The ability to use genetic information to inform clinical decision making has emerged as a new tool in clinical practice, with noteworthy examples across many area of medicine. Cardiology and anticoagulation in particular have led the way in the translation of genetic findings into actionable clinical recommendations, spurred by the addition of genetically guided dosing in the drug label for warfarin by the FDA. This review covers the pharmacogenomics related to warfarin therapy.
by Dr Minoli Perera
Pharmacogenomics is primarily aimed at identifying genetic variation that influences inter-individual differences in drug response. The guiding principle is “the right drug, at the right dose for the right person”. Its application promises to enable targeted drug administration, improve therapeutic outcomes, and inform drug development. Pharmacogenomic insights have also improved our understanding of the underlying pathways and mechanisms behind adverse drug reactions. Such adverse reactions account for approximately 100 000 deaths per year in the US, and markedly increase healthcare costs. Advances made over the last 30 years in molecular biology, molecular medicine, and genomics have had a major impact on the development of pharmacogenomics.
Currently a variety of approaches are used to associate genetic variants associated with drug response. Commonly used strategies include candidate gene approach and genome-wide association studies (GWAS). Candidate gene studies investigate single nucleotide polymorphisms (SNPs) that are correlated to drug response. These studies are usually restricted to genes or SNPs that have been shown to be involved in the pathway of drug action or drug clearance. Genome-wide association studies investigate up to 5 million SNPs spaced throughout the genome which are genotyped to identify genetic variants with the drug phenotype in an unbiased fashion. Each of these methods has advantages and disadvantages. Genome-wide studies comprehensively cover the entire genome, but their power to detect moderate associations is greatly limited by the multiple testing burden, which is a requirement for correction for false-positive associations. The candidate gene approach narrows the focus to a few important SNPs and therefore has higher power, but may miss the real causative SNP, and require a priori knowledge for the selection of SNPs/genes to study.
Both these methods have yielded important clinical findings that can immediately be used to reduce the incidence of adverse effects (many of which have been added to drug labels). Notable examples such as the SLCO1B1*5 polymorphism (associated with myopathy with statin use) and CYP2C19*2 (associated with clopidogrel non-response) have shown clinically meaningful outcomes related to genetic variants. However, the translation of these findings has been slower in coming and has many clinicians wondering about the utility of this new technology.
The “test case” for pharmacogenetics was thought to be pharmacogenetically guided warfarin dosing. In this review we will cover the genetic polymorphism effecting warfarin dose requirements and the currently available diagnostic tests, as a case study for the implementation of pharmacogenetics in the clinic.
Genes that affect warfarin dose
Numerous studies, predominately conducted in Caucasian and Asian populations, demonstrate that the CYP2C9 and VKORC1 genotypes contribute significantly to warfarin dose variability [1–3]. The role of these gene products can be seen in Figure 1. The CYP2C9 enzyme metabolises the more active S-enantiomer of warfarin to inactive 7-hydroxywarfarin. Warfarin inhibits the enzyme VKOR (encoded by the gene VKORC1) to prevent conversion of Vitamin K epoxide to its reduced form necessary for activation of the clotting factors II, VII, IX and X. Thus, SNPs in the CYP2C9 gene affect warfarin pharmacokinetics, whereas variation in the VKORC1 gene impacts warfarin pharmacodynamics.
The most extensively studied CYP2C9 variants are the CYP2C9*2 and CYP2C9*3 alleles, which lead to significant reductions in CYP2C9 activity. Compared with the people without this genotype, carriers of the *2 or *3 genotypes have S-warfarin clearance reduced to between 40 to 90% or normal levels. As a result, significantly lower doses are usually needed in individuals with a CYP2C9*2 or CYP2C9*3 allele. The CYP2C9 genotype is also implicated in the risk of bleeding during warfarin therapy, especially during the warfarin initiation period [4].
