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. Author manuscript; available in PMC: 2015 Jan 7.
Published in final edited form as: Stroke. 2014 Dec 4;46(1):182–189. doi: 10.1161/STROKEAHA.114.007221

Symptomatic carotid atherosclerotic disease: correlations between plaque composition and ipsilateral stroke risk

Dominic PJ Howard 1,#, Guus W van Lammeren 2,3,#, Peter M Rothwell 1, Jessica N Redgrave 1, Frans L Moll 3, Jean-Paul PM de Vries 4, Dominique PV de Kleijn 2, Hester M den Ruijter 2, Gert Jan de Borst 3, Gerard Pasterkamp 2
PMCID: PMC4285579  EMSID: EMS61430  PMID: 25477221

Abstract

BACKGROUND AND PURPOSE

For symptomatic patients with carotid artery stenosis the risk-benefit for surgical intervention may vary among patient groups. Various modalities of plaque imaging have been promoted as potential tools for additional risk stratification, particularly in patients with moderate stenosis. However, it remains uncertain to what extent carotid plaque components predict risk of future ipsilateral ischaemic stroke.

METHODS

In two large atherosclerotic carotid plaque biobank studies, we related histological characteristics of 1640 carotid plaques with a validated risk model for the prediction of individual 1- and 5-year stroke risk.

RESULTS

No significant heterogeneity between the studies was found. Predicted 5-year stroke risk (top versus bottom quartile) was related to plaque thrombus (OR=1.42, 95%CI 1.11-1.89, p=0.02), fibrous content (0.65, 0.49-0.87, p=0.004), macrophage infiltration (1.41, 1.05-1.90, p=0.02), high micro-vessel density (1.49, 1.05-2.11, p=0.03), and overall plaque instability (1.40, 1.05-1.87,p=0.02). This association was not observed for cap thickness, calcification, intra-plaque haemorrhage, or lymphocyte infiltration. Plaques removed within 30-days of most recent symptomatic event were most strongly correlated with predicted stroke risk.

CONCLUSIONS

Features of ‘the vulnerable carotid plaque’ including plaque thrombus, low fibrous content, macrophage infiltration and microvessel density correlate with predicted stroke risk. This study provides a basis for plaque imaging studies focused on stroke risk stratification.

Keywords: Acute Stroke, Carotid atherosclerotic plaque, Risk Prediction

Introduction

Approximately 15% of strokes are thought to be secondary to carotid artery atherosclerosis.1 Over the last 20 years, the degree of carotid artery stenosis has been the determining factor in selecting symptomatic patients for carotid endarterectomy (CEA).2 In symptomatic patients with carotid artery stenosis of 70% or more, CEA reduces ipsilateral stroke risk and has proven to be superior to best medical treatment.2 In symptomatic patients with lower degrees of stenosis, the benefit of CEA is less clear and is dependent on timing of surgery, the type of presenting event, gender, patient age, and co-morbidity.3 Recent improvements in intensive medical treatment and the slightly higher peri-operative risks in routine clinical practice compared to the large trials need also to be taken into account.4 This has led to a debate on risk-benefit ratios, correct patient selection, and the management of (moderate) symptomatic carotid stenosis. 5

To assess stroke risk in patients with symptomatic carotid stenosis more precisely, a risk prediction model has been developed using data of patients on best medical treatment in European Carotid Surgery Trial (ECST) that has been subsequently validated in the North American Symptomatic Carotid Endarterectomy Trial (NASCET).6,7 The model calculates individual ipsilateral stroke risk for recently symptomatic patients incorporating known significant risk predictors. Apart from the presence of an irregular/ulcerated plaque surface on conventional arterial angiography,8 no other carotid plaque features are included in the risk model as, to-date, their influence on future stroke risk are unknown. However, histopathological studies have revealed correlations between carotid plaque composition and several clinical key risk predictors. Male gender has been associated with lipid rich and inflammatory plaques,9 age has been linked with presence of large lipid cores,10,11 and plaques removed soon after last symptomatic event tend to be more inflammatory and show greater instability.12,13 Despite these associations, it remains uncertain whether any plaque feature can predict future stroke and risk-stratify patients independently of age, sex and more easily measured traditional vascular risk factors.

