Direct Comparison of Cardiac Myosin-Binding Protein C With Cardiac Troponins for the Early Diagnosis of Acute Myocardial Infarction

Supplemental Digital Content is available in the text.

O f the 130 million attendances to emergency departments (EDs) in the United States each year, ≈7 million (6%) are a result of acute chest pain. 1 The assessment and triage of such patients has become increasingly complex because now only a small proportion of those with acute myocardial infarction (AMI) have the diagnostic ECG change of ST-segment elevation. 2 Consequently, the identification of patients with AMI has become almost totally dependent on the measurement in the systemic circulation of cardiac troponin (cTn) I or cTnT. These biomarkers are released slowly. 3 To overcome this hurdle, the analytic performance of the cTn assays has been enhanced markedly to measure the lower concentrations achieved before the late peak. 4 Hence, the best assays can reliably measure cTn concentrations below the 99th percentile of the healthy population. These high-sensitivity (hs) assays are increasingly available and are the subject of national and international guidelines describing their use to achieve more rapid triage. 5,6 In particular, the European guidelines recommend the use of assays for hs-cTnI and hs-cTnT to rapidly rule in and rule out AMI. Algorithms using widely spaced decision limits based on concen-trations well below the population-defined 99th percentile (for rule-out) and above the 99th percentile (for rule-in) markedly improve the sensitivity of rule-out and specificity of rule-in. However, many patients presenting with chest pain have cTn concentrations that place them between these decision limits, in an indeterminate observation zone. These patients require repeat testing and subsequent second or third rounds of triage based on rates of change of cTn concentration over time. [6][7][8] European guidelines also do not support the use of rapid rule-out/rule-in pathways using hs-cTn in patients presenting too early after chest pain onsetonly after 3 hours is the rule-out threshold at the limit of detection guideline-compliant. 6 This introduces systemic delays in allocation of evidence-based treatments and prolongs stay in the pressured and precious environment of the ED.
Originally discovered by Offer et al 9 in 1973, the myosin-binding protein C family consists of 3 isoforms specific for slow skeletal, fast skeletal, and cardiac muscle, the latter being exclusively expressed in the heart from neonatal throughout human development. 10,11 Among others, [12][13][14][15] we have identified cardiac myosinbinding protein C (cMyC; Figure 1) as a new candidate biomarker of cardiac injury. 16 In common with cTnT and cTnI, cMyC expression is restricted to the heart but is more abundant. 17 Moreover, cMyC rises more rapidly in the systemic circulation than hs-cTnT after timed, iatrogenic AMI, 16 perhaps as a result of its higher myocardial concentration. 18 Using a recently developed hs assay for cMyC, 19 a pilot study in 26 patients presenting early with AMI suggested that cMyC may rise more rapidly than hs-cTnI. 20 The purpose of the current study is to compare the novel biomarker cMyC (measured on a research platform) against the most accurate currently available biochemical signals, hs-cTnI and hs-cTnT, for the early detection of AMI.

Study Design and Population
The APACE study (Advantageous Predictors of Acute Coronary Syndrome Evaluation) is an ongoing international multicenter diagnostic study (9 study centers in Switzerland, Spain, Poland, the Czech Republic, and Italy) designed to advance the early diagnosis of AMI. 4,21-23 All patients >18 years of age presenting to the ED with acute chest discomfort possibly indicating AMI were eligible for recruitment if the onset of or peak chest pain symptoms were within the preceding 12 hours. Enrollment was independent of renal function, whereas patients with terminal kidney failure on chronic dialysis were excluded. For this analysis, the following patients were excluded (Figure I in the online-only Data Supplement): patients presenting with ST-segment elevation myocardial infarction, patients with missing levels of cMyC at presentation, and patients in whom the final diagnosis