The VKORC1 genotype was originally recognised for causing warfarin resistance through mutations in the gene-coding region. More recently, common VKORC1 SNPs occurring in gene-regulatory regions and underlying usual warfarin dose variability were discovered [3]. Two such SNPs, one in the promoter region (-1639G>A) and one in intron 1 (1173C>T), show the strongest association and possible functional effects [5]. Thus, the majority of warfarin pharmacogenetic studies have focused on one of these two SNPs, which are in strong linkage disequilibrium across populations (meaning they are inherited together and strongly associated with each other). This means that only one of these SNPs needs to be taken into account for pharmacogenetic dosing of warfarin. Most investigators chose the VKORC1 -1639G>A as the predictive SNP in warfarin pharmacogenetic studies. This SNP explains approximately 20–28% of the overall variability in dose requirements in Caucasians, but only 5–7% of the variability in African–Americans, mainly due to the difference in allele frequency between populations [6, 7]. Unlike CYP2C9, the VKORC1 genotype does not appear to affect the risk of bleeding with warfarin treatment[4].
These findings have been confirmed in several genome-wide association studies (GWAS) in Caucasians and Asian individuals, showing that VKORC1 -1639G>A, CYP2C9*2 and CYP2C9*3 polymorphisms are the primary genetic determinants of warfarin dose requirements in these populations. The combination of VKORC1 -1639G>A, CYP2C9 (*2 and *3) and clinical factors (e.g., age, sex, weight and amiodarone use) explains approximately 55% of the total variance in warfarin maintenance dose in Caucasians, but only about 25% among African–Americans. With the exception of the CYP4F2 genotype, found in a GWAS study of Swedish patients [8], no other genetic variant has met genome-wide significance for association with warfarin dose requirements. Both genetic and non-genetic variables have been included in dosing algorithms that can be used to predict dose, such as WarfarinDosing.org.
Warfarin genetic testing and guidelines
Insurance companies consider genetic testing for genetic variants in CYP2C19 related to clopidogrel response and the HLA-B* 1502 allele for prediction of adverse effects related to carbamazepine as “medically necessary” and may therefore cover the cost of these tests. The same does not hold for warfarin testing, which is considered investigational and will only be covered in the context of a clinical trial.
In 2007, the US Food and Drug Administration modified the package insert for warfarin to include information on the relationship of safe and effective dosage to SNPs in CYP2C9 and VKORC1, including a table of recommended doses for each genotype combination [Table 1]. These recommendations give the range of doses that should be considered when dosing a patient that is a carrier of any of the tested SNPs. However, there is increasing evidence that additional alleles outside of CYP2C9*2 and *3 and VKORC1 -1639G/A may play a role in warfarin dose response. These SNPs are not included in the FDA dose recommendations and not all tests cover all these additional variants.
Currently, four warfarin pharmacogenetic tests are available as in vitro diagnostic devices [shown in Table 2]. All of these tests genotype for three loci: CYP2C9*2, CYP2C9*3 and one VKORC1 -1639G/A or 1173C/T (both of which give equivalent information because of the afore mentioned LD in all populations), with some including other known genetic variants that are associated with warfarin dose. All of the tests can be completed in 8 hours, including DNA extraction, with the fastest ones providing genotype results in less than 2 hours.
The Clinical Pharmacogenetics Implementation Consortium (CPIC) recently published guidelines on how to interpret and apply genetic test results to adjust warfarin doses [9]. These guidelines do not address when to order a genetic test, but rather how to dose warfarin when genetic test results are available. The guidelines strongly support the use of genetic information to guide warfarin dosing when genotype is known and recommend using either the International Warfarin Pharmacogenetics Consortium (IWPC) or Gage algorithm to do so.
Although the availability of FDA-cleared devices for warfarin pharmacogenetic testing makes genotype-guided warfarin initiation possible, several barriers to clinical adoption remain. First, many medical centres do not have warfarin pharmacogenetic testing available. In a recent survey, only 20% of hospitals in North America have testing available on site, suggesting the majority of the hospitals rely on outside commercial clinical laboratories. This outsourcing may make genotype-guided warfarin initiation impractical because of 3–7 days of turnaround time. Second, no professional organisation endorses warfarin pharmacogenetic testing in its guidelines because of the lack of the clinical utility data. Inclusion of a testing recommendation in professional guidelines has been identified as a factor influencing reimbursement of new technology. As such, the Centers for Medicare and Medicaid Services (CMS) and many commercial insurance plans generally do not reimburse the cost of testing ($300–500). Because of these barriers, warfarin pharmacogenetic testing is performed mainly for research purposes and for patients willing to pay the cost.