The concept of the “vulnerable plaque” was first introduced for coronary arteries following coronary plaque studies confirming that ruptured, inflammatory plaques with thin fibrous caps are often associated with acute coronary syndromes, whereas fibrous plaques tend to be associated with stable syndromes.14-16 This concept is less certain in the cerebral circulation, as only 50-60% of carotid plaques removed from symptomatic patients are ruptured17,18 and the pathophysiological mechanisms determining stroke secondary to large-vessel atherosclerosis (i.e embolism) differ greatly from those determining myocardial infarction (i.e. thrombosis). Despite this, there has been growing interest in the evaluation of carotid plaque morphology and functional characteristics, via the interpretation of various imaging modalities, biochemical markers, and embolic signal detection.19,20,21 Several plaque imaging studies have shown that development of intraplaque hemorrhage (IPH) is associated with carotid plaque progression, and recently it has also been associated with risk factors for future ipsilateral events.22-24 Among asymptomatic patients with moderate carotid artery stenosis (50-79%), several plaque characteristics including thinned or ruptured fibrous caps, intraplaque hemorrhage, and larger lipid and necrotic cores, and were associated with the occurrence of subsequent cerebrovascular events.25 However at present, the clinical utility of these techniques is still uncertain and current studies have lacked power to convincingly identify any influence of individual plaque features on future stroke risk.25-27 These studies have also been unable to follow-up patients with severe symptomatic carotid artery stenosis, as many undergo early intervention. The present study assesses the extent to which histological features in carotid plaques from symptomatic patients are related to individual predicted stroke risk.

Methods

We studied data from two large carotid plaque biobanks in the UK (Oxford Plaque Study) and the Netherlands (Athero-Express) in which plaques from consecutive patients undergoing carotid endarterectomy underwent detailed histological assessment with reproducible semi-quantitative scales. The Oxford Plaque Study included 481 plaques from symptomatic patients collected between 1975 - 2002 and the Athero-Express study included 1159 plaques from symptomatic patients collected between 2002 - 2012 (total n=1640). For each individual patient in both studies the predicted stroke risk was calculated and correlated to the carotid plaque histological features. Patients were excluded if they were undergoing surgery for asymptomatic stenosis, restenosis or radiotherapy-induced carotid stenosis.

Ipsilateral stroke risk

The stroke risk was calculated using a carotid stenosis risk prediction model, which has been described in detail previously and was validated against the NASCET patient database with a c-statistic of 0.67, 95% CI 0.63-0.72 (p<0.0001).6-7 The model can be accessed at www.stroke.ox.ac.uk/model/form1.html, and calculates 1-year and 5-year risk for ischaemic ipsilateral stroke based on clinical characteristics including patient age, sex, degree of stenosis, and relevant co-morbidities, together with type of primary symptomatic event and time from last symptomatic event. The characteristics at the time of CEA were used to calculate predicted stroke risks and the degree of stenosis was defined according to NASCET criteria.4 All patients in both studies had at least 50% carotid artery stenosis and were reviewed by a neurologist before consideration for CEA. The primary symptomatic event was defined as the most severe ipsilateral vascular event during the previous 6 months before CEA. The time since the last event refers to the most recent ipsilateral event. Diabetes was restricted to those cases receiving medical treatment, including insulin and oral medications. Previous myocardial infarction and peripheral vascular disease refer to previous confirmed clinical diagnoses. Treated hypertension referred to patients previously diagnosed with hypertension and actively treated with anti-hypertensives at the time of event. Monocular symptoms included amaurosis fugax or retinal artery occlusion. Transient ischemic attack (TIA) was defined as a symptomatic event lasting up to 24 hours, whereas minor strokes included symptoms lasting 24 hours to 7 days, and major strokes were non-disabling with residual symptoms present after 7 days.

