State of the Art: Newer biomarkers in heart failure

European Journal of Heart Failure (2015) 17, 559 569 doi:10.1002/ejhf.273 REVIEW State of the Art: Newer biomarkers in heart failure Rudolf A. de Boer 1, Lori B. Daniels 2,AlanS.Maisel 2,3, and James L. Januzzi Jr 4 * 1 University of Groningen, University Medical Center, Department of Cardiology, Groningen, The Netherlands; 2 Division of Cardiology, University of California at San Diego, La Jolla, CA, USA; 3 Division of Cardiology, Veterans Affairs San Diego Medical Center, La Jolla, CA, USA; and 4 Cardiology Division of the Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Received 8 January 2015; revised 11 March 2015; accepted 23 March 2015; online publish-ahead-of-print 16 April 2015 Since natriuretic peptides were successfully integrated into the clinical practice of heart failure (HF), the possibility of using new biomarkers to advance the management of affected patients has been explored. While a huge number of candidate HF biomarkers have been described recently, very few have made the difficult translation from initial promise to clinical application. These markers mirror the complex pathophysiology of heart failure at various levels: cell loss (troponin), fibrosis (ST2 and galectin-3), infection (procalcitonin), and renal disease (several renal markers). In this review, we examine the best emerging candidates for clinical assessment and management of patients with HF.... Keywords Biomarkers Heart failure Diagnosis Prognosis Monitoring Introduction With the successful integration of natriuretic peptides into clinical practice for heart failure (HF) care, the possibility that newer biomarkers to supplement BNP and its N-terminal equivalent (NT-proBNP) may further advance patient management is now being explored. Good rationale supports this effort: while the natriuretic peptides have proven their usefulness in diagnosis, prognosis, and risk stratification, and may have a role for therapy guidance, 1 the imperfections of natriuretic peptides (e.g. high biological variation and age dependence), along with the multiple co-morbidities of HF patients that can affect concentrations of BNP or NT-proBNP (e.g. sepsis, renal disease, and obesity) leave the door open for new biomarkers to supplement clinical judgement. The number of proposed biomarkers for use in HF can be overwhelming, 2 and only a select few are likely to succeed in translation to clinical use. 3 We therefore take this opportunity to discuss those biomarkers that appear to have most promise for the care of patients with HF. In each case, the appeal of these novel biomarkers relies not only on a synergism with natriuretic peptides, but also on their informing a unique pathophysiology that might allow specific selection and monitoring of treatments on the basis of their results.... Choice of biomarkers and analytical considerations The term biomarker does not necessarily indicate a blood-based marker, but in this work we have restricted ourselves to systemic biomarkers, i.e. circulating in the blood or urine. We did not include other (non-circulating and circulating) markers that could also be perceived as biomarkers, such as molecular imaging techniques using tracers, molecular markers, non-coding RNAs such as micrornas and long non-coding RNAs, and many more (bio-)markers. Herein, we considered circulating biomarkers that have progressed to a stage where the first hurdles of biomarker discovery and implementation have been overcome. Notably, for proper use of biomarkers, it is important that results are reliable, readily available, and easy to interpret. For all biomarkers we discuss in the current article, there are high quality assays available. Assays for ST2, galectin-3, and troponin, are FDA (Food and Dug Administration) cleared assays that have superior performance over other assays. 4 7 Several, but not all, markers can currently be measured using automated platforms, which greatly reduces turnaround time and costs. 6,8,9 The main characteristics of the biomarkers that will be discussed are summarized in Table 1. *Corresponding author: Massachusetts General Hospital, 32 Fruit Street, Yawkey 5984, Boston, MA 02114, USA. Tel: +1 617 726 3443, Fax: +1 617 643 1620, Email: jjanuzzi@partners.org These two authors contributed equally to this work.

