Elevated Serum Free Light Chains Predict Cardiovascular Events in Type 2 Diabetes

Predicting risk of cardiovascular disease (CVD) in type 2 diabetes (1–3) using algorithms achieves only moderate performance particularly in different ethnic populations (3). An association between increased polyclonal combined immunoglobulin free light chain (cFLC, summated FLCκ+FLCλ) concentrations and increased all-cause mortality has been reported (4,5); we assessed the potential of cFLC as a marker of CVD outcomes in south Asian patients with type 2 diabetes.

The United Kingdom Asian Diabetes Study was a cluster-randomized controlled trial aimed at reducing cardiovascular risk in south Asian patients with type 2 diabetes (6–8). The current study included 352 south Asian patients whose baseline fasting serum samples were available. Detailed clinical history including treatment, CVD history at baseline and over 2 years of follow-up, plus serum biochemistry at baseline (HbA_1c, lipids, and albumin-to-creatinine ratio [ACR]) were obtained. Serum cFLC (Freelite; The Binding Site Group Ltd, Birmingham, U.K.) and hsCRP (Siemens, München, Germany) levels were measured.

Baseline characteristics of patients with and without a nonfatal or fatal CVD event over 2 years of follow-up were compared using the t test, Mann–Whitney U test for nonnormally distributed variables, and χ² test for categorical variables as appropriate. Factors with a P value <0.1 were selected for inclusion in multivariate modeling using forward stepwise logistic regression analysis to identify significant predictors of CVD events, estimating odds ratio (OR) and 95% CIs. Intervention thresholds defined in other populations may not be appropriate for south Asian patients with type 2 diabetes (7,9), so receiver operator characteristic (ROC) analysis was used to determine the optimal prognostic value of individual risk factors for nonfatal or fatal CVD events. A simple risk score was generated by summing numbers of elevated risk factors for each patient. Age, ACR (>2.5 mg/mmol in men and >3.5 mg/mmol in women), diabetes duration, treatment with insulin, statins, or ACE inhibitors were included individually as confounding factors. Statistical analysis was performed using SPSS v19 (IBM, Armonk, NY).

Over 2 years, there were 7 fatal (myocardial infarction, 2; heart failure, 2; stroke, 2; and cardiac arrest, 1) and 23 nonfatal CVD events (angina, 10; myocardial infarction, 4; heart failure, 3; stroke, 2; and coronary artery bypass graft, 4). CVD events were associated with increased cFLC concentrations (50.7 vs. 42.8 mg/L; P = 0.004), HbA_1c (9.2 vs. 8.0% [77 vs. 64 mmol/mol]; P = 0.035), ACR (4.5 vs. 0.8 mg/mmol; P = 0.034), and age (64 vs. 55 years; P = 0.007), but not hsCRP (4.5 vs. 3.6 mg/L; P = 0.389). Patients with CVD events had longer duration of diabetes (10 vs. 6 years; P = 0.011) and were more likely to have previous history of CVD (34.5 vs. 15.1%; P = 0.008), treatment with insulin (41.4 vs. 21.4%; P = 0.014), statins (72.4 vs. 43.7%; P = 0.003), and ACE inhibitors (58.6 vs. 30.0%; P = 0.002).

The optimal concentrations for predicting CVD events in this population (determined by ROC analysis) were cFLC >57.2 mg/L, triglycerides >6.7 mmol/L, HbA_1c >9.2% [77 mmol/mol], and systolic blood pressure (SBP) >155 mmHg. In multivariate logistic regression analysis using these values and previous history of CVD, elevated cFLC (OR 3.25; P = 0.012), triglycerides (OR 18.30; P < 0.001), and SBP (OR 3.62; P = 0.004) were independently associated with CVD events (Table 1, Model 1). cFLC, triglycerides, and SBP remained significantly associated with CVD risk after individually adjusting for age (Table 1, Model 2), abnormal ACR (Table 1, Model 2), and treatment with ACE inhibitors (Table 1, Model 3) or statins (Table 1, Model 4).

A risk score based on cFLC, triglycerides, and SBP identified patients with 0 (reference), 1 OR = 5.7 (95% CI 2.1–15.0; P = 0.001), or 2 OR = 15.4 (4.8–49.3; P < 0.001) risk factors; no patients were abnormal for all three risk factors. A total of 23 (79%) patients with CVD events were abnormal for at least one risk factor. Individually, 13 (45%) patients with CVD events had elevated cFLC, 12 (41%) had abnormal SBP, and 6 (21%) had abnormal triglyceride concentrations.