Carotid plaque histopathology and phenotyping

The processing and assessment of carotid plaques has been described in detail for both studies previously.8,18,28,29 Briefly, carotid plaques were formalin fixed after endarterectomy and either the whole plaque (Oxford Plaque Study), or the portion of the plaque showing the highest plaque burden (Athero-Express), was paraffin embedded. After creating transverse sections, plaques were immunohistochemically stained with (1) hematoxylin and eosin (H&E) for assessment of overall plaque stability, lipid core, thrombus, and intraplaque haemorrhage, (2) elastin von Gieson (EVG) for fibrous plaque content, and (3) CD68 for plaque macrophage content. Both studies applied comparable 3-, or 4-point semi-quantitative scales for the assessment of overall plaque stability, lipid core, fibrous plaque content, micro vessel density (neovascularisation), macrophage infiltration, and calcifications, as defined previously (Supplemental Table I).8,18,28,29 However, to maximize comparability and overlap of definitions between studies, the scales were transformed into binominal variables (Supplemental Table I). In addition to the above, the Oxford Plaque Study scored the presence of plaque rupture, cap thickness and cap infiltration with macrophages and lymphocytes, whereas Athero-Express assessed for the presence of smooth muscle cells (alpha-actin). These histological characteristics that were recorded in only one of the two studies, were separately correlated with stroke risk for each study individually.

Analysis

SPSS version 18.0 (Chicago, IL, United States of America) was used for all statistical analyses. Baseline characteristics were compared between the two studies with students t-test and Mann-Whitney U test for parametric, and non-parametric continuous variables respectively. Baseline differences in nominal variables were examined with Pearson’s Chi-square test. The calculated 1-year and 5-year stroke risks were divided into quartiles. Associations between plaque characteristics and 1-year and 5-year stroke risks were assessed using odds ratios produced from binary logistic regression analysis. Regression analysis was also used to evaluate the strength of association between stroke risk and the presence of multiple plaque features. The key plaque features chosen for this analysis are those commonly thought to represent plaque vulnerability; presence of thrombus, large lipid core, low fibrous content, intraplaque haemorrhage, and inflammation (macrophage infiltration). As plaque stability is dependent on several characteristics, and a result of biological interaction of inflammation, plaque haemorrhage, and lipid metabolism; an additional analysis was performed to analyze the total number of vulnerable plaque characteristics per plaque in relation to predicted stroke risk. Heterogeneity between study cohorts for individual plaque features was assessed for using interaction terms in binary logistic regression. As no significant heterogeneity was found, binominal data from both studies were combined for pooled analysis. All such analyses were stratified by study cohort.

Results

The Oxford Plaque Study comprised 481 plaques and Athero-Express 1159 plaques, resulting is a pooled sample of 1640 symptomatic carotid plaques. Baseline characteristics for both studies are provided in table 1. In the Oxford Plaque study patients were younger, more frequently male, less frequently diabetic, less often on prior statin treatment, and had a longer time period between last event and CEA as compared with Athero-express patients. In addition, the Oxford Plaque Study included more patients with monocular events and major stroke, whereas Athero-Express included more patients with TIA and minor stroke. The 1-year predicted stroke risk was comparable between the two studies, but the 5-year stroke risk was marginally higher in Athero-Express (table 1).

Table 1. Patient characteristics in Oxford Plaque Study and Athero-Express.

Characteristic Oxford Plaque (n=481) Athero-Express (n=1159) P value ≤ 30-days (n=553) >30-days (n=1087) P value
Median 1year stroke risk [IQ range] 7.6 [5.1-12.0] 8.3 [5.4-11.8] 0.360 11.0 [8.2-15.2] 6.77 [5.0-9.9] <0.001
Median 5 year stroke risk [IQ range] 19.0 [12.2-29.4] 21.0 [14.0-29.0] 0.014 27.22 [20.8-36.1] 17.3 [11.7-24.8] <0.001
Age, years (±SD) 67.0 (±8.7) 69.0 (±9.4) <0.001 68.6 (±9.6) 68.3 (±9.0) 0.42
Male gender 71.9% (346/481) 65.9% (762/1158) 0.017 65.6 (363/553) 68.7 (746/1086) 0.21
Median time since last event, days [IQ range] 87 [34-150] 45 [18-95] <0.001 NA NA
Presenting event
  Monocular 27.0% (130/481) 16.7% (193/1159) <0.001 17.7 (98/553) 20.7 (225/1087) 0.15
  TIA , single 10.8% (52/481) 36.8% (426/1159) <0.001 26.6 (147/553) 30.5 (331/1087) 0.10
  TIA, multiple 28.9% (139/481) 15.4% (179/1159) <0.001 28.8 (159/553) 14.6 (159/1087) <0.001
  Minor stroke 7.3% (35/481) 17.6% (203/1159) <0.001 13.4 (74/553) 15.1(164/1087) 0.35
  Major stroke 26.0% (125/481) 13.5% (157/1159) <0.001 13.6 (75/553) 19.1 (208/1087) 0.005
Diabetes 10.8% (52/481) 17.9% (207/1159) <0.001 17.9 (99/553) 14.7 (160/1087) 0.10
Treated hypertension 58.2% (280/481) 59.2% (680/1149) 0.717 56.5 (310/549) 60.1 (650/1081) 0.16
Treated hypercholesterolaemia* 17.9% (86/481) 74.5% (828/1111) <0.001 62.9 (329/523) 54.7 (584/1068) 0.002
Previous myocardial infarction 12.1% (58/481) 9.6% (108/1121) 0.145 12.2 (65/531) 9.4 (101/1070) 0.08
Peripheral vascular disease 17.5% (84/481) 17.2% (194/1128) 0.898 19.7 (105/534) 16.1 (173/1074) 0.08
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Abbreviations: IQ, Interquartile; SD, Standard deviation; TIA, Transient ischemic attack.

