Editor's Note: Commentary based on Eapen DJ, Manocha P, Patel RS, et al. Aggregate risk score based on markers of inflammation, cell stress, and coagulation is an independent predictor of adverse cardiovascular outcomes. J Am Coll Cardiol 2013;62:329-37.

Background

The search continues for that elusive crystal ball, a predictive risk score which could accurately predict future adverse clinical cardiovascular events. Traditional clinical risk factors based on the Framingham Heart study while helpful in identifying patients at risk incompletely stratify patients at risk with underlying coronary artery disease (CAD). Abnormal lipoprotein profile including high LDL, low HDL, and abnormal LDL and HDL size density, are markers of atherosclerotic disease burden. Lipoprotein (a) levels and markers of lipid peroxidation (i.e., ox-LDL and ox-HDL) may further identify patients at increased risk. Elevation of various individual biomarkers such as high-sensitivity C-reactive protein (hs-CRP), brain natriuretic peptide (BNP), CK-MB, troponin, and D-dimer, in patients presenting with acute coronary syndromes have been shown to further stratify these population's risk of subsequent adverse events. Individually, the predictive power of each biomarker is modest. Thus, our ability to predict an individual patient's risk of subsequent cardiovascular events remains imprecise.

It is well established that acute coronary syndromes are mostly caused by acute plaque destabilization and rupture within the coronary vasculature.¹ At the anatomical and cellular level, the presence of high-risk markers such as thin cap fibroatheroma, large necrotic lipid core, and high macrophage content have been found to identify vulnerable, rupture-prone plaque. However, atherosclerosis is not a local but a systemic process that, in association with blood factors that may predispose to thrombosis and diseased myocardium that may be prone to fatal arrhythmias, define the so-called vulnerable patient. Various systemic processes including activation of inflammatory, coagulation, and cellular stress pathways are involved in the progression of atherosclerosis and the propensity for plaque rupture. The simultaneous evaluation of various biomarkers may allow a more accurate assessment of an individual patient's current state in any one of these dimensions.

Methods

Eapen and colleagues sought to determine an aggregate, pathway-specific, risk score for enhanced prediction of death and myocardial infarction (MI) by determining a risk score comprised of biomarkers involved in three of these different pathways.² The authors selected hs-CRP, fibrin degradation products (FDP), and heat shock protein70 (HSP70) as the three biomarkers to analyze. Serum levels of hs-CRP, FDP, and HSP70 were measured in 3,415 consecutive patients with suspected or confirmed coronary artery disease (CAD) undergoing cardiac catheterization. Survival analyses were performed with models adjusted for established risk factors.

Results

At a median follow-up of 2.3 years, each one of the biomarkers studied was associated with increased risk of adverse events. The hazard ratio (HR) of all-cause death and MI for a CRP level greater than 3.0 mg/l was 1.61 (p < 0.0001), for an HSP70 level greater than 0.625 ng/ml was 2.26 (p < 0.0001) and for an FDP level greater than 1.0 mg/ml was 1.62 (p < 0.0001). An aggregate biomarker score between 0 and 3 was calculated based on these cutpoints. Compared with the group with a 0 score, HRs for all-cause death and MI were 1.83, 3.46, and 4.99 for those with scores of 1, 2, and 3, respectively (p for each: <0.001). However, the prevalence of scores 2 or 3 were rather low (16.5% and 3.7%, respectively). Annual event rates were 16.3% in patients with a score of 3 and 2.4% in patients with a score of 0. The ability of a model based on clinical variables to discriminate patients with and without events (C-statistic) was 0.76. After the addition of the biomarker score, the C-statistic improved to 0.8.

Conclusions

Eapen and colleagues concluded that an aggregate score based on serum levels of CRP, FDP, and HSP70 predicts future risk of death and MI in patients with suspected or known CAD.²

Perspective

The current study provides valuable insights into combining biomarkers to assess a patient's cardiovascular risk profile in multiple dimensions. However, the results may not be generalizable, given that the study population, by definition, included patients with known or suspected CAD undergoing cardiac catheterization. Further, a proportion of this population presented with an acute coronary syndrome (ACS), which may increase these biomarkers acutely and is itself predictive of future events. Nevertheless, in this group of patients with suspected or with established CAD, the investigators demonstrated that a strategy using an aggregate risk score consisting of 3 biomarkers including CRP, HSP-70, and FDP could be used to further stratify individual patients' risks for death, MI, or other adverse cardiovascular outcomes.