560 R.A. de Boer et al. ST2 Biology and pathophysiology The proposed cardiovascular (CV) role of ST2 in myocardial biology has been demonstrated; two isoforms of ST2 are most relevant for CV disease: soluble (sst2) and the transmembrane ligand (ST2L). 10 The ST2 gene is highly expressed by cardiomyocytes and fibroblasts when strained; 11 in this setting ST2L becomes receptive for interleukin-33 (IL-33; also induced by cellular stretch). IL-33 has antihypertrophic and antifibrotic effects transduced by ST2L, while administration of sst2 blocks the favourable influence of IL-33. This suggests that sst2 may serve as a decoy receptor for circulating IL-33. Supporting this hypothesis, in an in vivo model of pressure overload, ST2 / mice demonstrated a greater degree of cardiomyocyte hypertrophy and fibrosis, with worse LV function than wild-type mice after 4 weeks of aortic banding. 12 Additionally, a role for ST2 in the development of vascular remodelling has also been suggested. 13 ST2 in acute and chronic heart failure Recent clinical practice guideline updates from the American College of Cardiology/American Heart Association (ACC/AHA) have given a Class IIb recommendation for sst2 measurement in acute decompensated HF (ADHF) (level of evidence A) and chronic HF (level of evidence B) for the purpose of risk stratification and prognostication in HF patients. 14 Data have suggested sst2 to be a strong contender for measurement in HF. In an analysis of 593 patients admitted to the Emergency Department with acute dyspnoea from the ProBNP Investigation of Dyspnea in the Emergency Department (PRIDE) study, 15 sst2 concentrations were higher in the 209 patients with acute HF. In fully adjusted analyses for predictors of death at 1 year, sst2 was the strongest biomarker examined in the PRIDE study, with a hazard ratio of 4.6, compared with 2.3 for NT-proBNP, and this observation was extended to outcomes at 4 years. 16 sst2 concentrations were linked to higher LV dimensions and volumes, poorer LVEF, worse right heart function, higher pulmonary pressures, as well as a more decompensated haemodynamic profile. 17 sst2 is equally prognostic in patients with HF with preserved ejection fraction (HFpEF) as it is in those with reduced ejection fraction (HFrEF). 18 In a separate study, 19 those with elevated sst2 and NT-proBNP values experienced the highest risk of death at 1 year (>40%), whereas, importantly, in those with a low NT-proBNP, an elevated sst2 level reclassified likelihood of death considerably, suggesting that sst2 provides useful information even in those with low natriuretic peptide values. Similar results were reported in an ADHF cohort, where multimarker testing with highly sensitive (hs) troponin T, NT-proBNP, and sst2 accurately stratified subjects from low (<5% of death) to very high (>50%) risk. 20 The comparative prognostic importance of sst2 compared with other biomarkers was demonstrated in several other analyses. In the PRIDE study, sst2 remained prognostic beyond several other biomarkers besides NT-proBNP, including inflammatory... biomarkers, troponins, and galectin-3. In a large multinational meta-analysis of patients with ADHF where an extensive number of biomarkers were compared relative to one another, 21 sst2 provided value comparable with the strongest prognostic biomarkers for predicting death (Figure 1), improving reclassification of risk beyond a clinical model containing other biomarkers and clinical variables. Additionally, among patients with advanced HF, only sst2 was predictive of 90-day death or transplantation (hazard ratio 5.53), while NT-proBNP, renal biomarkers, hs troponin, and inflammatory biomarkers were not. 22 Rather than relying on a single measurement for prognosis in ADHF, emerging data suggest a role for using serial levels of sst2 to monitor patients. 23,24 In one study, sst2 values >76 ng/ml following hospitalization identified those with a 50% risk for mortality, transplantation, or rehospitalisation. In ambulatory patients with chronic HF, sst2 has comparably strong prognostic value, again additive to natriuretic peptides and equally useful for prognosis in subjects with HFpEF and HFrEF. 25 In a recent head-to-head comparison of sst2 with galectin-3, sst2 had better discrimination, calibration, and reclassification for prognosis in ambulatory HF. 26 Much as in ADHF, serial measurement of sst2 in chronic HF added substantial prognostic information, showing superior prognostic performance compared with all other markers, including hs troponin T, growth differentiation factor-15 (GDF-15), and NT-proBNP. 27 32 ST2 in individuals at risk for heart failure Among 3428 subjects in the Framingham Heart Study, 33 elevated concentrations of sst2 were independently associated with incident HF, CV events, or death during a mean follow-up of 11 years; this predictive value may be explained by the ability of sst2 to predict onset of systolic hypertension, and consistent with a proposed role in vascular remodelling. 