*

As measured by prior statin use

Table 2 shows the odd ratios for the presence of individual plaque features in relation to 1-year and 5-year stroke risk analysed separately in each study. These were similar in the two studies with no significant heterogeneity found for any plaque characteristic. In the pooled analysis several statistically significant associations between plaque characteristics and stroke risk were observed. The presence of luminal thrombus, heavy macrophage staining, high micro-vessel density, overall plaque instability and a lower prevalence of fibrous content were significantly correlated with predicted stroke risk. The presence of large lipid-core was weakly correlated with stroke risk, whereas calcification and intra-plaque haemorrhage were not. Out of all non-overlapping histological plaque features, phenotyped separately in each study, none were significantly correlated with predicted stroke risk (table 3).

Table 2. Odds-ratios for the presence of individual plaque characteristics in the highest versus lowest quartile of stroke risk.

Plaque characteristic Oxford Plaque Study (n=481) Athero-Express (n=1159) Pooled Data (n=1640)
OR 95% CI P-value OR 95% CI P-value OR 95% CI P-value
1 YEAR STROKE RISK
Overall plaque instability 1.24 0.74-2.06 0.41 1.52 1.07-2.15 0.02 1.42 1.07-1.90 0.02
Thrombus 1.47 0.85-2.52 0.17 1.37 0.98-1.92 0.06 1.40 1.05-1.86 0.02
Heavy macrophage staining 1.78 1.05-3.00 0.03 1.19 0.85-1.67 0.30 1.39 1.04-1.84 0.03
High micro-vessel density 1.43 0.86-2.38 0.17 1.49 0.92-2.41 0.11 1.46 1.03-2.07 0.03
Large lipid core 1.22 0.74-2.02 0.44 1.34 0.93-1.97 0.11 1.31 0.97-1.76 0.08
Plaque haemorrhage 1.39 0.83-2.33 0.22 1.06 0.71-1.59 0.78 1.18 0.85-1.62 0.32
Fibrous plaque 0.63 0.37-1.07 0.09 0.66 0.47-0.93 0.02 0.65 0.49-0.87 0.004
Heavy calcification 0.91 0.56-1.49 0.71 0.82 0.59-1.14 0.24 0.84 0.64-1.11 0.23
5 YEAR STROKE RISK
Overall plaque instability 1.29 0.78-2.12 0.32 1.47 1.03-2.09 0.03 1.40 1.05-1.87 0.02
Thrombus 1.44 0.85-2.45 0.17 1.41 1.00-1.98 0.05 1.42 1.11-1.89 0.02
Heavy macrophage staining 1.83 1.10-3.05 0.02 1.26 0.89-1.76 0.19 1.41 1.05-1.90 0.02
High micro-vessel density 1.42 0.87-2.33 0.17 1.57 0.96-2.56 0.07 1.49 1.05-2.11 0.03
Large lipid core 1.26 0.77-2.07 0.35 1.31 0.90-1.92 0.16 1.29 0.96-1.75 0.09
Plaque haemorrhage 1.40 0.84-2.32 0.19 1.02 0.67-1.53 0.94 1.15 0.84-1.59 0.38
Fibrous plaque 0.58 0.35-0.98 0.04 0.68 0.48-0.97 0.03 0.65 0.49-0.87 0.004
Heavy calcification 1.03 0.64-1.67 0.90 0.81 0.58-1.13 0.22 0.88 0.67-1.16 0.35
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Abbreviations: OR, odds ratio; CI, confidence interval. Pooled data stratified by study cohort.