Future perspective and conclusions
There are substantial and convincing data supporting the clinical and analytic validity of warfarin pharmacogenetics. The CYP2C9 and VKORC1 genes are the primary determinants of warfarin dose requirements. There are several FDA-cleared tests available for CYP2C9 and VKORC1 genotyping. However, genotype-guided warfarin dosing has not yet become a reality in most medical centres despite the wealth of data supporting genetic influences of warfarin dose requirements. Many clinicians and third party payers are awaiting evidence of clinical utility and cost-effectiveness before adopting genetic testing for anticoagulation management in the clinic setting. Results from ongoing clinical trials (such as the NIH-sponsored COAG trial) are expected to address these issues and will likely determine the course of genotype-guided anticoagulant therapy. Whether pharmacogenetics will have a role in the treatment with newer anticoagulant agents has yet to be determined. However, the pharmacogenetics with these anticoagulants could be of great importance given the unavailability of routine monitoring parameters with these agents.
References
1. Wadelius M, et al. Blood 2009; 113: 784–792.
2. Klein TE, et al. N Engl J Med 2009; 360: 753–764.
3. Rieder MJ, et al. N Engl J Med 2005; 352: 2285–2293.
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9. Johnson JA, et al. Clinical pharmacology and therapeutics 2011; 90: 625–629.
10. Coumadin package insert. 2007. (Accessed October, 2007, at http://www.bms.com/cgi-bin/anybin.pl?sql=PI_SEQ=91.)
The author
Minoli A Perera, PharmD., PhD
Knapp Center for Biomedical Discovery
Room 3220B, University of Chicago, 900 E.
57th Street, Chicago, IL 60637, USA
E-mail: mperera@bsd.uchicago.edu
Primary hepatic tumours are one of the most aggressive and resistant forms of cancer. Early diagnosis of liver cancer and the development of more accurate markers for biological classification are crucial to improving the clinical management and survival of patients. This article discusses the emerging use of microRNAs for the diagnosis of liver cancer.
by Dr Luc Gailhouste and Dr Takahiro Ochiya
Liver cancer and diagnosis
Primary liver cancer is mainly represented by hepatocellular carcinoma (HCC) and accounts for almost 90% of primitive hepatic malignancies. Statistically, HCC is the third most common cause of death from cancer worldwide [1] and is generally encountered in patients exhibiting an underlying chronic liver disease such as hepatitis B virus (HBV) and/or C virus (HCV) infection, alcohol abuse, or liver steatosis. Chronic hepatitis leads to fibrosis and gradually evolves into cirrhosis. Global studies estimate that approximately 80–90% of all HCCs arise from cirrhotic livers. Despite great advances in the treatment of the disease, hepatic cancer exhibits one of the lowest remission rates (less than 10% after five years), mainly due to its late diagnosis and high resistance to the conventional agents of chemotherapy. Indeed, as such a disease tends to remain asymptomatic, approximately 50% of newly diagnosed patients already exhibit late advancement.
Common HCC diagnostic methods include liver imaging techniques such as triphasic computed tomography scanning, magnetic resonance imaging (MRI), and abdominal ultrasound [2]. A panel of serological biochemical markers, including aminotransferases ALAT and ASAT, has also been used for several decades to monitor liver pathologies in a non-invasive manner.
Until recently, imaging tests were frequently combined with the non-invasive measurement of serum alpha-fetoprotein (AFP). Normally produced by the fetal liver, AFP decreases soon after birth whereas its high level in adults can be correlated with the appearance of malignant hepatic disease. However, the American Association for the Study of Liver Diseases (AASLD), in its practice guidelines, discontinued the use of the blood tumour marker AFP for surveillance and diagnosis due to the limited sensitivity and specificity of the method. When uncertainty regarding the diagnosis persists, a percutaneous biopsy followed by histological examination of the nodule is indicated [3]. This technique remains the gold standard method for determining the degree of underlying fibrosis and shows appreciable sensitivity (more than 80%) for HCC diagnosis.