Clinical Perspective
What Is New?
• Cardiac myosin-binding protein C is a recently described novel biomarker of cardiac injury, and in small proof-of-concept studies its serum concentration rises and falls more rapidly than that of troponin T and I. • This is the first study to assess the diagnostic and prognostic value of cardiac myosin-binding protein C in patients presenting with possible acute myocardial infarction. • A rule-in/rule-out pathway using the novel biomarker was designed to compare discriminative power in a clinical setting.
What Are the Clinical Implications?
• Diagnostic accuracy of cardiac myosin-binding protein C for acute myocardial infarction was similar to that of high-sensitivity cardiac troponin T and highsensitivity cardiac troponin I in the entire cohort but superior for those with chest pain of <3 hours (early presenters) when compared with high-sensitivity cardiac troponin T. • Cardiac myosin-binding protein C has correctly triaged more patients to rule-out or rule-in groups than either high-sensitivity cardiac troponin I or high-sensitivity cardiac troponin T, leaving a much smaller proportion in the observation groups. This advantage may facilitate early discharge of low-risk patients.
remained unclear after adjudication and at least 1 hs-cTnT level was elevated. This group is comprised of patients triaged and discharged after a negative gold-standard test at the time of enrollment (on a conventional cTn assay), who were later found to have an elevated hs-cTn result (  Table II in the online-only Data Supplement. The protocol for routine clinical assessment is also described in the online-only Data Supplement.
To obtain follow-up data, patients were contacted 3, 12, 24, and 36 months after discharge via telephone, email, or letter. Additionally, information regarding death during follow-up was obtained from the patient's hospital notes, the family physician's records, and the national registry on mortality.
The study was carried out according to the principles of the Declaration of Helsinki and approved by the local ethics committees. Written informed consent was obtained from all patients. T.K., R.T., and C.M. had full access to all the data in the study and take responsibility for its integrity and the data analysis. The authors designed the study, gathered and analyzed the data according to the STARD guidelines (Standards for Reporting Diagnostic accuracy studies) for studies of diagnostic accuracy ( Table III in the online-only Data Supplement), vouch for the data and analysis, wrote the paper, and decided to publish.

Adjudicated Final Diagnosis
Adjudication of the final diagnosis was performed centrally according to the universal definition of MI, incorporating Structure of cardiac myosin-binding protein C and cardiac troponins in (A) healthy cardiomyocytes and (B) ischemia-induced cardiomyocyte damage. The highlighted N-terminal domain C0C1 is the binding site for the previously developed monoclonal antibodies used for detection of the cardiac-specific isoform of cMyC. 16 cMyC indicates cardiac myosin-binding protein C; cTnI, cardiac troponin I; and cTnT, cardiac troponin T. levels of hs-cTnT as the adjudicating biomarker. 24 It was based on extensive patient documentation derived from 2 sets of data. First, clinical data were derived from routine clinical investigations, including all available medical records (eg, patient history, physical examination, results of laboratory testing including serial local hs-cTn, radiological testing, ECG, echocardiography, cardiac exercise stress test, lesion severity and morphology at coronary angiography) pertaining to the patient from the time of ED presentation to 90-day follow-up. Second, study-specific assessment was collected, including 34 chest pain characteristics and serial hs-cTnT measurements to take advantage of the higher sensitivity and higher overall diagnostic accuracy offered by the more sensitive assays, as previously published. 4,21 In situations of disagreement about the diagnosis, cases were reviewed and adjudicated in conjunction with a third cardiologist. In brief, AMI was diagnosed when evidence indicated myocardial necrosis in association with a clinical setting consistent with myocardial ischemia. Myocardial necrosis was diagnosed by ≥1 (h)s-cTn value above the 99th percentile together with a significant rise or fall. [25][26][27] All other patients were classified into the categories of unstable angina, cardiac but noncoronary disease (eg, tachyarrhythmias, perimyocarditis), noncardiac chest pain, and symptoms of unknown origin.

Measurement of cMyC, hs-cTnI, hs-cTnT, and Standard-Sensitivity cTnI
Blood samples for the determination of cMyC, hs-cTnI, hs-cTnT, and standard-sensitivity (s) cTnI were collected into heparin plasma and serum tubes at presentation to the ED and serially thereafter (at time points 1 h, 2 h, 3 h, and 6 h). Serial sampling was discontinued when a diagnosis of AMI was certain and treatment required patient transfer to the coronary care unit or catheter laboratory. After centrifugation, samples were frozen at -80ºC until they were assayed in a blinded fashion in a dedicated core laboratory. cMyC was measured using the previously established hs assay on the Erenna platform performed by Millipore Sigma. 19 The assay has a limit of detection of 0.4 ng/L and a lower limit of quantification of 1.2 ng/L. The 99th percentile cutoff point determined previously (in patients without obstructive coronary artery disease on invasive angiography) is 87 ng/L. 19 Details of the assays used for hs-cTnI, hs-cTnT, and s-cTnI are described in the online-only Data Supplement.