No significant heterogeneity was found between the study groups for any individual plaque features

Table 3. Odds-ratios for the presence of non-overlapping individual plaque characteristics in the highest versus lowest quartile of stroke risk.

Plaque characteristic Oxford Plaque Study (n=481) Athero-Express (n=1159)
OR 95% CI P-value OR 95% CI P-value
1 YEAR STROKE RISK
Plaque rupture 1.33 0.81-2.20 0.26 NA NA NA
Foam cells 0.82 0.49-1.36 0.43 NA NA NA
Plaque lymphocytes 1.18 0.71-1.95 0.53 NA NA NA
Thin Cap 1.28 0.73-2.24 0.39 NA NA NA
Cap inflammation 0.82 0.44-1.52 0.52 NA NA NA
Smooth muscle cells NA NA NA 0.70 0.48-1.02 0.06
5 YEAR STROKE RISK
Plaque rupture 1.39 0.85-2.27 0.19 NA NA NA
Foam cells 0.81 0.49-1.33 0.40 NA NA NA
Plaque lymphocytes 1.21 0.74-1.99 0.44 NA NA NA
Thin Cap 1.17 0.68-2.02 0.57 NA NA NA
Cap inflammation 0.95 0.52-1.73 0.87 NA NA NA
Smooth muscle cells NA NA NA 0.72 0.50-1.05 0.09
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Abbreviation: NA, not available.

As there is current uncertainty as to whether statin therapy influences carotid plaque morphology30-32 and an affect could potentially bias our results, we performed the same analyses for patients on prior statin therapy (n=913) and those not so (n=678) (supplemental table II). Odd ratios for individual plaque features in these subgroups (in relation to stroke risk) were similar, except for plaque inflammation (heavy macrophage staining), which was less associated with predicted stroke risk when compared to non-users. The Oxford Plaque Study patients were largely non-statin users (18%), whereas Athero-express were mainly users (75%). However, for all other features analysed, prior statin therapy appeared to have minimal impact on their correlation with predicted stroke risk.

To investigate whether plaque stabilisation over time post event may also influence the association between plaque features and future stroke risk, we performed the same analyses on all patients who underwent CEA within 30 days of event (n=552) compared to those operated greater than 30 days from most recent event (n=1088). The odd ratios for the presence of most plaque characteristics in relation to stroke risk were substantially greater for the ≤ 30-day sub-group when compared to the > 30-day subgroup (table 4). In particular, the presence of plaque inflammation and large lipid core were highly correlated with predicted stroke risk in ≤ 30-day sub-group, but these relationships were lost in the > 30-day subgroup. In the > 30-day subgroup, plaques from patients with no prior statin use did reveal significant correlation between plaque inflammation and predicted stroke risk (OR 1.81, 95% CI 1.03-3.18, p=0.04) whereas those with prior statin use did not (0.91, 0.54-1.55, p=0.74). Patient characteristics, including age, gender, vascular risk factor profiles, and event types did not differ significantly between these two subgroups (table 1). Analyses were also performed for sub-groups separated by event type (monocular, TIA, and stroke events) with no significant differences found (data not shown.)

Table 4. Odds-ratios for the presence of individual plaque characteristics in the highest versus lowest quartile of 1-year stroke risk for the subgroups of patients operated ≤ and > 30 days from the last event.

