Plaque 18FDG Uptake on PET Is Associated with Plasma Lipid Levels in Acute Symptomatic

Serum lipids are associated with inflammation-related PET-FDG uptake in symptomatic carotid plaque

SUPPLEMENTARY DATA

Supplementary Methods

Definition of Stroke/TIA and Recurrent Events

Stroke was defined as per the World Health Organization definition, and TIA was defined according to the Oxfordshire Community Stroke Project (OSCP) clinical definition, (i.e. symptoms lasting <24 hours duration).1 2 Patients were assessed by a trained stroke physician, and all episodes of TIA, stroke, or retinal embolism recorded. Stroke severity, measured by National Institutes of Health Stroke Scale (NIHSS), and functional status, by modified Rankin Score (mRS), were assessed by trained stroke researchers.

Clinical stroke and TIA recurrence was defined as a new, sudden onset focal neurological deficit, in the same carotid territory, where examination findings were initially stable for at least 24 hours, in whom no alternative likely cause was identified. Any potential recurrences were reviewed by a trained stroke physician. Patients were censored at the time of CEA or stenting; recurrent events that occurred during/after revascularization were not included in the analysis.

All recruited patients underwent diffusion-weighted magnetic resonance imaging (DWI) at the time of medical presentation, except where a contraindication to MRI (e.g. MRI non-compatible implant) existed, or where MRI would have delayed therapeutic procedures (e.g. carotid endarterectomy [CEA]). MRI scans were read by a trained stroke neurologist and radiologist blinded to PET and lipid data.

Patients with Potential Cardioembolic Source of Stroke

We excluded patients with a potential cardioembolic source when evidence of acute infarction existed in multiple areas of the cerebral circulation (anterior circulation contralateral to the carotid stenosis, or posterior circulation) in addition to the stroke/TIA in the territory of the stenosed carotid artery. Patients with a cardioembolic source and evidence of only acute infarction ipsilateral to the carotid stenosis were included.

Definitions of Body Mass Index, Central Obesity and Metabolic Syndrome

Body mass index (BMI), waist circumference, and waist:hip ratio were measured at recruitment. Central obesity was defined as waist ≥94cm for men, ≥80cm for women.3 The WHO definition of waist:hip ratio was used.4 Metabolic syndrome was defined by both (a) Adult Treatment Panel III criteria and (b) WHO criteria:5

1 (a) Adult Treatment Panel III criteria: Three of: waist >102cm men/>88cm women, triglycerides 1.7mmol/l, HDL <1.04 men/<1.3 women, fasting glucose 6.1mmol/l, hypertension [BP 135/85]

(b) WHO criteria: DM or impaired fasting glucose [IFG] or impaired glucose tolerance [IGT], plus two of: hypertension or antihypertensive medication, triglycerides 1.7mmol/l, HDL <0.9 men/<1.0 women, BMI >30kg/m2 or waist:hip ratio >0.9 men/>0.85 women.

Albuminuria details were not available for our cohort. Venous glucose was measures as part of routine clinical care.

FDG Uptake as Measured by Standardised Uptake Values

18FDG uptake was quantified using standardised uptake values (SUVs, g/ml), a validated method that takes account of injected dose, isotope decay, and patient weight.6 SUV is calculated according to the formula:

SUV= (Mean voxel value within ROI [MBq/ml]) / (Activity injected [MBq]/ W) where for our analyses, W represented body weight (lean body mass or BSA may alternatively be used).6

Statistical Analysis

Parametric and nonparametric comparisons were performed using the χ2 test, Fisher’s exact test, t test and Wilcoxin rank-sum as appropriate. For multivariable regression analysis, where collinearity existed between candidate predictor variables, these were not included in the final model if inclusion did not change the model fit (assessed by pseudo-R2) or findings, i.e. where their inclusion was redundant.

Inter-rater variability for SUV measurement was quantified using the kappa statistic and the intra-class correlation coefficient, and has been previously reported.7

2 Supplementary Results

Missing Data

No data were missing for variables included in statistical analyses. There was no loss to follow-up at 90 days.

Statin Type

Amongst 54 patients on statin at the time of PET-CT, 85.2% (46/54) were on atorvastatin (dose range 10-80mg, 30/46 on ≥40mg), with a further 2 patients on rosuvastatin.

See also Supplementary Tables and e-Figures below.

Supplementary Discussion

Patients with Potential Cardioembolic Source of Stroke

Patients with a cardioembolic source and evidence of only acute infarction ipsilateral to the carotid stenosis were included (N=21). We cannot entirely exclude the possibility that the qualifying stroke/TIA was due to cardioembolism in these cases, although we consider this unlikely.