Early Guideline-Based Triage and Net Reclassification Improvement
The European Society of Cardiology (ESC) has published a rapid rule-in/rule-out pathway in the 2015 non-ST-segment elevation MI guidelines using hs-cTn at 0 hours and 1 hour to risk-stratify patients into rule-out, observe, and rule-in categories. 6 Such categorization did not drive clinical decisions in this cohort, but it was used to compare the potential clinical utilities of cMyC and hs-cTn as triage tools. For this purpose, we have compared the categorical discrimination of hs-cTnT, hs-cTnI, and cMyC at presentation only (without subsequent delta measurements). In brief, the ESC pathway classifies patients, based on the presentation sample at 0 hours, into rule-out with an hs-cTnT level <5 ng/L and hs-cTnI <2 ng/L and into rule-in (for both assays) at ≥52 ng/L. 6 The ESC advocates the use of the pathway only in patients with ≥3 hours since chest pain onset; for completeness, we have presented results for all patients, <3 and ≥3 hours since chest pain onset alone.
For cMyC we separated the cohort into derivation and validation cohorts (a randomized 3:7 split; for comparison see Table IV in the online-only Data Supplement). The rule-out threshold was derived from a predefined sensitivity of ≥99.5% and rule-in from a predefined specificity >95% for the goldstandard diagnosis of AMI. This resulted in a rule-out threshold of ≤10 ng/L and a rule-in threshold of >120 ng/L for cMyC ( Figure II in the online-only Data Supplement). These thresholds were then used in the validation cohorts to compare cMyC against both hs-cTnT and hs-cTnI. Net Reclassification Improvement (NRI) operates as follows. Each patient is first assigned a classification (rule-out, observe, or rule-in) based on cutoff values of hs-cTnI/hs-cTnT in the presentation blood sample (the initial model). The same cohort is then reclassified to the same 3 groups based on the cMyC cutoff values (the new model). This reclassification may correctly or incorrectly reallocate a patient (eg, a patient who went on to be diagnosed with an AMI may be correctly reclassified from observe to rule-in or incorrectly reclassified from observe to rule-out). The NRI analysis defines separate categorical NRI values for those patients who were ultimately diagnosed with AMI (NRI AMI ) and those who were not (NRI noAMI ; (range, -1 to +1). Dimensionless NRI reflects the unweighted net movement of all patients regardless of final diagnosis (range, -2 to +2). NRI AMI is positive if there is a net movement of patients with adjudicated AMI into higher risk classifications using cMyC (the new model). NRI noAMI is positive when a net movement of patients occurs without an adjudicated diagnosis of AMI into lower risk classifications using cMyC (the new model). 28 NRI calculations were performed for the validation cohort, early presenters (<3 hours since onset of chest pain; ESC guideline not applicable), and late presenters (≥3 hours since onset; ESC guideline applicable). Tables are presented in full where appropriate.

Statistical Analysis
All data are expressed as medians (first quartile, third quartile) or means (SD) for continuous variables (compared with the Mann-Whitney U test or Student t test) and for categorical variables as numbers and percentages (compared with Pearson χ 2 ). Hypothesis testing was 2-tailed, and P values <0.05 were considered statistically significant. No adjustment for multiple comparisons was performed.
Discrimination power was quantified by the area under the receiver-operating characteristics curve (AUC) for each biomarker with all cases available, using 1000 stratified bootstrap replicates to calculate confidence intervals (CIs). Logistic regression was used to combine cMyC levels with hs-cTnT, hs-cTnI, or s-cTnI values for the assessment of an incremental value using 2 biomarkers at presentation. Subgroup analysis was performed for patients presenting early, defined as chest pain onset ≤3 hours of presentation to the ED. This is a particular limitation of the published ESC guidance on the use of hs-cTn for risk stratification because the rapid rule-out/ rule-in algorithms are only applicable to patients with chest pain onset >3 hours.
Predictive value of the biomarkers during follow-up was assessed 2-fold: We calculated (1) Harrell's C statistic for each biomarker at presentation for end points AMI, death or the composite of AMI, and all-cause mortality during follow-up (excluding the index event), and a higher C index indicates a higher probability of an event occurring during follow-up with higher biomarker values 29 ; and (2) Kaplan-Meier survival curves. Cox regression analysis was performed as follows. All available biomarker levels were divided into quintiles and groups according to rule-out, observe, and rule-in classification. Unadjusted Cox proportional hazard regression models were fitted for 30-day and 3-year follow-up for each group with the lowest quintile (or risk group, respectively) normalized to a hazard ratio of 1 and assessed using the likelihoodratio test. Cox coefficients and thus hazard ratios were not calculated if the lowest risk group did not suffer any events, which would invalidate the regression model. NRI statistics were calculated as categorical values. 28,30 The integrated discrimination improvement values quoted reflect a categoryfree (positive or negative) change in model performance. CIs for cutoff thresholds, NRI, and integrated discrimination improvement statistics were derived using 1000 bootstrap replicates. All statistical analyses were performed using R, version 3.3.0 GUI 1.68 (The R Foundation for Statistical Computing), including packages ggplot2, R Markdown, RStudio, PredictAbel, survival, Hmisc, compare, and ROCR.