Plaque characteristic Operated ≤ 30 Days from Event (n=553) Operated > 30 Days from Event (n=1087) P-Value for Interaction
Total N (%) OR 95% CI P-value Total N (%) OR 95% CI P-value
Overall plaque instability 366 (66.9) 2.55 1.23-5.28 0.01 727 (67.2) 1.18 0.81-1.72 0.39 0.08
Large lipid core 398 (72.2) 2.80 1.32-5.94 0.007 776 (71.7) 0.90 0.62-1.33 0.61 0.02
High micro-vessel density 101 (33.8) 4.52 0.98-20.87 0.05 268 (32.0) 1.47 0.95-2.29 0.09 0.05
Fibrous plaque 180 (32.6) 0.43 0.21-0.89 0.02 347 (31.9) 0.84 0.57-1.22 0.35 0.07
Heavy macrophage staining 316 (57.6) 2.51 1.20-5.28 0.02 667 (61.9) 1.31 0.90-1.91 0.16 0.09
Heavy calcification 259 (47.0) 0.57 0.27-1.20 0.14 577 (53.1) 1.03 0.71-1.49 0.87 0.15
Plaque haemorrhage 123 (22.3) 0.99 0.40-2.43 0.98 279 (25.7) 1.40 0.93-2.10 0.11 0.75
Thrombus 219 (39.7) 1.50 0.71-3.17 0.30 433 (39.9) 1.41 0.97-2.05 0.07 0.96
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Abbreviations: N, number of cases; OR, odds ratio; CI, confidence interval. All data stratified by study cohort.

To evaluate the strength of association between overall carotid plaque vulnerability and predicted stroke risk, we performed regression analysis for the presence of multiple plaque features currently thought to represent plaque “vulnerability”. Odd ratios for the presence of 0 up to ≥4 plaque features increased from 1.0 to 5.7 in relation to 1-year predicted stroke risk for patients operated within 30 days of event (trend p=0.001) (table 5, figure 1). For the > 30 day subgroup, however, these ranged from 0.9 – 1.5 (trend: non-significant).

Table 5. Odd-ratios for the presence of multiple vulnerable plaque characteristics in relation to stroke risk (upper versus lower quartile) for patients operated ≤ 30 days and > 30 days of last event.

CEA ≤ 30 DAYS FROM EVENT 1 year stroke risk 5 year stroke risk
(n=553) OR 95% CI P-value OR 95% CI P-value
0 vulnerable plaque features 1.00 1.00
1 vulnerable plaque feature 2.79 0.74-10.60 0.13 3.06 0.71-13.21 0.13
2 vulnerable plaque features 2.28 0.75-6.89 0.15 2.27 0.72-7.18 0.16
3 vulnerable plaque features 3.60 1.21-10.69 0.02 3.13 1.04-9.45 0.04
≥4 vulnerable plaque features 5.71 1.80-18.13 0.003 4.31 1.39-13.35 0.01
CEA > 30 DAYS FROM EVENT 1 year stroke risk 5 year stroke risk
(n=1087) OR 95% CI P-value OR 95% CI P-value
0 vulnerable plaque features 1.00 1.00
1 vulnerable plaque feature 1.08 0.53-2.21 0.84 1.16 0.57-2.36 0.69
2 vulnerable plaque features 0.88 0.44-1.76 0.71 0.99 0.49-1.98 0.98
3 vulnerable plaque features 1.11 0.58-2.12 0.76 1.19 0.62-2.27 0.60
≥4 vulnerable plaque features 1.54 0.82-2.87 0.18 1.65 0.89-3.06 0.11
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All data stratified by study cohort. Test for Interaction for ≥4 vulnerable plaque features by CEA timing: p=0.05

Figure 1.

Figure 1

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The relationship between overall plaque vulnerability and future stroke risk is time-dependent: Odd-ratios for the presence of multiple vulnerable plaque characteristics in relation to stroke risk (upper versus lower quartile)

Discussion

In the largest-ever study of carotid plaque histology, we have shown that various histological carotid plaque features correlate with predicted stroke risk, including plaque thrombus, fibrous content, macrophage infiltration, high micro-vessel density, and overall plaque instability. This is in line with the classical view on associations between composition of the vulnerable plaque and the risk of ipsilateral cerebrovascular events.12,33-36 Several features, including plaque inflammation, large lipid core, and low fibrous content, appear to have a highly temporal association with future stroke risk, being correlated in plaques removed within 30 days of last event, and not so in those removed greater than 30 days of event. Other key features targeted by current imaging modalities, such as cap thickness (MRA), intra-plaque haemorrhage (Ultrasound, MRA), plaque rupture (MRA, Ultrasound) and calcification (CT Angiography (CTA), were not significantly associated with calculated stroke risk.

