Serum-based biomarkers have been integral to the advancement of the field of cardiology during the past half-century, and with better knowledge about their role in the pathophysiology of cardiovascular disease (CVD), their utility has expanded from merely aiding in the diagnosis of disease to being important in predicting risk and prognosis. Some, such as the natriuretic peptides, B-type natriuretic peptide (BNP) and its amino-terminal pro peptide equivalent, N-terminal-proBNP (NT-proBNP), also are being actively explored as a means of guiding therapy. Novel biomarkers continue to be evaluated, along with new lines of investigation, including genomics and metabolomics, which hold the potential for even greater insights into CVD and its treatments.
Though by any measure this progress has been significant, the best may be yet to come, according to Fred Apple, PhD, medical director of clinical laboratories at Hennepin County Medical Center and professor of laboratory medicine and pathology at the University of Minnesota School of Medicine, in Minneapolis. "Our world is expanding in the sense that predominantly we've been using proteins to follow diagnosis and are now slowly inching into following outcomes. In the protein world, the question is, are there going to be other biomarkers that complement or replace the current ones that are pretty solid," he said. "Now, looking down the road, there's clearly growth in genomics and metabolomics, and the questions will be, how will these emerging fields assist in detecting disease early on, and how will they improve patient outcomes and care?" Apple co-edited the January issue of Clinical Chemistry, which explores the impact of biomarkers, proteomics, and genomics in CVD.
The Latest in Heart Failure
One particularly promising avenue for evaluating novel biomarkers or considering established ones in new ways pertains to heart failure, a major burden for the healthcare system due to its prevalence and high morbidity, mortality, and costs. Though physicians encounter this condition often, it remains challenging to diagnose, and once identified, assessing patients' status and fine-tuning their medications also can be a bit of a guessing game.
"It turns out heart failure is a very heterogeneous group of different syndromes with many different causes. So it stands to reason that if there are a wide range of reasons for heart failure and different types of heart failure, that different biomarkers are going to provide different information from patient-to-patient," explained James Januzzi, Jr., MD, associate professor of medicine at Harvard Medical School and director of the Cardiac Intensive Care Unit at Massachusetts General Hospital in Boston. "As a consequence, up until now the greatest value of biomarkers in heart failure has been restricted to being used for diagnosis. But the thing people are asking is whether we can utilize biomarkers to establish risk in the syndrome, and ultimately, better manage heart failure patients."
Can BNP Guide Therapy?
BNP and NT-proBNP have emerged as the workhorse biomarkers to aide in diagnosing heart failure. They also have been established as useful in predicting outcomes. However, the Holy Grail of using BNP or NT-proBNP to guide heart failure treatment remains tantalizingly out of reach for now. After one promising study published a decade ago, a spate of investigations has produced decidedly mixed results. Some have linked BNP-guided management with significant clinical benefit, at least in younger patients, while others have found no particular change in clinical outcome or quality of life from assigning higher medication doses to patients with higher BNP levels.
Januzzi thinks this muddled picture has to do with the heterogeneity of heart failure coupled with the shortcomings not only of BNP itself and the studies involving BNP, but also existing cardiac medications. "We recognize now that the natriuretic peptides, while useful in a large sense for diagnosis, prognosis, and now, management, of heart failure, come with some limitations. They're fairly promiscuous in terms of what causes them to rise, so they're not very specific for one particular pathophysiology in heart failure," he explained.
Despite the prevalence of the disease, biomarker-guided heart failure trials have suffered not only from their size—about 3,000 patients in total during the past decade, according to Januzzi—but also their patient populations. "One of the things we're coming to recognize is that some forms of heart failure respond to therapies we have in our armamentarium already, while others are less likely to respond. Some of the trials selected patients who were not likely to respond to standard therapies, so why would the marker change anything?" he observed. These caveats aside, Januzzi's research team recently published the results of a successful NT-proBNP guided heart failure trial, in which therapy guided by NT-proBNP reduced hospitalization for heart failure by more than 50% (J Am Coll Cardiol 2011;58:1881–9). He and his colleagues are now in the process of proposing pivotal multicenter trials, with the expectation that they will address both the N challenge and patient selection issues.
