*Imperial College London Peter S. Sever*, Neil R Poulter, Choon L Chang, Aroon Hingorani, Simon McG Thom, Alun D Hughes, Paul Welsh, Naveed Sattar, on behalf of the ASCOT Investigators *Imperial College London . Evaluation of C-reactive protein, prior to and on-treatment, as a predictor of benefit from atorvastatin: Nested case control study from ASCOT

ASCOT CRP Analysis Background Lowering C-reactive protein (CRP) by statins has been shown to independently predict cardiovascular (CV) outcomes The ASCOT database was used to explore the relationship between circulating CRP prior to, and on-treatment, with statins and their association with CV events

ASCOT Study Design Placebo Atenolol ± bendrofluazide Amlodipine ± perindopril Atorvastatin 10 mg 19342 hypertensive patients randomized to antihypertensive treatment 10305 patients eligible and randomized in lipid-lowering arm TC ≤ 6.5 mmol/L (250 mg/dL) ASCOT-BPLA stopped after 5.5 yrs ASCOT-LLA stopped after 3.3 yrs Investigator-led, multinational randomised controlled trial conducted in hypertensive patients, 40 -79 yrs, with no prior history of CHD, but with 3 additional cardiovascular risk factors (male sex, > 55 yrs, smoking etc )

Nested Case-control Trial Profile ASCOT – Subjects in the UK & Ireland (n = 9098) On-treatment Analysis Population (ASCOT-LLA) LDL-C: 156 cases & 498 controls CRP: 166 cases & 522 controls LDL-C & CRP: 155 cases & 488 controls 14 cases & 37 controls were excluded because event occurred before blood samples were obtained. 66 cases & 252 controls did not have both baseline and on- treatment CRP & LDL-C or with missing values in covariates White European (n = 8217) 6549 subjects met entry criteria as potential cases/controls 235 cases & 777 controls participating in ASCOT-LLA 485 cases matched with 1367 controls (ASCOT-BPLA case- control population) Baseline CRP Analysis Population (ASCOT-BPLA) 452 cases & 1269 controls 490 cases & 5750 controls with valid lab data No controls available for 5 cases 33 cases & 98 controls with missing values in covariates

Study Subjects & Blood Samples 485 cases (355 CHD & 130 stroke) matched with 1367 controls Up to 3 controls from the same risk-set were matched to each case by age±1 year, sex and study entry time±90 days. Fasting serum HDL-C, triglycerides and total cholesterol Routinely measured at study visits CRP at baseline and 6 months Measured at the same time using stored serum samples By a high sensitivity method (Dade-Behring, the lower limit of sensitivity was 0.1 mg/L)

Statistical Methods t-test or Χ2 test for baseline characteristics comparison between cases and controls Odd ratio obtained from a conditional logistic regression model for the association between CRP and the risk of CV events (CHD or stroke) Loge CRP (baseline) as a continuous variable Categorizing CRP into tertiles with the lowest as reference Conditional logistic regression model Model 1: unadjusted Model 2 (Framingham CV risk factors + randomised treatment ) Model 3 (extended CV risk factors): as in Model 2 plus Body mass index (BMI) ,Loge transformed fasting glucose, Family history of coronary heart disease (FHCHD) ,Creatinine and Educational attainment

Baseline Characteristics Variable Cases (n = 485) Controls (n = 1367) P value Male (%) 409 (84.3) 1159 (84.8) 0.81 Age, years 64.80 (7.83) 64.65 (7.66) 0.71 Current Smokers (%) 127 (26.2) 297 (21.7) 0.04 Diabetes (%) 150 (30.9) 356 (26.0) 0.038 SBP, mmHg 164.61 (17.81) 161.70 (17.48) 0.002 DBP, mmHg 92.95 (10.37) 92.13 (9.83) 0.12 Total cholesterol, mmol/L 5.99 (1.07) 5.92 (1.07) 0.21 LDL- C, mmol/L 3.92 (0.97) 3.79 (0.96) 0.02 HDL-C, mmol/L 1.26 (0.33) 1.30 (0.34) Loge triglyceride, mmol/L 0.52 (0.46) 0.50 (0.49) 0.44 Loge CRP, mg/L 1.13 (0.98) 0.95 (0.98) 0.0006 Loge glucose, mmol/L 1.82 (0.31) 1.78 (0.28) 0.03 Creatinine, mmol/L 102.34 (19.31) 99.23 (16.57) 0.0008 Amlodipine (%) 227 (46.8) 694 (50.8) 0.13 Atorvastatin* (%) 101 (43.0) 407(52.4) 0.01 Values are mean (SD) or n (%) Missing: LDL-C:121, triglycerides: 79, fasting glucose: 84, CRP: 4, creatinine: 45 *235 cases and 777 controls participated in ASCOT-LLA