High resolution magnetic resonance imaging is currently the most reliable and reproducible modality for the evaluation of carotid plaque morphology.21,37-38 It provides greater accuracy and atherosclerotic definition when compared to Colour Doppler ultrasonography and CT angiography, without the drawbacks of user-dependency in ultrasound and both radiation and contrast loads in CTA. MRA also enables the acquirement and combination of multiple different contrast weightings to distinguish tissue composition within the arterial wall. This modality has been validated with histology and shown to accurately quantify several key carotid plaque characteristics, including lipid-rich necrotic core,39-42 intraplaque hemorrhage,41-43 calcification,21,39 and cap rupture.43-44

Intraplaque hemorrhage is a frequently studied plaque feature, since it has been described to be associated with plaque progression. Turc et al showed that intraplaque hemorrhage detected with MRI in symptomatic patients was associated with the degree of stenosis, the type of cerebrovascular event, and the time from ischaemic event. These parameters are also important risk factors for future ipsilateral stroke risk. However in our study, intraplaque hemorrhage was not significantly associated with future stroke risk. The results are consistent throughout the two studies, which suggests that once important risk factors for ipsilateral stroke risk are taken into account, intraplaque hemorrhage is probably less useful in predicting ipsilateral stroke risk. An explanation for this might be found in the greater understanding of the different types and stages of intraplaque hemorrhage. For example, it has been shown that organized plaque hemorrhages with newly formed microvessels are associated with vulnerable plaque characteristics. Newly formed microvessels contain newly formed endothelium, which is frequently insufficient, leading to micro-vascular leakage, and thereby potentially resulting in plaque hemorrhage and progression. 45-47 This association may not be valid for other types of intraplaque hemorrhage. We could not analyze subgroups of intraplaque hemorrhage, because these data were not available for both studies.

At present the clinical utility of carotid plaque imaging is uncertain and no studies to-date have found a non-invasively identified plaque feature that is able to risk stratify patients. Our results suggest that out of the plaque features that are currently able to be imaged reliably, only the presence of plaque thrombus and large lipid-core (when assessed within 30 days of event) correlate significantly with predicted stroke risk. When considering the recent progress made in attempts to image and quantify features such as inflammation, neovascularisation, and cap thickness, we have also shown that out of these features, histological plaque inflammation (and not isolated cap inflammation) and high micro-vessel density correlate significantly with increased predicted stroke risk. It was not possible to perform an adjusted multivariate analysis for individual plaque characteristics in relation to stroke risk, as the features are too mutually correlated for a standard multivariate model to run effectively.

The strength of association between carotid plaque morphology and future predicted stroke risk appears to be greatest when the plaque is characterised shortly after the last symptomatic event with no significant plaque feature associations being found in plaques removed greater than 30 days from event. This has several important implications. First, this highlights a temporal relationship between plaque morphology and future stroke risk. Second, as our stroke risk model is based on the presence of traditional vascular risk factors in conjunction with details of event timing, type, and degree of carotid artery stenosis, it can also be inferred that plaque composition is related to these factors in a highly temporal manner. The reason for this is likely to be plaque stabilization over time after an event, as previously described.10,12,32 The plaques stabilise gradually after a cerebrovascular event and therefore it is likely that the results of patients that underwent surgery within 30 days most accurately reflect the situation preceding any event. Third, asymptomatic carotid plaques have mostly stable morphology35 and when considering time to intervention, can be viewed in a similar light to symptomatic plaques that are removed at a delayed point in time from presenting event. Our results could therefore also suggest that there will be limited clinical utility in imaging asymptomatic plaque morphological features in an attempt to risk-stratify these patients. Due to the lack of a valid prediction tool for asymptomatic patients with carotid stenosis we could not perform subgroup analyses, to answer the question whether the presented results can be extrapolated to this patient group.

When focusing on the overall strength of association between plaque morphology and future stroke risk, we can conclude that the number “vulnerable” plaque features present increases as predicted stroke risk rises, and this relationship is very strong in plaques assessed within 30 days of last event, and completely diminished those assessed greater than 30 days from event. Of interest for future prognostic imaging studies, the presence of 4 or more vulnerable plaque features is highly correlated with increased predicted stroke risk in plaques assessed within 30 days of event.