An important breakthrough in the clinical management of liver cancer would come from the accurate correlation of the alterations of cancer-related genes and the tumour phenotype. Although HCC lesions can be broadly distinguished by histological or immunological assessment, their prognosis and clinical evolution vary greatly from one individual to another. The discovery of innovative and effective biomarkers ensuring an early diagnosis of the disease correlated with the etiology, the pathogenic tendency, and the malignancy of the tumour could significantly enhance the molecular assessment of HCC and its classification in order to maximize the positive response of therapeutics.
MicroRNAs: biogenesis and mechanism of action
MicroRNAs (miRNAs) constitute a group of evolutionary conserved small non-coding RNAs of approximately 22 nucleotides that accurately regulate gene expression by complementary base pairing with the 3’-untranslated regions (3’-UTRs) of messenger RNAs (mRNAs) [4]. These post-transcriptional regulators were first evidenced in C. elegans by Ambros and co-workers who discovered that lin-4, a gene known to control the timing of nematode larval development, did not code for a protein but produced small RNAs that specifically bind to lin-14 mRNA and repress its translation.
miRNA biogenesis is a multistep process that has been reviewed extensively [Figure 1]. An essential feature of miRNAs is that a single miRNA can recognize numerous mRNAs, and, conversely, one mRNA can be recognized by several miRNAs. These pleiotropic properties enable miRNAs to exert wide control over a plethora of targets, attesting to the complexity of this mechanism of gene expression regulation. Several reports have described the key role of these post-transcriptional regulators in the control of diverse biological processes such as development, differentiation, cell proliferation, and apoptosis. The alterations of miRNA expression have also been reported in a wide range of human diseases, including cancer [5].
In HCC, the atypical expression of miRNAs frequently contributes to the deregulation of critical genes known to play an essential role in tumorigenesis and cancer progression. The current consensus is that cancer-related miRNAs function as oncogenes or tumour suppressors [6]. As for other malignancies, two situations can occur in HCC: (i) tumour suppressor miRNAs can be downregulated in liver cancer and cause the upregulation of oncogenic target genes repressed in normal hepatic tissues, increasing cell growth, invasion abilities, or drug resistance; (ii) oncogenic miRNAs, also called oncomirs, can be upregulated in HCC and can downregulate their target tumour suppressor genes, potentially leading to hepatocarcinogenesis.
miRNA as a diagnostic tool
As miRNA signatures are believed to serve as accurate molecular biomarkers for the clinical classification of HCC tumours, the availability of consistent technologies that enable the detection of miRNAs has become of interest for both fundamental and clinical purposes. The most current detection methods commonly used are microarray and real-time quantitative polymerase chain reaction (RT-qPCR).
Microarray analysis presents the advantage of offering a high speed of screening by employing various miRNA probes within a single microchip. However, the technique has lower sensitivity and specificity than RT-qPCR, which is the most widely used method.
miRNA RT-qPCR is based on the use of stem–loop primers, which can specifically bind to the mature miRNA during reverse transcription, granting a high degree of accuracy to the method [7]. Analysis of miRNAs by RT-qPCR is a cost-effective technique and, due to its efficiency, a valuable way to validate miRNA signatures. Moreover, the development of RT-qPCR protocols has improved the sensitivity of miRNA detection down to a few nanograms of total RNAs. This amount can be easily and routinely obtained by extracting total RNAs from a small fragment of a hepatic percutaneous biopsy.
A plethora of studies have already reported various miRNA profiles potentially reflecting HCC initiation and progression that could be employed as specific cancer biomarkers [8]. Comparative analysis of bibliographic data provides evidence of the persistent augmentation of miR-21 in cancer, regardless of the tumour origin. In the HCC, miR-21 is also frequently overexpressed where it acts as an oncogenic miRNA. The major overexpression of miR-21 is associated with the inhibition of the tumour suppressor PTEN and the poor differentiation of the tumour. The use of an miRNA-based classification correlated with the etiology and the aggressiveness of the tumour appears very promising, as it could significantly enhance the accuracy of the molecular diagnosis of HCC and its classification, leading to the consideration of more appropriate therapeutic strategies.