Baseline CRP and Risk of CV Events (CHD or Stroke) Cases Control OR P value Trend Per 1 SD increase in loge CRP 452 1269 1.21 0.0004 Tertile 1 CRP: <1.74 mg/L 131 448 1 (ref) 0.005 Model 1 Tertile 2 CRP: 1.74-4.09 mg/L 153 417 1.26 0.08 Tertile 3 CRP: >4.09 mg/L 168 404 1.45 Unadjusted Per 1 SD increase in loge CRP 452 1269 1.16 0.009 Tertile 1 CRP: <1.74 mg/L 131 448 1 (ref) 0.057 Model 2* Tertile 2 CRP: 1.74-4.09 mg/L 153 417 0.29 Tertile 3 CRP: >4.09 mg/L 168 404 1.30 0.06 Per 1 SD increase in loge CRP 452 1269 1.19 0.006 Tertile 1 CRP: <1.74 mg/L 131 448 1 (ref) 0.05 Model 3 Tertile 2 CRP: 1.74-4.09 mg/L 153 417 1.25 0.14 Tertile 3 CRP: >4.09 mg/L 168 404 1.35 Adjusted as for in Model 2 plus BMI, loge-glucose, family history of CHD, creatinine and educational attainment Odd ratio (95% CI) *Model 2: Adjusted for current smoking status, diabetes mellitus, randomized BP treatment (atenolol/amlodipine), randomized atorvastatin/placebo/not in LLA, left ventricular hypertrophy, baseline SBP, total cholesterol and HDL-C

Modified Framingham Model (Model 2) Predictive Ability of Baseline CRP in CVD Prediction Beyond Classical Risk Factors - 1 Modified Framingham Model (Model 2) Full Model (Model 3) Performance Measure Without CRP With CRP Discrimination Area under ROC (95% CI) 0.592 (0.562, 0.621) 0.600 (0.571, 0.630) 0.620 (0.591, 0.650) 0.627 (0.598, 0.656) P-value 0.20 0.18 Calibration Hosmer-Lemeshow, χ² ,deciles 5.94 10.88 2.83 2.97 0.65 0.21 0.94 Adjusted for age, sex, smoking status, diabetic mellitus, baseline SBP, total cholesterol, HDL-C, randomized BP treatment, randomized statin/placebo/not in LLA and LVH Full model: as for reduced model plus loge glucose, family history of CHD, educational attainment, creatinine and BMI

Predictive Ability of Baseline CRP in CVD Prediction Beyond Classical Risk Factors - 2 Net Reclassification Improvement (NRI) for model including CRP over model without CRP Of 485 cases the Framingham model with CRP improved risk classification in 54 but worsened classification in 39 In 1367 controls CRP improved classification in 116 and worsened classification in 131 In Model 2, NRI=0.021 (p=0.32) In Model 3, NRI=0.03 (p=0.17) Estimation of Integrated Discrimination Improvement (IDI) When CRP was included In Model 2, IDI increased 0.38% (P=0.015) In Model 3, the increase in IDI was 0.49% (P=0.013) It suggests that the addition of CRP to the model with established risk factors very modestly improved the discriminatory property of the model for risk prediction.