34 More recent data from a population-based Finnish study comprising 8444 subjects with a follow-up of 15 years showed that baseline sst2 levels did not predict incident HF or CV events in adjusted models, but did predict all-cause mortality. 35 Beyond community-based subjects, sst2 also predicts onset of HF among those with acute coronary syndromes. 36 38 For example, among 4426 patients with non-st segment elevation acute coronary syndromes, sst2 > 35 ng/ml was associated with an increased risk of HF complications at 30 days and 1 year, even after adjustment for clinical covariates and biomarkers (hazard ratio 1.90),withcon- siderable reclassification improvement from adding sst2 to a base model. Potential role for ST2 in heart failure management Several significant interactions between sst2 and therapeutic interventions for HF have been recently suggested; all of these suggestions are based on retrospective data. In chronic HF patients, sst2 concentrations fell after treatment with higher doses of beta-blockers, and, when titrated in the context of an

Newer biomarkers in heart failure 561 Table 1 Summary of main characteristics of the discussed biomarkers Biomarker Chronic HF Meta-analysis, chronic HF ADHF Meta-analysis, ADHF Subjects at risk Therapy guidance, Therapy guidance, FDA/EMA cleared Increases in response to retrospective prospective assay co-morbidities... Prognosis Prognosis Prognosis Prognosis Prediction ST2 ++ 0 ++ + + ++ 0 + COPD, sepsis Galectin-3 + + ++ + ++ + 0 + Age, renal Hs troponin ++ 0 ++ 0 ++ + 0 + (EMA), 0 (FDA) Age, renal GDF-15 + 0 + 0 + + 0 0 Renal Procalcitonin 0 0 0 0 + ++ 0 + Sepsis, infection NGAL + 0 + 0 0 0 0 0 Renal ADHF, acute decompensated heart failure; EMA, European Medicines Agency; FDA, Food and Drug Administration; GDF-15, growth differentiation factor-15; HF, heart failure; HS, high sensitivity; NGAL, neutrophil gelatinase-associated lipocalin. +, one positive study addressing the item, or multiple studies with variable outcomes where aggregate evidence is positive; ++ multiple studies providing evidence for the item; 0 no such studies conducted. Figure 1 The relative importance of soluble ST2 (sst2) compared with other biomarkers for reclassifying risk for mortality in patients with acutely decompensated heart failure. Compared next to other established or emerging biomarkers, sst2 predicted death independent of clinical variables, and strongly reclassified risk beyond clinical and biochemical measures. CI, confidence interval; CRP, C-reactive protein; IDI, integrated discrimination improvement; MR-proADM, Mid-regional pro-adrenomedullin; MR-proANP, mid-region of the propeptide for atrial natriuretic peptide, NRI, net reclassification index. Reproduced with permission from Lassus et al. 21 sst2 > 35 ng/ml, the benefit of high dose beta-blockers was substantially higher than in the setting of lower sst2 concentrations. 28 Treatment with valsartan in the Valsartan HF Trial (Val-HeFT) resulted in lower sst2 concentrations compared with those treated with placebo, 29 while Maisel and colleagues 30 suggested a treatment interaction between elevated sst2 and benefit from mineralocorticoid receptor antagonist (MRA) therapy; in contrast, no such interaction was found between natriuretic peptides... or galectin-3. Concentrations of sst2 also predict implantable cardioverter/defibrillator (ICD) therapies and sudden death in chronic HF. 31 sst2 measurement may also have a role for detecting rejection as well as for prognostication after heart transplantation. 32 Lastly, preliminary data suggest that measurement of sst2 in patients with acute myocardial infarction (MI) may inform therapy decision-making: Weir and colleagues reported that elevated sst2 concentrations appeared to identify

562 R.A. de Boer et al. those patients post-mi at higher risk for remodelling who showed greater benefits from treatment with eplerenone, 39 a finding not reproduced in an analysis of galectin-3. 40 Galectin-3 Biology and pathophysiology Galectin-3 (LGALS3) is a lectin generally expressed at low levels; however, upon injury or stress, its production is substantially increased, with roles in cell adhesion, inflammation, and tissue fibrosis. 41,42 Accordingly, galectin-3 is markedly regulated in HF: in HF-prone transgenic rats, galectin-3 gene expression was strongly up-regulated. 43 Recent studies have confirmed the consequent up-regulation of galectin-3 in multiple animal models of myocardial, 44 vascular, 45 and renal 46 fibrosis and dysfunction. The presence or absence of galectin-3 is not an epi-phenomenon of heart failure: administration of galectin-3 results in myocardial fibrosis and HF, 43,47 while genetic disruption and pharmacological inhibition of galectin-3 prevents cardiac fibrosis, remodelling, and HF development. 