Current theory of vulnerable plaque holds that various factors and pathways likely contribute to acute plaque rupture.¹ The study findings support the multiple pathways theory of atherosclerotic plaque destabilization, rupture, and subsequent myocardial infarction. Each pathway holds only a small piece of the larger puzzle. The authors selected three circulating biomarkers, hsCRP, FDP, and HSP70. Each is involved in different signaling pathways involving inflammation, coagulation, and stress-induced cellular responses, respectively, and has been shown to be associated with adverse cardiovascular events and plaque instability.

The role of inflammation in CAD has been well documented.³ There are various inflammatory biomarkers which have been noted or associated with acute cardiac events including hsCRP, lipoprotein a, CD40L, ICAM-1, VCAM-1, and P-selectin. Perhaps the best studied inflammatory biomarker in population-based studies is hsCRP. This marker has been shown to add to risk prediction above standard risk factor assessment allowing reclassification of intermediate risk populations. Even though hsCRP can identify an increased risk for an acute coronary event independent of LDL cholesterol,⁴ the magnitude of this effect is only marginal in subjects free of known CAD. Moreover, hsCRP is a nonspecific marker of systemic inflammation and, by itself, has low predictive accuracy for acute cardiac events in individual patients.

Platelet activation and the coagulation cascade are central to the process of arterial thrombosis. Markers of blood hypercoagulability such as fibrinogen, D-dimer, and factor V Leiden may be indicative of increased risk.^1,4,5 Certain gene polymorphisms of platelet gp IIb/IIIa, Ia/IIa, and Ib/IX receptors that result in increased platelet activation and aggregation are also markers of increased risk. Increased coagulation factors (e.g., factors V, VII, and VIII; von Willebrand factor; and factor XIII) and decreased anticoagulation factors (e.g. proteins S and C, thrombomodulin, and antithrombin III) as well as markers of decreased endogenous fibrinolysis activity (eg, reduced t-PA, increased PAI-1, certain PAI-1 polymorphisms) may all contribute to the risk. In the current study, an FDP immunoassay was selected to assess coagulation products and the full complement of fibrin and degradation products. Interestingly, it was not a predictor as a continuous variable but had a threshold cut-off value for FDP (>75th percentile of the population mean) above which it is associated with increased risk.

Heat shock proteins (HSPs) play complex roles in the function of the immune system. They can activate humoral and cellular immune responses, as well the complement system. Immune reactions against heat shock proteins have been implicated in the pathogenesis of atherosclerosis.^6,7 While increased HSP70 expression has been noted at different sites of atherosclerosis, the exact role of HSP70 in atherosclerosis and CAD remains conflicted. A previous study assessed the association between extracellular human Hsp70, anti-Hsp70 antibody and acute MI, acute coronary syndrome and stable angina.⁸ After adjustment for traditional CHD risk factors, increasing levels of HSP70 were significantly associated with increased risk and severity of ACS. In patients with AMI, HSP70 levels decreased rapidly from days 1-7 after onset, whereas anti-HSP70 antibody levels increased. Increasing levels of anti-HSP70 antibody were associated with a decreased risk of ACS.⁸ These findings suggest that higher HSP70 levels or lower anti-HSP70 antibody levels are independently associated with a higher risk of ACS.⁸

For the selected biomarkers, the authors defined certain cutpoints. For each elevated biomarker above these defined cut-off values, the risk of future cardiovascular events significantly increased. Further, the value of these biomarkers was additive such that the patients with greater number of elevated markers were at higher risk. In comparison to patients with a biomarker score of zero, patients with one biomarker elevation experienced a 1.5-fold elevation in event rates, and those with two elevated markers had a threefold increase in event rates. Patients who had a risk score of 3 (<5% of the population) experienced over a 5-fold increased risk of all-cause death or MI within 1 year, with an annual rate of more than 16%.² Therefore, while individually each of these markers may be weakly predictive, in combination, their predictive accuracy improves.

