by the enzymes of the cell and therefore accumulates in proportion to metabolic activity.

Reports of uptake of FDG within human atheroma in vivo were first made by Yun et al. (13) who described that up to 50% of patients undergoing FDG-PET for cancer staging were incidentally found to have high FDG uptake in large arteries, in particular in patients with strong risk factors for atherosclerosis. This confirmed earlier animal studies which showed that FDG was taken up by atherosclerotic lesions and that the degree of uptake appeared to be related to macrophage activity within plaques (14).

Rudd et al. (15) were the first to demonstrate the uptake of FDG within carotid artery atherosclerotic plaques in the setting of stroke and TIA. They imaged eight patients with recently symptomatic severe carotid stenosis with FDG-PET and showed FDG accumulation in all eight symptomatic carotid plaques at 3 h post-FDG injection.

II. Aims of the project

The primary aim of the EUCAPS study is:
To identify new stroke risk markers in patients with carotid artery disease.

The Secondary aims of EUCAPS are:
1. To determine if plasma levels of GrB, the CXCR2 ligand GROa or soluble CD36 can
    predict the risk for stroke in patients with carotid stenosis.
2. To identify genes and proteins that are specifically over-expressed in patients with
    symptomatic carotid artery plaques (new risk markers) and which also can be detected
    in the patients' serum.
3. To determine if accumulation rates of FDG on PET studies can predict the risk
    for stroke in patients with carotid stenosis.
4. To determine if a thin or ruptured fibrous cap, as identified by MRI can predict the risk
    for stroke in patients with carotid stenosis.
5. To determine if there is a relationship between plasma levels of GrB, GROa and soluble
    CD36, as well as plasma levels of new identified markers, and:
       a. Accumulation rates of FDG on the PETstudies.
       b. Risk for stroke while waiting for endarterectomy or during the first two years.
       c. New asymptomatic cerebral ischemic lesions ipsilateral to the carotid stenosis on 
          DWMRI at 2 years.
6. To determine if there is a relationship between accumulation rates of FDG on the PET
    studies and
       a. The patients risk for stroke while waiting for endarterectomy or during the first two
       b. New asymptomatic cerebral ischemic lesions ipsilateral to the carotid stenosis on
           DWMRI at 2 years.

IV. Patients and Methods

This study will include a total of 500 patients. The number of patients is based on power calculations using a test of trend in a logic regression setting. Exposure is divided in quartiles. The overall response probability is set to 0.25 and the effect of exposure equivalent to OR=2.0 for comparison of quartile 4 with quartile 1. The total number needed with a power of 0.8 is 496. By dividing the exposure in two groups instead of four, we may accept a lower response probability or a lower OR. Two hundred patients will have recent carotid territory TIA or minor stroke (< 30 days prior to the start of the study) and a "culprit" internal carotid artery stenosis of =70% who will be treated with endarterectomy, 100 patients with asymptomatic carotid artery stenosis of = 70%, who for various reasons, decided by their treating physician, will be treated with endarterectomy, and 200 patients with asymptomatic carotid artery stenosis of = 70% who will be treated medically.

Clinical information and follow-up
A full clinical history and neurological status will be made on all 500 patients when they enter the study. It will be repeated on the day before and the day after carotid endarterectomy and on out-patient follow-up for all patients at 6 and 12 and 24 months. An ABCD Score (16) assessment will be made for patients who present with a transitory ischemic attack (TIA). All cerebrovascular events will also be registered when the patients attend the hospital's out-patient controls at 6, 12 and 24 months.

Serum markers and mediators of carotid plaque instability
Venipuncture of a forearm vein will be performed, on all patients, on the same day as the ultrasound examination except for those patients scheduled for carotid endarterectomy (CEA) when it will be performed within 2 days prior to CEA. Platelet-poor plasma and serum will be analyzed for levels of Granzyme B (GrB), CXCR2 ligand growth-related oncogene, soluble transmembrane glycoprotein CD36 and genes and proteins that are specifically over-expressed in patients with symptomatic carotid artery plaques (new risk markers) as described previously (8,10,12).

Structural Imaging and inflammation
Ultrasound of the precerebral arteries
The degree of stenosis and echogenicity of the plaques will be assessed at the start of the study and after 1 and 2 years, in all patients, using Color Duplex examinations as described previously (8).

CT angiography
The carotid stenoses will also be classified by CT angiography, in all patients, according to consensus criteria (17). This assessment will be made by experienced radiologists at the respective hospitals.