44,47 Galectin-3 in acute and chronic heart failure Recent clinical practice guideline updates from the ACC/AHA have given a Class IIb recommendation for measurement of galectin-3 in ADHF (level of evidence A) and chronic HF (level of evidence B), for the purpose of risk stratification and prognostication in HF patients, stages C D. 14 The first human study reporting the value of galectin-3 as a biomarker of HF was an analysis from the PRIDE study; in this analysis comprising 209 patients with ADHF, galectin-3 was a stronger prognostic marker for 60-day mortality, compared with NT-proBNP. 48 In another ADHF study, the Coordinating study evaluating Outcomes of Advising and Counseling in Heart Failure (COACH), galectin-3 was associated with 18-month mortality and HF rehospitalization. 49 A recent pooled analysis from three ADHF studies showed that galectin-3 appeared to be a consistent and strong predictor of short- and middle-term (30 120 days) rehospitalization and mortality, 50 with significant addition to clinical models, and significant reclassification indices. In the PRIDE study, it was observed that galectin-3 correlated particularly with echocardiographic parameters of diastolic function. 51 In concert with this, the prognostic value of galectin-3 appeared stronger in patients with HFpEF, as compared with those with HFrEF. 49,52 Accordingly, the potential usefulness of galectin-3 in HFpEF has raised considerable interest. 53 In chronic ambulatory systolic HF, several analyses, e.g. from the Val-HeFT, 54 the COntrolled ROsuvastatin multinational trial in heart failure (CORONA), 55 the CArdiac REsynchronization in Heart Failure (CARE-HF), 56 the Heart Failure: A Controlled Trial Investigating Outcomes of Exercise TraiNing (HF-ACTION), 57 and other, smaller studies, 58,59 reported the univariable predictive value of galectin-3 in ambulatory systolic HF. On average, patients in... the highest galectin-3 groups have an excess risk for mortality and HF rehospitalization of about two- to three-fold compared with patients in the lowest galectin-3 groups. Several studies reported that the prognostic value of galectin-3 was lost when renal function or natriuretic peptides were added to the statistical models. 55,57 In these studies, on top of extensive base models, typically combining 10 20 clinical and biochemical variables, galectin-3 appeared to add less or little additional prognostic value. However, a recent meta-analysis, pooling data from 8419 patients with acute and chronic HF with a follow-up of 1 8.7 years, showed that an increase of 1% in circulating galectin-3 was associated with a 28% increased risk for all-cause mortality and a 59% increase in CV mortality, in fully adjusted models including estimated glomerular filtration rate and NT-proBNP. 60 Still, the interaction with renal function appears particularly important. 61,62 Elevated galectin-3 concentrations precede chronic kidney disease in the general population, 63 and galectin-3 was reported to have stronger prognostic value in subjects with renal impairment than in subjects with normal renal function. 64 Galectin-3 in individuals at risk for heart failure The predictive value of galectin-3 for disease development in apparently healthy individuals (HF stages A B) was confirmed in several recently published large studies. In the Prevention of Renal and Vascular End-stage Disease (PREVEND) study of 7968 participants, individuals with the highest galectin-3 levels had a five-fold higher likelihood of dying in the 10 years of follow-up, which remained significant after correction for clinical and biochemical covariates. 65 In the Framingham offspring cohort (3353 subjects), galectin-3 was an independent predictor of new-onset HF and all-cause mortality over 10 years of follow-up, also when corrected for BNP and kidney function, and galectin-3 significantly reclassified subjects at risk vs. not at risk for HF. 66 Additionally, among community-dwelling older adults without CV disease at baseline, elevated galectin-3 levels were independently associated with all-cause and CV mortality over 11 years of follow-up. 67 Potential role for galectin-3 in heart failure management Although galectin-3 is considered a stable marker, changes in galectin-3 may reflect changes in severity of HF. In a proportion of HF patients ( 25%) galectin-3 levels do change over the course of 3 6 months and serial measurements of galectin-3 provide specific and independent prognostic information beyond baseline measurements, as was shown by three recent reports. 54,68,69 In CORONA and COACH, a 15% increase in galectin-3 over 3 6 months was associated with a 50% increased risk of death and HF rehospitalization (Figure 2). 68 This suggests that increasing levels of galectin-3 identify a subgroup of HF patients with changing risk; whether the risk identified by rising galectin-3 can be attenuated is unknown. As with ST2, there are no prospective studies that have addressed whether galectin-3 could have a role in guiding therapy.