The goal of refining predictive scores is to ultimately identify treatment strategies and recommendations. Whether treatments aimed at reducing activity of these pathways can positively alter the disease course remains to be determined. It is beyond the scope of our commentary to address this in depth, but it is of interest to note some of the previous experience and controversy surrounding systemic therapy with statins, known to have pleiotropic, anti-inflammatory effects. Statin therapy has been shown to decrease hs-CRP levels.^9,10 The JUPITER trial subsequently enrolled more than 17,000 apparently healthy subjects without hyperlipidemia but with elevated hsCRP levels. In addition to reducing LDL cholesterol levels by 50%, rosuvastatin also reduced hs-CRP by 37%, and significantly reduced the absolute risk of heart attack, stroke, and death at one year by 0.2-0.6%.¹¹ A Mendelian randomization study identified polymorphisms in 5 genetic loci which were strongly associated with CRP levels.¹² However, there was a lack of concordance between the effect on coronary heart disease risk of CRP genotypes and CRP levels, suggesting that CRP is not directly causative of cardiovascular events. While likely not causative, the bulk of the data still suggests that there exists an association between inflammation as documented, among other things by CRP level elevation, and the risk of CHD events.

The authors conclude an aggregate risk score based on various biomarker levels, specifically CRP, HSP70, and FDP appears to better predict and risk stratify patients compared to a single marker strategy. However, whether the combination of these three markers is the ideal biomarkers, or whether there is a different combination of other markers which may hold greater predictive power remains to be seen, as biomarkers and potential targets continue to be identified. There are plenty of other biomarkers currently available which are also predictive of adverse cardiac events. Other studies have yielded different and failed to demonstrate the predictive power of CRP. For example, in the Women's Health Initiative study including more than 27,000 post-menopausal women, among the 18 biomarkers measured, CRP level did not significantly improve CHD prediction either alone or in combination with other biomarkers. In this study, five of the 18 biomarkers studied demonstrated additional improvement in the C-statistic with a net reclassification improvement of 6.45% compared with the traditional risk factor model. These markers were IL-6, D-dimer, coagulation factor VIII, von Willebrand factor, and homocysteine.¹³ Elevated levels of BNP or NT-proBNP are indicative of increased left atrial pressures and hemodynamic stress and are prognostically valuable. Further, it is well established that myocardial enzyme elevation including CK, CK-MB, and troponins are associated with increased myocardial necrosis, infarct size, and subsequent events. A 10 biomarkers profile was tested in 3,209 participants attending a routine examination as part of the Framingham Heart Study which included CRP, BNP, NT pro–BNP, aldosterone, renin, fibrinogen, d-dimer, PAI-1, homocysteine; and the urinary albumin-to-creatinine ratio. In this study, the multi-marker scores resulted in only small increases in the ability to classify risk in comparison to conventional risk factors, as measured by the C statistic.¹⁴ The authors of the present study were primarily focused on biomarkers that may suggest plaque instability and subsequent vascular events secondary to plaque destabilization and/or rupture. While the authors solely looked at cardiovascular events, given the degree of overlap with cerebrovascular disease and the systemic nature of these processes, it would be interesting to note the observed event rate in regards to non-cardiac events. Further, whether the risk of subsequent events can be modified and reduced by systemic therapy will need to be addressed in large, randomized, prospective trials. The crystal ball may be a little clearer, but some uncertainty still remains.

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References

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11. Ridker PM, Danielson E, Fonseca FA et al. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med 2008;359:2195-207.
12. Elliott P, Chambers JC, Zhang W et al. Genetic Loci associated with C-reactive protein levels and risk of coronary heart disease. JAMA 2009;302:37-48.
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Keywords: Acute Coronary Syndrome, Arrhythmias, Cardiac, Atherosclerosis, Lipids, Macrophages, Myocardium, Necrosis, Thrombosis, Plaque, Atherosclerotic

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