Magnetic resonance imaging (MRI)
1. Cerebral magnetic resonance imaging (MRI) with DWI will be carried out in all patients, on entry to the study and repeated at two years. They will be assessed using standard criteria (18) by experienced radiologists who will be blinded to the clinical status of the patients
2. Identification of Fibrous Cap Rupture with MRI
The MRI scans of the carotid plaques will be performed in all patients, within 2 days before endarterectomy in symptomatic patients and at the beginning of the study in asymptomatic patients. A standardized protocol will be used to obtain 4 different contrast-weighted image sets (three-dimensional (3D) TOF axial source images and T1, proton density (PD), and T2 weighted images of the carotid arteries for each patient.
The appearance of the fibrous cap will be categorized as intact and thick (rating I), intact and thin (rating II), or ruptured (rating III) on the basis of the following definitions: intact and thick cap: a uniform, continuous dark band adjacent to the lumen on the TOF image and a smooth lumen surface on TOF and T1-, PD-, and T2-weighted images; intact, thin cap: no visible dark band adjacent to the lumen on the TOF image and a smooth lumen surface on TOF and T1-, PD-, and T2-weighted images; and ruptured cap: disrupted or no visible dark band adjacent to the lumen on TOF images, irregular lumen boundary on TOF and T1-, PD-, and T2-weighted images, and a hyperintense, bright signal adjacent to the lumen. The fibrous cap state will be categorized by experienced radiologists who will be blinded to the clinical status of the patient.

Fluorine-18-labelled fluorodeoxyglucose PET (FDG-PET)
FDG-PET will be carried out, at the start of the study, on a total of 100 patients at Oslo University Hospital, Haukeland University Hospital, the Department of Clinical stroke Research University of Munster, Sourasky University Medical Centre, Tel Aviv, Israel, Tel Aviv Sourasky University Medical Centre, University of Medicine and Pharmacy "Iuliu Hatieganu", Cluj-Napoca and the University of Debrecen Medical School. Blood glucose will be controlled to make sure that it is normal. PET images will be acquired in 3D mode 90 minutes after the IV administration of 370 Mob of FDG. After an unenhanced attenuation-corrected CT from the base of the skull to the upper thorax with the arms at the patient's side, the PET emission scan data sets will be acquired in the same anatomical position under quiet breathing. Thereafter, a subsequent contrast medium enhanced diagnostic CT volume will be obtained according to normal routine through the neck. The patients will receive an intravenous injection of iodinated contrast medium (100 ml visipaque 320 mg I/ml, GE Healthcare, Oslo, Norway) by means of an automated power injector.

A semiquantitative assessment of FDG uptake (Standard Uptake Value) will be carried out by a specialist in nuclear medicine blinded to the patient's clinical details. For each patient, regions of interest will be drawn around all of the atheromatous plaques seen on and then transferred onto the corresponding co-registered PET image to enable FDG uptake values (kBq/ml) to be calculated. The same process will be carried out for 5 segments of vessel wall with normal appearance on CT angiography. The uptake value for each plaque will then divided by the average of the normal vessel wall values to give an uptake ratio. This will be done in order to normalize for inter-patient variations in FDG delivery to the plaque and basal metabolism.

Quality Control
Random examples of ultrasound, CT, MRI and PET examinations from the various centers will be transferred electronically to a central EUCAPS quality control center (Oslo University Hospital) to assure that all assessments adhere to the study protocol. The main facility in Oslo will also assist in the handling and assessment of laboratory and image modalities for centres which lack these facilities.

Assessment of Carotid plaques after endarterectomy.
Histology and Immunohistochemistry
After endarterectomy, the intact carotid artery segments, will be formalin-fixed, sectioned in 5-mm transversal slices and decalcified for 30 minutes in 10% formic acid in PBS, and embedded in paraffin. Subsequently, 4-µm sections will be subjected to histo-immunological analysis of plaque phenotype (HE staining) and macrophage content (CD68 immunostaining). The number of macrophages (CD68-positive cells) will be assessed by an investigator on a scale from 0 to 4, with 0 indicating no; 1, hardly any; 2, some; 3, many; and 4, very many CD68-positive cells. In addition, for each tissue section, plaque stage will be independently determined by 2 investigators according to the morphological criteria of the American Heart Association (19) Stable lesions (type IV and V) will be characterized by an intact and thick fibrous cap. These plaques contain either a large lipid core (type Va), calcification (type Vb), or fibrous tissue (type Vc). A disrupted fibrous cap and the presence of a thrombus characterize the ruptured lesion (type VI). Type Va lesions with a very thin fibrous cap will be considered as rupture-prone lesions.