Newer biomarkers in heart failure 563 Figure 2 The importance of increasing galectin-3. The figure shows data of 1329 CORONA patients; the green line indicates patients with a 15% increase; the black line indicates patients with stable levels; the red line indicates patients with a 15% decrease in galectin-3 levels. In the CORONA (and the COACH) study, 18% of heart failure (HF) patients had an increase in circulating galectin-3 of 15%, but these patients had a 50% increased in all-cause mortality and HF rehospitalizations. Reproduced with permission from van der Velde et al. 68 In the CORONA study, it was observed that patients with low galectin-3 levels had lower rates of the primary endpoint of CV death, MI, or stroke from adding rosuvastatin, while patients with elevated galectin-3 did not. 55 In the Val-HeFT study, addition of valsartan appeared to reduce HF hospitalizations only in patients with low galectin-3 levels. 54 Very high galectin-3 levels may therefore identify patients with advanced disease who are less likely to respond to pharmacotherapy, while patients with lower levels still have a window of opportunity, and are amenable to pharmacotherapy aimed at certain disease pathways. Concentrations of galectin-3 have been shown to predict ICD therapies and sudden death. 70,71 Contradictory results with respect to response to CRT exist; in the CARE-HF 56 trial, benefit was not related to baseline galectin-3 levels, but in MADIT-CRT patients with elevated galectin-3 had a strong response related to resynchronization. 70 It remains to be addressed whether elevated galectin-3 may be targeted by specific (antigalectin-3) therapies such therapies exist and are in clinical trials for other fibrotic diseases. 72 Cardiac troponins Cardiac troponin is a sensitive and specific indicator of cardiac myonecrosis and plays a key role in the diagnosis of acute MI. However, troponin levels can be elevated even in the absence of an acute coronary event. Patients with HF, whether acute or chronic, frequently have elevated levels of troponin which can complicate the diagnosis of an acute MI. Nonetheless, troponin levels in HF... patients provide important clinical and prognostic information, even in the absence of coronary events. Biology and pathophysiology Troponins are part of the contractile apparatus in striated muscle and help modulate the actin myosin interaction that leads to muscle contraction. The skeletal forms of troponin T and troponin I have different genes and structures from the cardiac forms; thus, measurement of cardiac troponin is thought to be nearly specific for cardiomyocyte injury. 73 With the improving sensitivity of cardiac troponin assays, detection of circulating troponin concentrations in the general population and even more so in the HF population has increased. Emerging hs troponin assays (currently approved in Europe, but not yet in the USA) can, by definition, detect troponin in most of the normal population. 74 The 99th percentile cut-off point varies with each troponin assay, but is rendered somewhat irrelevant in the setting of HF, especially with the newer hs assays since a very significant percentage of HF patients chronically have levels greater than this. 20,75 Troponin levels in HF patients need to be interpreted within a broader clinical context, and used as a continuous instead of a binary variable especially when an acute coronary ischaemia aetiology for a HF exacerbation is suspected. A number of potential mechanisms for troponin elevation in HF patients have been proposed. Myocardial ischaemia is always an important consideration, but studies have demonstrated elevated levels of troponin even in HF patients with normal epicardial coronary arteries. 76 Subendocardial ischaemia leading to myocyte necrosis has been proposed, as has direct toxicity from neurohormones, inflammatory cytokines, or oxidation by-products. Other potential mechanisms include cardiomyocyte apoptosis and/or autophagy due to increased wall stretch; proteolysis of the contractile apparatus, possibly triggered by increased preload; and troponin release from injured but viable myocardium due to increased permeability of the plasma membrane with leakage of the cytosolic pool of troponin. 77 Additionally, plasma membrane shedding of vesicular blebs containing troponin has been documented in cardiomyocytes subjected to stress. Troponin in acute and chronic heart failure Recent clinical practice guideline updates from the USA have given a Class I recommendation for measurement of troponin in ADHF (level of evidence A) and a Class IIb recommendation for its measurement in patients with chronic HF (level of evidence B). 14 The recommendation for ADHF is based on the fact that acute MI is a common cause of decompensation of HF. Because HF patients often have chronically elevated troponin levels, guideline-recommended cut-off points for diagnosing MI need to be assessed within the broader clinical context. 77 The prevalence of elevated troponin levels in HF patients varies widely based upon the population studied and the troponin assay used (Figure 3). In the Acute Decompensated Heart Failure National Registry (ADHERE) of 67 924 patients hospitalized