Effect of Atorvastatin on CVD events by Tertile of Baseline CRP Odd Ratios (95% CI) by Tertile of Baseline CRP Low Middle High Interaction* CVD 0.60 (0.33, 1.10) 0.77 (0.41, 1.46) 0.94 (0.51, 1.78) P=0.54 Adjusted for current smoking status, diabetes mellitus, randomized BP treatment (atenolol/amlodipine), randomized atorvastatin/placebo/not in ASCOT-LLA. Left ventricular hypertrophy, baseline SBP, total cholesterol, HDL-C, BMI, loge-glucose, family history of CHD, creatinine and educational attainment and baseline LDL-C. * Interaction between statin treatment and tertile baseline CRP

Effects of Atorvastatin on LDL-C and CRP (median and interquartile range) 40.3% reduction 27.4% reduction 3.55 2.52 2.12 1.83 On-treatment change in CRP was independent of baseline classical risk factors, baseline CRP and change in LDL-C (P=0.02) In ASCOT-LLA, in those assigned atorvastatin, age-adjusted Spearman’s correlation between percentage change in CRP and percentage change in LDL-C was modest (r=0.19 P=0.0006)

Baseline Profiles by 6 Month Median CRP Variable On-treatment CRP ≥1.83mg/L (n = 195) CRP <1.83mg/L (n = 193) P value Male (%) 171 (87.7) 175 (90.7) 0.34 Age (Years) 64.69 (7.67) 65.44 (6.86) 0.31 Current Smokers (%) 53 (27.2) 31 (16.1) 0.008 Alcohol (%): Never 45 (23.1) 35 (18.1) <=14/21 units/week 109 (55.9) 125 (64.8) >14/21 units/week 41 (21.0) 33 (17.1) 0.20 SBP (mmHg) 161.59 (16.95) 163.63 (19.04) 0.27 DBP (mmHg) 91.70 (9.17) 92.47 (9.52) 0.42 BMI (Kg/m2) 29.76 (4.43) 27.95 (4.07) <0.0001 Total Cholesterol (mmol/L) 5.53 (0.71) 5.51 (0.75) 0.83 LDL-C (mmol/L) 3.53 (0.66) 3.50 (0.71) 0.69 HDL-C (mmol/L) 1.23 (0.30) 1.30 (0.36) 0.04 Loge Triglyceride (mmol/L) 0.46 (0.44) 0.35 (0.47) 0.03 Loge glucose (mmol/L) 1.77 (0.31) 1.78 (0.25) 0.73 Loge CRP (mg/L) 1.41 (0.91) 0.49 (0.89) Creatinine (mmol/L) 101.29 (19.43) 99.99 (14.62) 0.47 Diabetes (%) 61 (31.3) 41 (21.2) 0.02 Family history CHD (%) 87 (44.6) 61 (31.6) Amlodipine (%) 31 (15.9) 23 (11.9) 0.26 Values are mean (SD) or n (%) Missing: fasting glucose: 24; creatinine: 12; LDL-C: 27; triglyceride: 22

On-treatment LDL-C (6 Month in Trial) and Risk of CV Events (CHD or Stroke) Cases Control OR P value Placebo 89 232 1 (ref) Atorvastatin LDL-C ≥2.1 mmol/L 44 126 0.97 0.87 Model 1 LDL-C <2.1 mmol/L 23 140 0.45 0.001 LDL-C <2.1 vs. ≥2.1 mmol/L 0.40 0.002 Unadjusted Placebo 89 232 1 (ref) Atorvastatin LDL-C ≥2.1 mmol/L 44 126 1.09 0.71 Model 2 LDL-C <2.1 mmol/L 23 140 0.45 0.003 LDL-C <2.1 vs. ≥2.1 mmol/L 0.41 Adjusted for baseline LDL-C and loge baseline CRP Placebo 89 232 1 (ref) Atorvastatin LDL-C ≥2.1 mmol/L 44 126 1.10 0.68 Model 3* LDL-C <2.1 mmol/L 23 140 0.45 0.003 LDL-C <2.1 vs. ≥2.1 mmol/L 0.41 0.004 Odd ratio (95% CI) *Model 3: Current smoking status, diabetes mellitus, randomized BP treatment (atenolol/amlodipine), left ventricular hypertrophy, baseline SBP, HDL-C, BMI, loge-glucose, family history of CHD, creatinine, educational attainment, baseline LDL-C or total cholesterol and loge baseline CRP