Baseline Characteristics
A total of 1954 unselected patients eligible for this analysis were enrolled (Figure I in the online-only Data Supplement). Median age was 62 years, 31% were women, and 36% had a prior history of coronary artery disease (Table 1). Overall, 1469 patients (75%) had no significant electrocardiographic abnormalities at presentation to the ED. Median time since onset of chest pain was 5 hours (interquartile range [IQR], 3, 12), with a median of 3 hours (IQR 2, 7) since peak chest pain severity.
The adjudicated final diagnosis was AMI in 340 (17%) patients, unstable angina in 10%, symptoms of cardiac origin other than coronary artery disease in 14%, noncardiac symptoms in 54%, and symptoms of unknown origin in 5%.

Distribution of Biomarker Concentrations
As shown in Figure  Overall, blood concentrations of cMyC in relation to the limit of detection were higher than those of hs-cTn in all diagnostic categories (Table V in the online-only Data Supplement). Noncardiac sources of cMyC variation were previously investigated in an ambulatory cohort. 19 Results of comparison within the groups with AMI and noncardiac symptoms have been displayed in Tables VII and VIII in the online-only Data Supplement.
All data for the groups of early (<3 hours of chest pain) and late presenters (≥3 hours of chest pain) are presented in Tables XI and XII in the online-only Data Supplement. In short, in early presenters, cMyC demonstrates higher sensitivity than hs-cTnT (100% versus 98.8%) and greater specificity (46.4% versus 33.3%) at

Risk Group Reclassification
The distribution of patients in risk groups rule-out, observe, and rule-in based on the initial blood test (either hs-cTnT, hs-cTnI, or cMyC) is displayed in Figure 4 (validation cohort, n=1368, AMI in 17%

Prognostic Performance of cMyC
As quantified by Harrell's C statistic calculated from the presentation sample (Table XIV in   cMyC was significantly better at predicting AMI, death, or the composite end point when compared with cTnI. For the calculation of cumulative hazard ratios for all-cause mortality using a Cox regression model, each biomarker was separated into quintiles. The hazard ratios for hs-cTnT at 3-year follow-up were 2.3 (95% CI, 0.6-9.0) in the second quintile, 7.7 (95% CI, 2. 3-25.8) in the third quintile, 17.7 (95% CI, 5.5-57.1) in the AMI indicates acute myocardial infarction; AUC, area under the curve; cMyC, cardiac myosin-binding protein C; hs-cTnI, highsensitivity cardiac troponin I; hs-cTnT, high-sensitivity cardiac troponin T; and s-cTnI, standard-sensitivity cardiac troponin I. *P value for direct comparison between biomarkers. †P value for direct comparison between AUC for combination (cMyC with cTn) and respective cTn on its own.