In the meantime, research is quite active in looking at candidate biomarkers in heart failure that are involved in various aspects of the disease's pathophysiology (See Table, below). Of the more than 60 potential markers, Januzzi believes soluble ST2 and galectin 3 hold considerable promise. "We and others have shown that concentrations of these markers are very prognostic, and are additive to the natriuretic peptides for prognosis, and that sets the stage for looking at their additive value for managing heart failure," he said. "If we find that specific drugs and markers work better together, it may be that we use one marker to make adjustments in beta blockers, for example, and another to adjust aldosterone antagonists. So the marker will be used to guide the biologically driven application of medications."
Candidate Biomarkers in Heart Failure
More than 60 biomarkers, reflecting the many different pathophysiologies in heart failure, have been proposed. Here are some examples.
- C-reactive protein
- Tumor necrosis factor α
- Interleukins 1, 6, 10 and 18
- Oxidized LDL
- Urinary biopyrrins
- MMP2, MMP3, MMP9
- Collagen propeptides
- Angiotensin II
- Arginine vasopressin, copeptin
Myocyte Injury and Apoptosis
- Troponins I and T
- Myosin light-chain kinase I
- Heart-type fatty-acid binding protein
- Creatine kinase MB fraction
- BNP, NT-proBNP, MR-proANP
- Cystatin-C, β-trace protein
- NGAL, NAG, KIM-1
Legend: lipoprotein-associated phospholipase A2; low-density lipoproteins; matrix metalloproteinases; tissue inhibitor of metalloproteinases; B-type natriuretic peptide; N-terminal-proBNP; mid-regional pro-atrial natriuretic peptide; soluble ST2, Growth differentiation factor 15; Neutrophil gelatinase-associated lipocalin; N-acetyl-β-D-glucosaminidase; Kidney Injury Molecule-1.
Adapted from: van Kimmenade RRJ, Januzzi JL. Emerging Biomarkers in Heart Failure. Clin Chem 2012;58:127–138.
Januzzi also is bullish on the role of the cardiac troponins, cTn I and T, in heart failure. "While troponins often are regarded in the context of acute MI and should always trigger suspicion for acute MI, it's very common for them to be elevated in patients with heart failure. So while that may complicate use of high-sensitivity troponin in evaluating possible acute coronary syndrome, it opens a remarkable prognostic opportunity, because patients who have elevated troponin in heart failure have a worse prognosis, clearly, than those who don't have elevated values."
The Clinical Trial Connection
As the situation with heart failure illustrates, biomarkers and CVD-related clinical trials have been inextricably linked, with information flowing in both directions. On the one hand, biomarkers have helped advance knowledge about CVD pathophysiology and disease prognosis, and on the other, clinical trials have brought new insights into both existing and novel biomarkers. This synergy will continue in the coming years, but with a few important twists, according to Sharif Halim, MD, a cardiology fellow at the Duke Clinical Research Institute in Durham, N.C. "We've gone full cycle from some biomarkers we use all the time today being developed in the early days in the context of clinical trials. Now, biomarkers are a key factor not only in defining the target population, but once again, in helping identify patients who may benefit the most from new therapies that were developed over the past few years," he said.
Halim recalled that one of the first studies that suggested cTn T might be a more sensitive indicator of myocardial cell injury than creatine kinase MB (CK-MB) was published in 1992, and that two 1994 trials, put cTn on the map in terms of its prognostic importance in acute coronary syndrome (ACS). As evidence about cTn mounted, eventually it replaced CK-MB as a tool for diagnosing and risk-stratifying patients with suspected ACS. A series of subsequent trials have used cTn as an inclusion criterion to enrich their study populations, identifying patients at high risk for events who would be most likely to benefit from treatment. cTn also has been incorporated in trials assessing or validating risk scores, and now, with high-sensitivity assays moving from research into clinical practice, another round of clinical trials are poised to find new value for cTn in non-ACS populations.