High-throughput gene expression profiling and proteinomics.
The plaques will be examined using high-throughput gene expression profiling (microarray) and proteinomics (mass spectrometry) in order to identify genes and proteins that are specifically over-expressed in patients with symptomatic carotid artery plaques.
Biochemical and immunological methods:
a. Methods for quantification of a wide range of cytokines/chemokines and their receptors (enzyme immunoassay, bioassays, flow cytometry, real-time RT-PCR, RNase protection assay).
b. Immunohistochemistry for studying the expression of various inflammatory mediators in human carotid plaques. Immunohistochemistry will be supplemented by other methods for protein quantification such as western blotting.
c. Microarray and proteinomic technology will be used to detect new genes and proteins associated with the degree of plaque instability, trying to identify the signature(s) of unstable plaques.

Participating centers, patient inclusion and data collection.
All stroke centres in Norway and interested centres in Europe (To date: Munster, Tel Aviv, Debrecen, Riga, Cluj-Napoca, Belgrade and Padova have pledged their participation) are invited to participate in EUCAPS. The majority of centers will contribute with clinical data, blood tests, ultrasound and MRI data and those centers that carry out carotid endarterectomy with carotid plaques. The PET studies will be carried out at university hospitals where these examinations are available. However, cooperation within the infrastructure will encourage the inclusion of patients from both university and non-university hospitals to the MRI and PET studies.

All of the blood samples and plaque tissue will be sent to and assessed at Oslo University Hospital, Rikshospitalet (The Research Institute for Internal Medicine and The Department of Pathology). All other data will be electronically entered into the part of the ECRI data base designated to EUCAPS by the local investigators.

Analysis of results:
A statistical comparison will be made with regard to:
1. Plasma levels of GrB, GROa or soluble CD36, as well as plasma levels of new identified markers the risk for
stroke in patients with carotid stenosis (on entry to the study i.e. symptomatic v asymptomatic patients and new symptoms at the 6 and 12 and 24 month follow-ups).
2. The Identification of genes and proteins that are specifically over-expressed in patients with symptomatic carotid artery plaques (new risk markers) and which also can be detected in the patients' serum.
3. Accumulation rates of FDG on the PET studies and stroke risk.
4. Thin or ruptured fibrous cap, as identified by MRI, and stroke risk.
5. Plasma levels of GrB, GROa and soluble CD36, as well as plasma levels of new identified markers, and
5.1 Accumulation rates of FDG on the PET studies.
5.2 Risk for stroke while waiting for endarterectomy or during the first two years.
5.3 New asymptomatic cerebral ischemic lesions ipsilateral to the carotid stenosis on DWMRI at 2 years.
6. Accumulation rates of FDG on the PET studies and
6.1 The patients risk for stroke while waiting for endarterectomy or during the first two years.
6.2 New asymptomatic cerebral ischemic lesions ipsilateral to the carotid stenosis on DWMRI at 2 years.
All the statistical analyses will be carried out independently by the Department of Medical Biostatistics, Oslo University Hospital, Rikshospitalet under the supervision of Professor Hein Stigum (Section for Preventive Medicine and Epidemiology, University of Oslo).

V. References:
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4 Pendlebury ST et al. Underfunding of Stroke Research: A Europe-Wide Problem. Stroke 2004; 35:2368.
5 Rothwell P. The high cost of not funding stroke research. Lancet 2001; 357: 1612-16.
6 Halliday A, Mansfield A, Marro J et al. Prevention of disabling and fatal strokes by successful carotid endarterectomy in patients without recent neurological symptoms: randomised controlled trial. Lancet 2004; 363:1491–502.
7 Skjelland M, Michelsen AE, Krohg-Sørensen K, Tennøe B, Dahl A, Bakke S, Brosstad F, Damås JK, Russell D, Halvorsen B, Aukrust P. Plasma levels of granzyme B are increased in patients with lipid-rich carotid plaques as determined by echogenicity. Atherosclerosis 2007; 195:142-6.
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11 Handberg A, Skjelland M, Michelsen AE, Sagen EL, Krogh-Sørensen K, Russell D, Dahl A, Ueland T, Øie E, Aukrust P, Halvorsen B. Soluble CD36 in plasma is increased in patients with symptomatic atherosclerotic carotid plaques and is related to plaque instability.Stroke 2008 ;39: 3092-3095
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17 Wahlund LO, Barkhof F, Fazekas F, et al. A new rating scale for age-related white matter changes applicable to MRI and CT. Stroke 2001; 32:1318 –22
18 Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb Vasc Biol. 1995; 15: 1512–1531.