3 Supplementary Table I. Association between FDG uptake, expressed as TBR, and each lipid moiety. P-values for (i) Univariate regression analysis, (ii) test for trend across tertiles of each lipid moiety, (iii) Multivariable regression analysis, adjusted for age, gender, degree of stenosis, and smoking.

TBRmax TBRROImax TBRmean

(i) Univariate regression analysis (p-values) LDL 0.02 0.03 0.01 Total Cholesterol 0.048 0.06 0.04 HDL 0.03 0.02 0.10 Triglycerides 0.03 0.02 0.28

(ii) Test for trend across lipid tertiles (p-values) LDL 0.08 0.12 0.02 Total Cholesterol 0.03 0.07 0.04 HDL 0.02 0.01 0.18 Triglycerides 0.04 0.04 0.12

(iii) Multivariable regression analysis (p-values) LDL 0.02 0.02 0.008 Total Cholesterol 0.02 0.03 0.02 HDL 0.04 0.41 0.42 Triglycerides 0.095 0.15 0.43

4 Supplementary Table II. Association between FDG uptake in the asymptomatic

(contralateral) ICA, expressed as SUVmax, SUVROImax, and SUVmean, and each lipid moiety. P-values for (i) Univariate regression analysis, (ii) Multivariable regression analysis, adjusted for age, gender, smoking and statin use.

SUVmax SUVROImax SUVmean

(i) Univariate regression analysis (p-values) LDL 0.002 0.006 0.002 Total Cholesterol 0.006 0.02 0.004 HDL 0.005 0.018 0.14 Triglycerides 0.001 0.016 0.03

(ii) Multivariable regression analysis (p-values) LDL 0.004 0.005 0.025 Total Cholesterol 0.005 0.007 0.03 HDL 0.58 0.88 0.81 Triglycerides 0.10 0.30 0.27

5 Figure e-1. Generation of measures of FDG uptake, SUVmax, SUVmean, SUVROImax, for the Segment of Interest (SOI), comprised of 10 Regions of Interest (ROI). Anatomic image representing fused PET and CT angiogram.

S OI R OI

7.5mm 1.5mm

10 R OI, each 1.5mm F or each R egion of Interest (R OI): -Max S UV reading 7.5mm -Mean SUV reading

S ingle highest S UV max reading = S UV R OImax

S UV mean for Segment of interest = (S um of Mean S UV each R OI)/10 S UV max for Segment of interest = (S um of Max S UV each R OI)/10

6 Figure e-2. Carotid plaque FDG uptake, expressed as SUVROImax, by tertile of each of lipid moiety (P-values for test for trend) (a) By tertile of LDL cholesterol (tertile 1: 2.0mmol/l, tertile 2: 2.1-3.0mmol/l, tertile 3: 3.1mmol/l) (b) By tertile of total cholesterol (tertile 1: 3.8mmol/l, tertile 2: 3.9-4.9mmol/l, tertile 3: 5.0mmol/l) (c) By tertile of HDL cholesterol (tertile 1: 0.90mmol/l, tertile 2: 0.91-1.20mmol/l, tertile 3: 1.21mmol/l) (d) By tertile of triglycerides (tertile 1: 1.0mmol/l, tertile 2: 1.01-1.44 mmol/l, tertile 3: 1.45mmol/l)

(a) SUVROImax by tertile of LDL cholesterol

7 (b) SUVROImax by tertile of total cholesterol

(c) SUVROImax by tertile of HDL cholesterol

8 (d) SUVROImax by tertile of triglycerides

9 Figure e-3. Carotid plaque FDG uptake, expressed as SUVmean, by tertile of each of lipid moiety (P-values for test for trend) (a) By tertile of LDL cholesterol (tertile 1: 2.0mmol/l, tertile 2: 2.1-3.0mmol/l, tertile 3: 3.1mmol/l) (b) By tertile of total cholesterol (tertile 1: 3.8mmol/l, tertile 2: 3.9-4.9mmol/l, tertile 3: 5.0mmol/l) (c) By tertile of HDL cholesterol (tertile 1: 0.90mmol/l, tertile 2: 0.91-1.20mmol/l, tertile 3: 1.21mmol/l) (d) By tertile of triglycerides (tertile 1: 1.0mmol/l, tertile 2: 1.01-1.44 mmol/l, tertile 3: 1.45mmol/l)

(a) SUVmean by tertile of LDL cholesterol

10 (b) SUVmean by tertile of total cholesterol

(c) SUVmean by tertile of HDL cholesterol

11 (d) SUVmean by tertile of triglycerides

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