Halim sees this pattern playing out for other established and novel biomarkers, especially given new tools and lines of discovery that are coming to the fore. "We have the opportunity to focus on improving our ability to detect people at risk for disease by incorporating all the new laboratory techniques such as the ‘omics'. This will help improve not only our identification of people at risk for future events but also how best to guide therapy. We may be looking at multiple panels of biomarkers to help identify new mechanisms of disease that have yet to be established. This will lead to novel therapies," he predicted.
A Tougher Proving Ground
Even as the role of biomarkers in clinical trials evolves, Halim cautioned that clinical trials in the future will be an even tougher proving ground for biomarkers than they have been in the past. With genome-wide association studies (GWAS) and other techniques making it easier to identify potential phenotype-disease associations, these sometimes tenuous links will have to pass higher hurdles. "The major thing that will have to be demonstrated is not only do you have to better identify people at risk but also show that all the testing and money incorporated into that leads to better outcomes." Several research teams have proposed criteria for assessing the utility of CVD-related biomarkers (See Box, below).
The Ultimate Test for Cardiac Biomarkers
Novel biomarkers have to clear a high hurdle to be adopted in clinical practice. Numerous experts have proposed criteria or features for evaluating the utility of cardiac biomarkers.
- Does the biomarker measure a specific pathology with known reference limits and high myocardial specificity?
- Can the marker be measured with high accuracy, precision, and reproducibility?
- Does the biomarker have high sensitivity and specificity?
- Are results available with high throughput and short turnaround time?
- Can the biomarker be measured at reasonable cost?
- Have pre-analytical issues been evaluated and are measurable—is the marker stable?
- Has a linear relationship between a change in the marker and a change in pathology been demonstrated in multiple studies?
- Is the marker superior to existing tests in predicting risk, diagnosing disease earlier, or correlating with disease?
- Have the decision limits been validated in more than one study?
- Has the marker been evaluated in diverse populations?
- Does a change in the marker alter patient management?
- Is there evidence that biomarker-guided triage or monitoring improves care?
- Does the marker aide in selecting therapy, stratifying risk, monitoring response to therapy or disease progression, detecting sub-clinical disease?
- Will knowledge of the test result bring better patient understanding of their disease or risk?
- Will knowledge of the test result prompt healthier patient behaviors?
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Other experts echoed Halim's sentiments. "It's important that we rest what we're doing on important scientific principles so that we move the field forward," said Robert Gerszten, MD. "These studies are only as good as their weakest link. You can have great technology but bad or poorly annotated samples, or phenomenal samples but a mediocre technology that doesn't give you enough breadth or depth of coverage and you're not going to find anything that has lasting importance." Gerszten is associate professor of medicine at Harvard Medical School and director of the clinical and translational research program at the Massachusetts General Hospital Heart Center in Boston.
What's the Outcome?
Halim also cautioned researchers not to be beguiled by using biomarkers as surrogate outcomes measures in clinical trials. "We've been burned several times in relying on biomarkers and not necessarily coordinating them with large clinical endpoints that are more meaningful," he said. He cited the unexpected finding from the Women's Health Study that post-menopausal women on hormone replacement therapy did not realize a protective benefit despite having lower low-density lipoprotein cholesterol and higher high-density lipoprotein cholesterol.
The Achilles Heel?
Halim is not alone in his view that biobanks and informatics will be essential to identifying and validating novel biomarkers. "From all these clinical, population-based cohort data, there's increasingly broad and complex information that first of all needs to be made available for researchers. We need access to clinical biosample repositories and online databases for the epigenomics of disease associations," said Renate Schnable, MD. "Then we need the opportunity to integrate these data and assess interactions between the genetic and environmental factors. It's an emerging field, and we can't assess what we'll know in the near future because there's so much information." She is an assistant professor of medicine at Hamburg University in Hamburg, Germany.
Schnabel believes biobanked samples and the field of computational biology will be particularly important in enabling researchers to understand the considerable portion of CVD attributable to genetics that has yet to be described, despite considerable effort. "We recognize that CVD is a heritable condition with heritability estimates of up to 60 percent for coronary heart disease. With completion of the Human Genome Project and widespread availability of GWAS data, the thinking evolved that with common genetic variants we might be able to find disease associations very quickly and easily, but that has not been achieved," according to Schnabel. "While we've now identified more than 25 different regions of the genome related to CVD, we've only explained about 10 percent of variation."