564 R.A. de Boer et al. Figure 3 Approximate prevalence of detectable and elevated cardiac troponin (Tn; contemporary and high sensitivity) in various heart failure (HF) populations. ADHF, acute decompensated heart failure. Prevalence data were extracted from several studies; 20,78,79,83 90,110 in categories with discordant values over multiple studies, an average value was used. with HF, 75% had detectable troponin I or T levels, but only 6.2% had levels over the upper reference limit, reflecting the insensitivity of conventional assays. 78 With hs troponin assays, nearly all patients with HF have measurable and/or elevated levels. 20,75 Illustrating this point, among 4053 patients with chronic, stable HF from the Val-HeFT study, troponin T was detectable in only 10% using a contemporary assay, while it was detectable in 92% using the hs assay. 79 Detectable troponin in patients with HFpEF seems common as well, though, compared with those with HFrEF, concentrations may be somewhat lower. 80 In more advanced chronic HF patients referred for cardiac transplantation, conventional troponin I was detectable in 49%. 81 In each of these studies, higher levels of troponin were associated with a worse prognosis, even after adjusting for other clinical characteristics. With hs assays, even previously undetectable levels of cardiac troponin have been shown to carry important prognostic information, 79 particularly when troponin concentrations are in a range that is associated with imprecision of conventional methods. Similar to the natriuretic peptides and sst2, the dynamic pattern of hs troponin in ADHF patients carries important prognostic information. In a study of ADHF patients (excluding those with MI), those with increasing troponin levels had increased 90-day mortality compared with patients whose levels were stable or declined. 75 Across a wide spectrum of HF syndromes and regardless of mechanism and assay, elevated troponin is independently associated with adverse events and a worse prognosis, independent of natriuretic peptide levels. While specific therapies are not yet identified to mitigate the risk of HF patients with chronically elevated troponin levels, present recommendations are to ensure such... patients are being treated with optimized, guideline-directed medical therapy. Closer monitoring and follow-up may also be reasonable; there is some evidence that serial troponin levels modestly improve risk prediction. 82 Troponin in individuals at risk for heart failure Detectable levels of cardiac troponin have been demonstrated among apparently healthy individuals in the general population (including stage A HF), as well as asymptomatic individuals with stable CV disease (stage B). With contemporary assays, the prevalence of detectable troponin in stage A/B HF is in the 1 5% range, 83,84 while with hs assays most studies have found that at least 66% of asymptomatic individuals have measureable levels, 85 87 and frequently the percentage is even higher. 88 90 In both at-risk normal populations and those with structural heart disease, higher troponin levels are independently associated with known HF risk factors including diabetes, LV hypertrophy, chronic kidney disease, and elevated natriuretic peptide levels, but not with prior MI or coronary calcium. 83,84,87 The elevated troponin levels generally reflect chronic processes rather than acute ischaemia, and have stronger associations with future risk of HF than with risk of ischaemic events. 86 Serial measurements may improve risk classification. 85 Potential role for troponin in heart failure management While no particular therapy is yet indicated for asymptomatic individuals with elevated troponin levels, a careful review of cardiac risk factors is warranted, along with patient education about symptoms of CV disease. Additionally, elevated troponin levels could be used to motivate individuals to adhere to diet and exercise recommendations, and probably justify aggressive targeting of blood pressure and cholesterol goals. On the horizon: emerging biomarkers in heart failure While the scope of this review precludes an exhaustive review of the multitudes of candidates for measurement in patients with suspected or proven HF, we will emphasize, in rapid-fire fashion, those biomarkers most eligible for further development and clinical use. Growth differentiation factor-15 Growth differentiation factor-15 is a stress-response cytokine that is a distant member of the transforming growth factor (TGF)-beta family. In cardiomyocytes, GDF-15 is produced and released in the setting of oxidative stress or in response to stimulation with cytokines or angiotensin II. Outside the heart, GDF-15 is produced by macrophages, vascular smooth muscle cells (VSMCs), endothelial cells, and adipocytes, and, as such, GDF-15 confers information both from cardiac and extra-cardiac disease. Increased GDF-15 has been associated with worse outcomes and new-onset HF in