In this regard, Budhu and collaborators defined a combination of 20 miRNAs as an HCC metastasis signature and showed that this 20-miRNA-based profile was capable of predicting the survival and recurrence of HCC in patients with multinodular or single tumours, including those at an early stage of the disease [9]. Remarkably, the highlighted expression profile showed a similar accuracy regarding patient prognosis when compared to the conventional clinical parameters, suggesting the relevance of this miRNA signature. Consequently, the profiling of aberrantly expressed cancer-related miRNAs might establish the basis for the development of a rational system of classification in order to refine the diagnosis and the prediction of HCC evolution.
Tumour suppressor miRNA: the case of miR-122
The case of miR-122 is of prime interest, first, because it represents by itself more than half of the total amount of miRNAs expressed in the liver [10]. Remarkably, miR-122 is a key host factor required for HCV replication. A phase 2 clinical trial was recently initiated that reported the world’s first miRNA-based therapy targeting miR-122 in HCV-infected patients using the locked nucleic acid (LNA)-modified antisense oligonucleotide miravirsen [11]. Thus, a four-week miravirsen treatment by subcutaneous injection provided long-lasting antiviral activity and was well tolerated.
However, the experimental silencing of miR-122 resulted in increased expression of hundreds of genes normally repressed in normal hepatocytes. The miR-122 knockout mouse model displays hepatosteatosis, fibrosis, and a high incidence of HCC, suggesting the tumour suppressor role of miR-122 in the liver. In primary liver carcinoma, the existence of an inverse correlation was demonstrated between the expression of miR-122 and cyclin G1, which is highly implicated in cell cycle progression.
Regarding the potential of miR-122 as a diagnostic biomarker in liver cancer, numerous studies have already reported the significant and specific downregulation of miR-122 expression in both human and rodent HCC models. Obviously, miR-122 was shown as downregulated in more than 70% of the samples obtained from HCC patients with underlying cirrhosis as well as in 100% of the HCC-derived cell lines [12].
To illustrate this statement, we analyzed the expression levels of miR-122 in 20 patients who exhibited HCC using RT-qPCR. Following RNA extraction from biopsies with the miRNeasy Mini Kit (Qiagen), 100 ng of total RNA was reverse-transcribed using the Taqman miRNA Reverse Transcription Kit (Applied Biosystems). The expression levels of mature miR-122 were determined in each sample by RT-qPCR with Taqman Universal PCR Master Mix in a 7300 Real-Time PCR System from Applied Biosystems. The expression levels of miRNAs were normalized with respect to the endogenous levels of RNU6B. RT-qPCR data were obtained easily and rapidly by a routinely conventional method used in our laboratory. As a result, miR-122 expression was reduced more than threefold in HCC biopsies relative to the normal liver group (median 0.935 and 3.495, respectively; P<0.0001, Mann–Whitney U test) [Figure 2]. These data suggest that cancer-related miRNAs, such as miR-122, which are deregulated in HCC tissues, could be relevant with regard to the development of new diagnostic tools and the clinical management of liver cancer patients.
Conclusions and emerging approaches
The expression profile of specific miRNAs has been found to reflect the biological behaviour of HCC tumours, such as aggressiveness, invasiveness, or drug resistance. As a consequence, miRNA investigations may offer opportunities to determine miRNA signatures that would provide valuable information to stratify and refine HCC diagnosis in terms of prognosis, response to treatment, and disease relapse. Recently, tumour-derived miRNAs have been efficiently detected in the serum of patients and characterized as potential non-invasive biomarkers for HCC.
The concept that miRNAs could serve as potential plasma markers for liver diseases is, thus, gaining attention. Due to its frequent deregulation in viral hepatitis, cirrhosis, and cancer as well as its specific and massive expression in the liver, the assessment of serum miR-122 could represent one reliable strategy for the non-invasive diagnosis of chronic liver pathologies. Although the process of assessing serum miRNAs remains under improvement, cancer-related circulating miRNAs represent an exciting and promising field of investigation for the development of more accurate technologies for the early diagnosis of HCC.
References
1. Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 2006; 6: 674–687.
2. Befeler AS, Di Bisceglie AM. Hepatocellular carcinoma: diagnosis and treatment. Gastroenterology 2002; 122: 1609–1619.
3. Ryder SD. Guidelines for the diagnosis and treatment of hepatocellular carcinoma (HCC) in adults. Gut 2003; 52(Suppl 3): iii1–8.
4. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.
5. Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; 6: 857–866.