Next Generation Sequencing and Beyond
If GWAS has not unlocked the mystery of CVD heritability as much as many thought it would, the latest genomic technologies are well-positioned to do so, according to Schnabel. "Next generation sequencing has become much easier, it's quicker than conventional techniques, and the output is much larger each year, so we can get a much more detailed map of the genome. However, at present it's still too costly. That means it will be reduced to a few individuals or that we need to put the emphasis on exome sequencing because exomes are the genetic parts that are going to be transcribed and potentially translated into proteins of interest."
Schnabel also sees promise in nascent analyses of structural variance—particularly copy number variants—and in epigenetics and transcriptomics. Early reports have investigated insertions and deletions involved in hypertension and coronary heart disease, while an insertion/deletion in the angiotensin I converting enzyme has been identified and repeatedly associated with CVD and its risk factors. Similarly, investigations involving DNA methylation, histone modifications, and nucleosome positioning are elements of epigenetic modifications that look promising, but which for now remain in the basic science stage. In the realm of transcriptomics—also still in its infancy—Schnabel thinks microRNA expression profiling could be particularly informative. "Eighteen of the 940 known mature human microRNAs have been shown to dominate cardiac microRNA expression, and they can be detected during physiological cardiac development and in pathological states of cardiovascular disease," she said. "Intriguingly, microRNA can be easily and effectively antagonized."
While she remains quite enthusiastic, Schnabel cautions that new, validated genetic biomarkers for CVD are a ways down the road. "We've gained tremendous insights in a short time, including identifying genetic loci across the genome in regions we would never have related to cardiac disease from candidate genes based on a pathophysiological standpoint," she said. "We've really gained deep insights into biology but the clinical impact at this point is limited."
The Next Frontier
The field of metabolomics also is far from everyday clinical practice, but has game-changing potential, according to Gerszten. "Metabolites are downstream of genetic variation, transcriptional, and proteomic changes, so we would argue that they're the most proximal reporters of disease pathology," he said. "Small molecule profiling really integrates genetics and the environment in a way that some other molecules can't."
Advancements in magnetic resonance spectroscopy and mass spectrometry have opened the investigation of metabolites, and Gerszten's lab is on the vanguard of these efforts. Last year, after analyzing stored samples from Framingham Heart Study participants, he reported that 12 years before they developed diabetes, individuals had a pattern of elevated amino acids in comparison to controls. Those in whom phenylaline, valine, tyrosine, leucine, tryptophan, and isoleucine were at the highest levels were 400% more likely to develop diabetes (Nat Med 2011;17:448–53).
Gerszten's team also is looking at immediate metabolic changes from exercise and in planned heart attacks. The latter involves taking blood samples before, during, and after an alcohol septal ablation procedure to correct hypertrophic obstructive cardiomyopathy. Alcohol injected into thickened heart muscle destroys the excess tissue in a way that mimics the myocardial damage during MI. "We're doing this study because we're trying to beat existing markers of myocardial injury, which don't change until about four hours after someone's had a heart attack," Gerszten explained. "We wanted to find markers that demonstrate injury within minutes of the onset of disease, and this was a nice opportunity to do that."
Although these early experiments with patients serving as their own controls are far from being bedside-ready, Gerszten thinks metabolomics tests could be coming to clinical labs in the not-too-distant future. "I would posit that within the next decade there will be a couple of new metabolic tests for complex diseases," he said. "Remember, we already do metabolic profiling in newborns for certain types of inborn errors of metabolism. We're trying to apply the same types of signals to complex metabolic diseases."
Given the interest in narrowing diagnostic windows and finding better ways to guide therapy and assess prognosis in CVD, the hunt for better, more sensitive biomarkers will go on with full force. Though much is subject to change, laboratorians will do well to follow developments closely, according to Apple. "We still have a lot to learn, but laboratorians should be thinking about how they're going to be bringing these assays into their practice. There's going to be a great evidence-based platform for convincing administrators, for example, about which tests are worth the investment."