Newer biomarkers in heart failure 565 the general population, 33 and with worse outcomes in patients with acute coronary syndromes as well as acute and chronic HF. 91 GDF-15 offers additional prognostic information to clinical risk factors, and on top of natriuretic peptides. There are few data with respect to the utility of GDF-15 to guide specific therapy. Mid-regional pro atrial natriuretic petide Novel assays for the mid-region of the propeptide for atrial natriuretic peptide (MR-proANP) have been recently explored in patients with HF. Although this marker is another natriuretic peptide, MR-proANP provides both diagnostic and prognostic information that is additive to BNP or NT-proBNP, as was shown in the Biomarkers in Acute Heart Failure (BACH) and PRIDE studies. 92,93 The future use of MR-proANP alone or together with a BNP assay remains to be explored. Procalcitonin The chief complaint of shortness of breath requires a rapid and accurate assessment with regards to aetiology, with HF and pneumonia being top considerations that require timely treatment. However, misdiagnoses may result in delayed or erroneous treatment, potentially increasing adverse outcomes, mortality, and cost. 92,94 Accordingly a biomarker to elucidate the presence of concomitant bacterial infection in patients with HF presenting with dyspnoea would be welcome. Procalcitonin (PCT) is a protein whose expression in parenchymal tissue is induced by bacterial infection, either directly by endotoxin or indirectly via cytokines (e.g. IL-6). PCT concentrations may be useful to diagnose and guide antibiotic therapy (initiation and cessation) in lower respiratory tract infections, pneumonia and sepsis, 95,96 and may have value for the evaluation and management of the dyspnoeic patient with ADHF. In a secondary, post-hoc analysis of the BACH study, simple models using PCT were valuable in identifying when pneumonia was likely, and showed that PCT could possibly help inform therapy decision-making. 97 Patients with the lowest quintile of PCT (indicating that bacterial infection was unlikely) who were given antibiotics had a worse outcome than those not given antibiotics. Conversely, patients with the highest quintiles of PCT who were not given antibiotics faired much more poorly than those treated with antibiotics (Figure 4). More recently, in a prospective study of HF patients presenting with respiratory symptoms, PCT-guided treatment resulted in reduced antibiotic usage and improved outcomes (admission to the IntensiveCareUnitormortalityat30days). 98 A prospective trial evaluating whether PCT may help in guiding antibiotic therapy in heart failure has recently been launched (IMPACT-EU; ClinicalTrial.gov identifier: NCT02392689). Renal biomarkers Acute kidney injury (AKI) is a sentinel event in the course of HF. With the increasing complexity of HF patients, AKI is increasing in incidence and prevalence; its prediction and detection are thus of special importance.... Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding protein expressed by neutrophils and various epithelial cells, whose expression is up-regulated in kidney injury. Various cohorts of chronic HF patients have been found to have significantly higher levels of both serum and urine NGAL when compared with control subjects, despite having only modest reductions in estimated glomerular filtration rates. 99 NGAL levels significantly correlate with neurohormonal and clinical measures of disease severity in chronic HF. Measurement of serum NGAL may be useful for predicting the onset of worsening renal function in patients with HF, and levels are generally prognostic for adverse outcomes, particularly when combined with measurement of a natriuretic peptide. 100 Serum NGAL levels have also been shown to predict incident CV disease and mortality in apparently healthy community-dwelling adults. 101 Other markers have also shown promise for predicting renal dysfunction and outcomes in HF patients. Urinary measures of kidney injury molecule-1 (KIM-1) and n-acetyl-β-d-glucosaminidase appear to identify those patients likely to develop worsening renal function whose risk for adverse outcome is considerable. 102 Similarly, both (serum) cystatin C and beta trace protein (biomarkers of renal function) appear to be more prognostic in HF than standard renal function measures such as creatinine. 