6. Gailhouste L, Ochiya T. Cancer-related microRNAs and their role as tumor suppressors and oncogenes in hepatocellular carcinoma. Histol Histopathol 2012.
7. Chen C, Ridzon DA, Broomer AJ, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005; 33: e179.
8. Gailhouste L, Gomez-Santos L, Ochiya T. Potential applications of miRNAs as diagnostic and prognostic markers in liver cancer. Front Biosci 2013; 18: 199–223.
9. Budhu A, Jia HL, Forgues M, et al. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology 2008; 47: 897–907.
10. Girard M, Jacquemin E, Munnich A, et al. miR-122, a paradigm for the role of microRNAs in the liver. J Hepatol 2008; 48: 648–656.
11. Lindow M, Kauppinen S. Discovering the first microRNA-targeted drug. J Cell Biol 2012; 199: 407–412.
12. Gramantieri L, Ferracin M, Fornari F, et al. Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma. Cancer Res 2007; 67: 6092–6099.
The authors
Luc Gailhouste PhD and
Takahiro Ochiya PhD
Division of Molecular and Cellular
Medicine, National Cancer Center Research Institute, Tokyo, Japan
Plasma levels of heart-type fatty acid binding protein (H-FABP) have been shown to rise early after the onset of acute myocardial infarction (AMI). Recent evidence suggests combining H-FABP with troponin gives superior diagnostic accuracy compared to the alternative ‘early markers’ of myocardial necrosis, creatine kinase-MB (CK-MB) and myoglobin. However, using a single measurement at the time of presentation to the Emergency Department (ED), H-FABP is unlikely to have sufficient sensitivity to safely ‘rule out’ AMI, even when combined with a standard troponin assay. With the advent of high sensitivity troponin assays which have higher diagnostic sensitivity at the time of presentation, it is possible that H-FABP could be combined with levels of high sensitivity troponin and potentially with other clinical information to enable safe ‘rule out’ of AMI using a single blood test at the time of presentation. Further work in this area is needed.
by Dr Richard Body
Background
Suspected cardiac chest pain accounts for approximately one quarter of acute medical admissions, although only a minority of the patients admitted will ultimately be diagnosed with an acute coronary syndrome [1]. Meanwhile, up to 2% of patients with acute myocardial infarction (AMI) have that diagnosis missed and are inadvertently discharged, leading to a worse prognosis [2]. There is therefore tremendous potential to reduce unnecessary hospital admissions in this patient group, although advances in diagnostic technology are clearly necessary in order to do so.
High sensitivity troponin
Cardiac troponins are regulatory proteins contained within the myofibrillar apparatus of cardiac myocytes. They are released into the bloodstream following myocardial necrosis and their detection allows highly sensitive and specific diagnosis of AMI. Indeed, the detection of a rise and/or fall of cardiac troponin in serum or plasma is integral to the diagnosis of AMI. With the advent of high sensitivity troponin (hs-cTn) assays, which have greater analytical and diagnostic sensitivity than standard assays, it is tempting to believe that the hunt for an ‘early rule out’ strategy for acute coronary syndromes is over. Standard troponin assays lack the diagnostic sensitivity to enable safe exclusion of acute myocardial infarction (AMI) when measured at the time of presentation. This creates a period of ‘troponin blindness’, when patients with AMI still have low circulating troponin levels prior to the development of a late troponin rise. Hs-cTn assays have been shown to improve diagnostic sensitivity at the time of initial presentation to the Emergency Department (ED). While this reduces the magnitude of our problem with ‘troponin blindness’, it does not overcome the problem completely. Even hs-cTn assays fail to identify approximately 10% of patients with AMI at the time of presentation [3, 4]. With hs-cTn assays it may be possible to reduce the time taken to confidently ‘rule out’ AMI with serial sampling from 6 to 9 hours after arrival (or 10–12 hours from symptom onset) to as little as 3 hours after arrival [4, 5]. This approach still needs to be validated against a hs-cTn reference standard, however, and there are a few other reasons to be cautious. The sensitivity of the Siemens troponin I Ultra assay (a sensitive assay but not high sensitivity), which had a diagnostic sensitivity of 100% at 3 hours after presentation in Keller et al.’s original study (evaluated against the reference standard of testing 6 hours after arrival), was actually only 94.5% at 6 to 12 hours from symptom onset [4]. Further, high sensitivity troponin T (hs-cTnT) has been shown to have a sensitivity of only 92.2% when measured 2 hours after presentation, which is still some way from a satisfactory rule out strategy [6]. Using the new Abbott Architect high sensitivity troponin I assay, sensitivity for AMI is 98.2% (with 95% confidence intervals extending down to 96.9%), again using a standard troponin assay as the reference standard [5]. Even if we accept that no rule out strategy will be 100% sensitive and consider this 3-hour troponin to be a satisfactory rule out strategy, that still means an anxious wait for patients and would still, in health systems like the United Kingdom, necessitate admission to an inpatient ward for investigation.