To our knowledge, this is the first report of a specific association between cFLC and increased risk of CVD events in type 2 diabetes. Our results are consistent with recent reports showing that high cFLC levels were prognostic for all-cause mortality where circulatory disease was a predominant cause of death (4,5). A number of factors are traditionally associated with CVD risk in western European populations (1–3,7). In our study, HbA_1c, SBP, triglycerides, and CVD history were all associated with poor outcomes; however, only cFLC, SBP, and triglycerides were independently associated with increased risk of CVD events. An association between elevated hsCRP levels and increased cardiovascular risk has been reported in European populations (1,3). Although both cFLC and hsCRP are elevated in south Asians with type 2 diabetes (8,9), in this study, we found that cFLC was a better predictor of CVD risk than hsCRP. Healthy south Asians have higher hsCRP levels than white Europeans (9), so the lack of association observed in our study may simply reflect disease-specific and ethnic variations and support the need for more ethnic-specific studies.

In our study, the association between cFLC and CVD outcome was unaffected after adjusting for age or for diabetes duration. However, diabetes occurs at a younger age in south Asian populations (7), and age at diagnosis may be a more appropriate risk marker than current age (2). Furthermore, cFLC remained independently associated with CVD risk after adjustment for insulin, statins, or ACE inhibitors. Although these treatments have been successful in reducing the risk of CVD in diabetes, the residual risk remained significant and may result from elevated triglyceride levels and chronic inflammation, which have been proposed as additional therapeutic targets (10,11). In this study, a simple risk model including cFLC, SBP, and triglycerides identified the majority of patents suffering CVD events and could therefore be useful in identifying high-risk patients that may benefit from such novel treatments. Microalbuminuria is an important risk factor for CVD (12). In this study, an abnormal ACR was not independently associated with CVD outcome and adjusting for abnormal ACR did not alter the association of cFLC with CVD risk. The lack of association between ACR and CVD may simply reflect the aggressive risk-factor management in those with microalbuminuria but needs further verification.

Serum cFLC levels represent the balance between B-cell lineage production and clearance/metabolism that occurs principally in the kidneys (13,14). cFLC concentrations are raised by chronic inflammation or renal insufficiency (13,14), which are both features of type 2 diabetes. cFLC levels also correlate strongly with the severity of renal impairment (15). Previous work from our group demonstrated that cFLC concentrations are elevated in patients with diabetes compared with healthy control subjects and were seen before overt signs of renal disease (8). Increasing cFLC concentrations may therefore provide an early indication of CVD risk in patients with diabetes (12).

While our study is limited by its size, follow-up time, and few events, the consistent association of cFLC with adverse CVD outcomes suggests that cFLC could be an important marker in the estimation of CVD risk. Further studies involving larger cohorts are needed to examine the validity of these findings in other ethnic groups and to determine whether cFLC measurement would improve existing risk models, such as QRISK2 or the United Kingdom Prospective Diabetes Study (2). Further investigations are also required to examine the influence of factors such as obesity, hypertension, and diabetes treatments on cFLC concentrations. cFLC elevation may aid in the identification of additional therapeutic targets in patients with high residual risk.

Funding. The Binding Site Group Ltd. provided assays and assayed samples for the study free of charge.

Duality of Interest. The United Kingdom Asian Diabetes Study is supported by unrestricted grants from Pfizer, Sanofi, Servier Laboratories UK, Merck Sharp & Dohme/Schering-Plough, Takeda UK, Roche, Merck Pharma, Daiichi-Sankyo UK, Boehringer Ingelheim, Eli Lilly and Company, Novo Nordisk, Bristol-Myers Squibb, Solvay Health Care, and Assurance Medical Society UK. S.B. and A.H.B. have received research grants and lecture fees from the companies that sponsored this study. J.M.F. and L.K.A. are employees of and S.J.H. is a director of The Binding Site Group Ltd. C.A.H. received research funding from The Binding Site Group Ltd. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. All authors contributed to the study design, writing the manuscript, data interpretation, had full access to all data in the study, and had final responsibility for the decision to submit for publication. S.B. had the original idea for the study, was responsible for data collection and analysis, and undertook the detailed statistical analysis. J.M.F. and N.T.R. were responsible for data collection and analysis and undertook the detailed statistical analysis. L.K.A. was responsible for data collection and analysis. C.A.H., S.J.H., and A.H.B. had the original idea for the study. S.B. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. This paper was presented in poster form at the American Society of Nephrology Kidney Week, Atlanta, GA, 5–10 November 2013.