The current study was the largest-ever of carotid plaque histology and although we believe our results to be valid, certain limitations need to be addressed. First, the Oxford Plaque study examined the entire specimen along the longitudinal axis with 3mm intervals, whereas the Athero-Express study examined the culprit lesion, adjacent segments being processed for protein extraction.

However, both studies have shown previously that differences in sampling and sectioning technique do not have a major impact on categorisation of plaque histology.9,28 Second, many plaques from the Oxford study were collected in the 1980s and 1990s, since when time from symptoms to surgery has been reduced and use of antiplatelet agents and statins has increased. The time from event to intervention was included in the stroke risk prediction model and will therefore have been adjusted for in our analyses. Third, the carotid stenosis risk prediction model was developed based on patients on best medical treatment within ECST, which was conducted in the pre-statin era. This might, therefore, lead to an overestimation of stroke-risk, as some strokes may have been prevented if patients were on statin therapy. However, in our sub-group analysis, we have confirmed minimal statin influence on the majority of individual plaque feature – stroke risk correlations. Prior aggressive antiplatelet treatment may potentially have had an impact on these correlations. Unfortunately, however, we do not have accurate data on single versus dual antiplatelet use prior to event for a large proportion of cases and so we were unable to add perform additional analyses.

Fourth, the analyses of patients operated within 30 days after the last event is a subgroup analysis and therefore hypothesis generating. Nonetheless, we feel that the subgroup analysis has sufficient statistical power as it encompassed 553 patients. Since early carotid endarterectomy within two weeks of indicative event is part of current best medical practice, our subgroup results are relevant for current stroke management and provide important messages for carotid plaque imaging and risk-stratification studies. Imaging of plaques, including MRI, is ideally performed shortly after the event. Our results also suggest a stronger association between plaque characteristics and stroke risk, if the plaque is analysed shortly after an event. In our population the median time between the last event and surgery was 87 and 45 days, for the 2 studies respectively. This time is decreasing strongly over time due to current guidelines stating that patients with significant symptomatic carotid stenosis should be operated within 14 days of the last event. Due to this trend, future analyses on plaque composition and ipsilateral stroke risk could therefore find stronger correlations. One must note, however, that the risk prediction model is validated for the calculation of future ipsilateral stroke risk for cases who are 7-180 days post-event. In order to calculate stroke risk in patients who are within 6 days of event, further calibration and validation of the model would be required.

Fifth, the performance of the validated risk prediction model against actual stoke risk in the external NASCET cohort produced a c-statistic of 0.67, 95% CI 0.63-0.72 (p<0.0001). Therefore, as with all models, the risk prediction model is not perfect and it is therefore feasible that the plaque features found not to be associated with predicted stroke risk in our study could be associated with actual stroke risk in vivo. Future prognostic studies should focus on the follow-up of asymptomatic patients and those suffering symptomatic events who are believed to be at low future stroke risk using predictive tools such as the model used in this study. These studies should be guided by our findings and will be ideally placed to confirm if plaque features that are identifiable on non-invasive imaging correlate with future stroke risk in vivo.

Summary

This study shows that specific “vulnerable” carotid plaque characteristics correlate with predicted stroke risk. Plaques removed within 30-days of last symptomatic event are most strongly correlated with predicted stroke risk whereas those removed greater than 30-days reveal no such relation. Although, it remains uncertain whether any individual plaque feature can predict stroke independently of age, sex and more easily measured traditional vascular risk factors, this study provides a basis for plaque imaging studies focused on stroke risk stratification.

Supplementary Material

1

Acknowledgements

We are grateful to the staff of the Departments of Vascular Surgery and Histopathology at the John Radcliffe Hospital, Oxford and University Medical Center Utrecht and St Antonius Hospital, Nieuwegein, The Netherlands for the provision of plaques for this study.

Sources of Funding

Dominic Howard and the Oxford Plaque study are supported by the National Institute for Health Research (NIHR) Biomedical Research Centre Programme (Oxford). PMR has a Wellcome Trust Senior Investigator Award and an NIHR Senior Investigator Award. The views expressed are those of the authors and not necessarily those of the NIHR or UK Department of Health.

Footnotes

Disclosure:

None

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