103 Lastly, the ratio of the tissue inhibitor of matrix metalloproteinase-2 to insulin-like growth factor-binding protein 7 in the urine was recently approved for use to predict risk for AKI. 104 Conclusions and future directions The incorporation of BNP and NT-proBNP into routine clinical care was the first step into the realm of biomarker-supported evaluation and management of patients with HF, and the use of these markers has far exceeded initial expectations. 105 However, residual risk for HF patients remains unacceptably high, which probably reflects the complex pathophysiology of HF. Given this layered pathophysiology, HF represents a prime area where new and emerging biomarkers may considerably raise the bar beyond the natriuretic peptides for accurate, high quality care. Such care would be based on individual phenotype, rather than the one-size-fits-all approach describing modern HF care an approach that may overlook specific treatment opportunities. Using biomarkers in this vein, one could envisage not only employing therapies that are likely to improve outcomes, but also deciding on avoiding use of therapies that are likely not to. We realize that currently these concepts have not been prospectively tested, and these issues must be adequately evaluated before any new biomarker can become a mainstay in HF management. Clearly, a HF patient with an elevated BNP or NT-proBNP concentration is at higher risk. Whether intended lowering of BNP or NT-proBNP with adjustments in therapies such as beta-blockers, vasodilators, or MRAs will result in better outcomes compared with treating patients without specifically aiming to lower natriuretic peptide levels remains a hotly investigated subject. The Guiding Evidence Based Therapy Using Biomarker Intensified Treatment (GUIDE-IT; ClinicalTrials.gov identifier:

566 R.A. de Boer et al. Figure 4 Procalcitonin (PCT) values as a function of infection in patients with acute dyspnoea. Among those with high PCT concentrations, treatment with antibiotics (Abx) was associated with superior outcomes, while in those with low concentrations, conservative management had better outcome. Data derived from Maisel et al. 97 NCT01685840) is an ongoing trial that addresses this issue. 106 The next logical step will be understanding how newer biomarkers will enhance care beyond the care triggered with or without BNP and NT-proBNP. Indeed, in order to be clinically useful, the bar is set considerably higher than simply showing prognostic merit. A major prerequisite for moving forwards from a single-marker strategy to one involving application of multiple biomarkers will be to establish whether an additional biomarker adds to the value of the natriuretic peptides, whether such a marker has a therapeutic implication, and whether it may be used in an easily deployed, cost-effective manner. 107 If of value, an informatics approach that integrates clinical information with results of biomarker and other objective testing may be the optimal method to deploy such advanced care, given the complexity involved. 108 Most focus has been on sst2, galectin-3, and the troponins for this approach, with most convincing data for sst2. In contrast, rather than using biomarkers as an indirect target for HF therapy adjustment, as noted, specific targeting of biomarker function using specific inhibitors may be a useful approach; direct galectin-3 inhibitors have been developed and proof-of-concept has been shown. 72 Finally, while substantial focus has been placed on the use of markers in patients with established HF, we believe that the prognostic merit of these biomarkers in community-based subjects at risk for HF may be even more important. Recent data supporting... use of BNP to prevent HF onset in at-risk community subjects provides enthusiasm to explore use of other biomarkers to identify and address incipient risk for HF more precisely, before the diagnosis has manifest. 109 Such precision medicine approaches represent the future of this area of research. Funding J.L.J. is supported in part by the DeSanctis Clinical Scholar Endowment, and R.A.d.B. by a Netherlands Organization for Scientific Research VIDI grant (917.13.350). Conflict of interest: R.A.d.B. reports receiving speaking fees from Novartis and Medcon; and research support from AstraZeneca. L.B.D. reports receiving consulting fees from diadexus; speaking fees from Critical Diagnostics and Roche; and research supplies from BG Medicine and Critical Diagnostics. A.S.M. reports receiving grant support from Alere, Abbott, BG Medicine, and Thermo Fisher; consulting fees from Alere, Critical Diagnostics, Sphingotec, BG Medicine, and EFG Diagnostics; speaking fees from BG Medicine and Alere; and has ownership interest in Cardero Therapeutics and My Life Diagnostics. J.L.J. reports receiving grant support from Siemens, Singulex, and Thermo Fisher; and consulting fees from Roche, Critical Diagnostics, Amgen, Zensun, Novartis, and Sphingotec.

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