Interest in ‘early markers’ of myocardial necrosis
There has been interest in the role of ‘early markers’ of myocardial necrosis for many years. As troponin is predominantly an intracellular constituent and levels do not peak for 12 to 24 hours after the onset of infarction [7], many have investigated the value of biomarkers with release kinetics suggesting that they may enable earlier identification of AMI. Thus, the measurement of creatine kinase-MB (CK-MB) and myoglobin levels in combination with troponin were shown to improve early diagnosis of AMI as early as 2001 [8]. More recently, the ASPECT study from 14 countries in the Asia-Pacific region examined the value of CK-MB, myoglobin and troponin I (using assays from Alere, San Diego, CA, USA) measured at presentation and 120 minutes later in patients with a Thrombolysis In Myocardial Infarction (TIMI) score of 0/7. The authors found that 9.8% of patients could be discharged using this strategy with a 0.9% incidence of adverse cardiac events within 30 days [9]. Around the same time, the Randomised Assessment Using Panel Assay of Cardiac Biomarkers (RATPAC) study demonstrated that serial evaluation of CK-MB, myoglobin and troponin I over 90 minutes led to an increase in the proportion of patients successfully discharged from the ED, although this came at a cost of rebound-overuse of Coronary Care resources, perhaps as a function of the lack of specificity of myoglobin and CK-MB. The strategy was found to be not cost effective [10].
Heart-type fatty acid binding protein
Heart-type fatty acid-binding protein (H-FABP) is a cytosolic protein that is abundantly expressed in human myocardial cells, where it facilitates intracellular fatty acid transport within cardiac myocytes [11]. Plasma H-FABP levels rise early after the onset of AMI. McCann et al. evaluated H-FABP (Hycult Biotechnology ELISA) and troponin T (cTnT; Roche Elecsys, 4th generation) in 415 patients who were admitted to an acute cardiology unit on suspicion of an acute coronary syndrome. They demonstrated that H-FABP had superior sensitivity to troponin in patients who presented early (<4h) after symptom onset [Figure 1] [12]. A meta-analysis of 16 studies including 3,709 patients with suspected AMI demonstrated a pooled sensitivity of 84% [95% confidence intervals (CI) 76–90%] and a pooled specificity of 84% (95% CI 76–89%), although there was significant heterogeneity between studies [13]. It is clear that measurement of H-FABP alone cannot enable safe ‘rule out’ of AMI.
Combining H-FABP with troponin will, however, yield a higher diagnostic sensitivity. Body et al. [14] demonstrated that the combination of H-FABP and troponin I offers both superior sensitivity and superior specificity to the combination of CK-MB, myoglobin and troponin I [Figure 2].
A systematic review by Carroll et al. demonstrated that, in 4 studies, the combination of H-FABP and troponin had an overall sensitivity of between 76 and 97% [15]. Two of these studies did, however, use insensitive troponin assays with diagnostic sensitivities of 42% and 55% respectively. The use of more sensitive troponin assays may be expected to yield higher diagnostic performance. Indeed, in the study by Body et al., the sensitivity of the combination of H-FABP and troponin increased from 82% to 87% when a more sensitive troponin assay was used [14, 16]. If only low risk patients (using the modified Goldman risk stratification tool) who had normal H-FABP and normal cTnT were considered for early discharge, a sensitivity and negative predictive value of 99% could be achieved, although this strategy may have a specificity as low as 19%, meaning that only a minority of patients would be eligible for early discharge while 1% of AMIs would still be missed [16].
