Circulating Inflammatory Markers and the Risk of Vascular Complications and Mortality in People With Type 2 Diabetes and Cardiovascular Disease or Risk Factors: The ADVANCE Study

Gordon Lowe; Mark Woodward; Graham Hillis; Ann Rumley; Qiang Li; Stephen Harrap; Michel Marre; Pavel Hamet, Anushka Patel; Neil Poulterl; John Chalmers


Diabetes. 2014;63(3):1115-1123. 

In This Article

Research Design and Methods

The design and results of the ADVANCE randomized clinical trial have been previously published.[2,3,20] Participants were men and women in 20 countries from Asia, Australasia, Europe, and North America, aged ≥55 years, who had received a diagnosis of type 2 diabetes after the age of 30 years. In addition, they were required to have a history of CVD (stroke, myocardial infarction,s transient ischemic attack, unstable angina, coronary or peripheral revascularization, amputation, macroalbuminuria, proliferative retinopathy or photocoagulation, macular edema, or blindess in one eye) or one or more additional cardiovascular risk factors (T2DM duration >10 years, age ≥65 years, current cigarette smoking, total cholesterol >6.0 mmol/L, HDL cholesterol <1.0 mmol/L, or microalbuminuria).[20] The study made two randomized comparisons: a double-blind assessment of the efficacy of fixed combination therapy with perindopril-indapamide (2 mg/0.625 mg for 3 months increasing, if tolerated, to 4 mg/1.25 mg) versus placebo, and an open-label evaluation of an intensive glucose-lowering regimen using modified release gliclazide, with a target HbA1c level of ≤6.5%, versus standard guideline-based glycemic control. A total of 11,140 participants were randomized, and the median duration of follow-up was 5 years.

Nonfasting blood samples were taken at baseline, anticoagulated with EDTA, and stored centrally at −80°C for a median of 7.8 years prior to transportation to the University of Glasgow coagulation laboratory. Samples were available from all countries involved in the ADVANCE Study, except China and India, giving a total population of 7,376 trial participants who contributed samples. Using a nested case-cohort study design,[21] a random subcohort of 3,500 samples was selected plus samples from all additional individuals who had experienced a macrovascular event or a microvascular complication, or had died during the follow-up period (n = 697). High-sensitivity CRP and fibrinogen levels were assayed by immunonephelometry (ProSpec; Dade Behring, Milton Keynes, U.K.) and high-sensitivity IL-6 levels by ELISA (R&D Systems, Oxford, U.K.). Intra-assay and interassay coefficients of variation were 4.7 and 8.3%, 2.6 and 5.3%, and 7.5 and 8.7%, respectively.

Major macrovascular events were cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke. Major microvascular events were new or worsening nephropathy (i.e., development of macroalbuminuria, doubling of serum creatinine level to at least 200 μmol/L, need for renal replacement therapy, or death due to renal disease) or retinopathy (i.e., development of proliferative retinopathy, macular edema, diabetes-related blindness, or retinal photocoagulation therapy).

Hazard ratios for linear effects of each of the three inflammatory biomarkers on each of the three studied outcomes (major macrovascular events, worsening nephropathy, and worsening retinopathy) were obtained from weighted Cox regression models using the STSELPRE procedure for case-cohort analyses in the Stata package. CRP and IL-6 were log-transformed to remove the effects of their skewness. Linearity of relationships among log CRP, log IL-6, and fibrinogen was verified using restricted linear splines, and by dividing each variable into ordinal groups according to fifths and testing for linearity and nonlinearity.[22] To enable direct comparison among the three biomarkers, results were produced for a 1 SD increment (based on the entire sample). Three models, with different sets of potential confounding variables, were fitted for each of the nine inflammatory biomarker/outcome combinations: model 1 with age, sex, and randomized treatment; model 2 with, in addition to the variables in model 1, duration of diabetes, current smoking, systolic blood pressure, BMI, albumin/creatinine ratio (AC ratio), estimated glomerular filtration rate (eGFR), glycated hemoglobin (HbA1c), plasma glucose, total and HDL cholesterol, and triglycerides; and model 3 with, in addition to the variables in model 2, the other two biomarkers. For the variables found to be independent predictors in model 2, the ability to discriminate risk[23,24] and reclassify risk—using the integrated discrimination index (IDI)[25] and the net reclassification improvement (NRI)[25]—were assessed using methods suitable for survival data and applied to the subcohort.