On-treatment CRP (6 Month in Trial) and Risk of CV Events (CHD or Stroke) Cases Control OR P value Placebo 93 245 1 (ref) Atorvastatin CRP ≥1.83 mg/L 41 137 0.86 0.47 Model 1 CRP <1.83 mg/L 32 140 0.61 0.03 CRP <1.83 vs. ≥1.83 mg/L 0.70 0.17 Unadjusted Placebo 93 245 1 (ref) Atorvastatin CRP ≥1.83 mg/L 41 137 0.83 0.39 Model 2 CRP <1.83 mg/L 32 140 0.71 0.14 CRP <1.83 vs. ≥1.83 mg/L 0.86 0.56 Adjusted for baseline LDL-C and loge baseline CRP Placebo 93 245 1 (ref) Atorvastatin CRP ≥1.83 mg/L 41 137 0.82 0.38 Model 3* CRP <1.83 mg/L 32 140 0.70 0.15 CRP <1.83 vs. ≥1.83 mg/L 0.86 0.60 Odd ratio (95% CI) *Model 3: Current smoking status, diabetes mellitus, randomized BP treatment (atenolol/amlodipine), left ventricular hypertrophy, baseline SBP, HDL-C, BMI, loge-glucose, family history of CHD, creatinine, educational attainment, baseline LDL-C or total cholesterol and loge baseline CRP

Risk of CV Events (CHD or Stroke) by On-treatment (6 Month in Trial) LDL-C and CRP* Cases Control OR P value Placebo 88 230 1 (ref) LDL-C ≥2.1 & CRP ≥1.83 27 65 1.28 0.40 LDL-C ≥2.1 & CRP <1.83 17 55 0.99 0.98 ASCOT Medians LDL-C <2.1 & CRP ≥1.83 11 62 0.43 0.02 LDL-C <2.1 & CRP <1.83 12 76 0.49 0.05 Placebo 88 230 1 (ref) LDL-C ≥1.8 & CRP ≥2 30 81 1.05 0.85 LDL-C ≥1.8 & CRP <2 27 94 0.93 0.80 JUPITER Cut-offs LDL-C <1.8 & CRP ≥2 3 36 0.21 0.01 LDL-C <1.8 & CRP <2 7 47 0.42 0.06 Placebo 88 230 1 (ref) LDL-C ≥1.8 & CRP ≥1 46 126 1.08 0.75 LDL-C ≥1.8 & CRP <1 11 49 0.76 0.48 JUPITER Cut-offs LDL-C <1.8 & CRP ≥1 5 53 0.23 0.003 LDL-C <1.8 & CRP <1 30 0.53 *LDL-C in mmol/L and CRP in mg/L Odd ratio (95% CI) Adjusted for current smoking status, diabetes mellitus, randomised BP treatment (atenolol/amlodipine), , left ventricular hypertrophy, baseline SBP, total cholesterol, HDL-cholesterol, BMI, loge-glucose, family history of CHD, creatinine, educational attainment, and baseline LDL or total cholesterol and loge baseline CRP

Summary Baseline LDL-C and loge transformed CRP were correlated and predicted CV events respectively Inclusion of baseline CRP into a modified Framingham risk model in the whole cohort very modestly improved risk prediction Baseline CRP was not an indicator of the magnitude of the effect of atorvastatin on CV outcome of those assigned atorvastatin At 6 months, atorvastatin reduced median LDL-C by 40.3% and median CRP by 27.4% In those randomized to atorvastatin Lower on-treatment LDL-C at 6 months was associated with a highly significant reduction in subsequent CV events By contrast, lower CRP at 6 months was not associated with CV events Consequently, addition of on-treatment CRP to on-treatment LDL-C did not improve prediction of statin efficacy

Conclusion In ASCOT-LLA, neither baseline nor on-treatment CRP provide useful information about the efficacy of statin treatment to reduce CV events beyond LDL-C reduction The results do not support current proposals to measure CRP in the clinical setting either to assign statins to individuals on the basis of an elevated CRP alone, or to monitor CRP levels as an indicator of the efficacy of statin treatment