H-FABP and high sensitivity troponin
It is clear that neither H-FABP nor troponin (even using a high sensitivity assay) can be used to safely exclude a diagnosis of AMI when measured at the time of presentation to the ED. The combination of H-FABP and standard troponin assays improves overall diagnostic sensitivity but is still unable to ‘rule out’ this important diagnosis. By combining H-FABP with high sensitivity troponin assays, it may be possible to further increase sensitivity and thus achieve an effective early rule out strategy. Evidence in this area is still limited. However, Aldous et al. did evaluate the combination of H-FABP (Hycult Biotech) and hs-cTnT in a cohort of 384 patients presenting to the ED with suspected acute coronary syndromes. This combination had a sensitivity of 90.0% for AMI and a specificity of 73.5%. Notably, the sensitivity of the H-FABP assay alone was particularly low in this study (50.0%), which may be a function of the high diagnostic cut-off employed (60ng/ml) when compared to the cut-off employed by McCann et al. using the same assay (5ng/ml) [12, 17]. Using this high diagnostic cut-off, however, the combination of H-FABP and hs-cTnT measured at the time of presentation may help to ‘rule in’ the diagnosis of AMI, with a specificity of 99.4% (95% CI 97.9–99.9%) [17].
Inoue et al. also evaluated both hs-cTnT and H-FABP (DS Pharma Biomedical, Osaka) in 432 ED patients with suspected acute coronary syndromes. In this study, H-FABP had a similar area under the receiver operating characteristic (ROC) curve (AUC) to hs-cTnT (0.83 versus 0.82), although hs-cTnT had a higher sensitivity at the diagnostic cut-off (87.9% vs. 78.5%) [18]. The authors do not report the diagnostic value of the combination of both biomarkers. Meanwhile, in 1,818 patients with suspected acute coronary syndromes, Keller et al. reported that H-FABP had an AUC of 0.89, which rose to 0.97 when combined with high sensitivity troponin I (Abbott Architect STAT high sensitive troponin) [5]. This implies that the combination has high diagnostic accuracy, although the sensitivity and negative predictive value of the strategy were not reported.
H-FABP and prognosis
H-FABP levels may also have prognostic value in patients with suspected acute coronary syndromes. Viswanathan et al. studied 1,080 consecutive patients presenting with suspected acute coronary syndromes [19]. They measured both H-FABP (Randox Evidence Biochip) and troponin I using a sensitive assay (Siemens Advia troponin I Ultra) and followed patients for a median of 18 months. H-FABP predicted death or AMI occurring during follow up, even in troponin negative patients and after adjustment for age and serum creatinine. For predicting death or AMI, H-FABP had an AUC of 0.79 (95% CI 0.74–0.84)
compared to 0.77 (95% CI 0.72–0.82) for troponin I.
Future work
Further work is still needed to determine whether the combination of H-FABP and high sensitivity troponin will enable safe rule out of acute coronary syndromes in the ED. Combination with other clinical information available from risk stratification tools (such as the modified Goldman or TIMI scores) or the ECG may further increase sensitivity, enabling AMI to be safely excluded in a proportion of patients presenting to the ED. Further, with the increase in false positive results given by high sensitivity troponin assays, H-FABP may help to ‘rule in’ the diagnosis of AMI in patients with troponin elevations at the time of presentation, before the results of serial testing are available. This will facilitate early treatment and triage to an appropriate level of care in the hospital, while avoiding the risks of unnecessary treatment for those patients with false positive elevations.
Conclusions
H-FABP is a promising biomarker for use in patients with suspected acute coronary syndromes. Used alone or in combination with a standard troponin assay, sensitivity will be insufficient to safely ‘rule out’ AMI. Further work is needed to determine whether combination with a high sensitivity assay can enable safe ‘rule out’ for a proportion of patients, and to evaluate whether H-FABP may have a role in the differentiation between ‘true positive’ and ‘false positive’ troponin elevations at the time of initial presentation.
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The author
Richard Body, MB ChB MRCSEd(A&E) FCEM PhD
Emergency Department,
Manchester Royal Infirmary,
Oxford Road, Manchester, M13 9WL, UK
e-mail: richard.body@manchester.ac.uk
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