DISCUSSION
To our knowledge, cMyC is the first cardiac-restricted protein to be analyzed as a diagnostic test for AMI since cTn. In this diagnostic multicenter study, we compared its diagnostic performance to cTnI and cTnT, measured using the leading hs assays recommended in current practice guidelines, 6 in a well-characterized and large cohort of patients presenting with symptoms suggestive of AMI. Discrimination for MI with cMyC was similar to that of hs-cTnT and hs-cTnI and superior to s-cTnI.
In patients presenting <3 hours of chest pain onset,  AMI indicates acute myocardial infarction, based on the adjudicated gold-standard diagnosis; CI, confidence interval; IDI, integrated discrimination improvement; NPV, negative predictive value; NRI, net reclassification improvement; and PPV, positive predictive value.
cMyC was superior to hs-cTnT, despite the latter's use as the adjudicating biomarker. Using cutoffs for cMyC calibrated against those recommended in the guidelines, 6 cMyC correctly and safely rules out and rules in AMI in a greater proportion of patients than either hs-cTnT or hs-cTnI. These findings indicate that cMyC may be better able to triage patients presenting to the ED with suspected AMI.
cTnT and cTnI have transformed the management of patients with suspected AMI, and their importance is enshrined in the Universal Definition of Myocardial Infarction. 31 Consequently, AMI events are identified/ adjudicated based on a significant rise or fall in cTnT/I blood concentration. This definition has harmonized clinical care and clinical research but also has introduced an inherent bias in favor of cTnT/cTnI versus novel diagnostic biomarkers in studies such as ours. cMyC is not part of the troponin complex and has a distinct location within the cardiac sarcomere ( Figure 1). For these reasons, our findings regarding the performance of cMyC Data are based on European Society of Cardiology guideline 2015 6 for hs-cTnT and hs-cTnI and newly developed cutoff thresholds for cMyC at ≤10 ng/L for rule-out and >120 ng/L for rule-in of myocardial infarction. AMI indicates acute myocardial infarction; cMyC, cardiac myosin-binding protein C; hs-cTnI, high-sensitivity cardiac troponin I; and hs-cTnT, high-sensitivity cardiac troponin T. against the hs-cTnT and hs-cTnI gold standard are notable. Because cMyC was not measured through the patients' journey, it is a matter of speculation whether the outcome would have been different with cMyC as the adjudicating biomarker.
After iatrogenic or spontaneous AMI, cMyC appears more rapidly in the systemic circulation than either hs-cTnT or hs-cTnI. 16,20 This finding is probably a result of a combination of cMyC's greater myocardial abundance, distinct sarcomeric location, and loose association with myosin and actin. 16 This biological distinctiveness of cMyC likely underpins the diagnostic advantage we observed over hs-cTnT/hs-cTnI in patients presenting <3 hours of symptom onset. Moreover, the more rapid appearance of cMyC in the systemic circulation after cardiac injury is also likely to explain the net reclassification improvement over both hs-cTnT and hs-cTnI.
No large prospective clinical trials compare the effect of different biomarkers of cardiac necrosis on clinical outcome. Nonetheless, it is interesting to speculate what effect the improved classification of events by cMyC could have in clinical practice. The current guidelines identify 3 risk groups, where only hs-cTn concentrations at the limit of detection or significantly above the 99th percentile clearly triage patients toward ruleout or rule-in of AMI, respectively. 6 This outcome leaves a significant proportion of patients within the observe zone of clinical uncertainty requiring repeat cTn measurement and further investigation. 32 In the current study, of the patients who ultimately did not have AMI, the proportion in the observe zone after the first measurement at presentation was 55.2% using hs-cTnT, 63.2% using hs-cTnI, and 46.1% using cMyC. It is expected that the greater diagnostic certainty afforded on a single-presentation blood draw by cMyC may reduce median time to discharge and costs of investigations.
As yet, near-patient, point-of-care devices have not been able to rule out AMI because they have struggled to achieve the required analytic sensitivity to measure low concentrations of cTnT or cTnI. In addition, the development of reliable large platform-based hs-cTn assays has proved more challenging than expected. Until now, only 2 manufacturers have overcome the difficulties of developing and introducing hs-cTn assays into clinical practice, 6 of which 1 had major quality issues initially. [33][34][35] These uncertainties and concerns have led to delays in the approval of these assays for clinical care in the United States. 36 The US Food and Drug Administration has only recently ratified the use of the fifth-generation hs-cTnT assay. 37 Because cMyC is more abundant and rises more rapidly, migration to a point-of-care format may be less challenging. Risk prediction appears grossly similar when comparing hs-cTn and cMyC and could therefore be performed on either. Notably, a cMyC level <10 ng/L (the threshold resembling 25 times the limit of detection) offers both high negative predictive value and 30-day mortality rates approaching 0.
Our study has a number of limitations. First, the diagnostic cutoffs for cMyC require external validation. Despite its size, a single cohort cannot entirely safeguard against calibration issues and is inherently subject to potential institutional bias. We have attempted to mitigate these risks by using both randomization and bootstrapping, but in an ideal scenario the findings require validation in an independent cohort. Second, the analyses within this manuscript are confined to the concentration of the necrosis biomarker on first blood draw. We have not analyzed the effect on the gray zone of repeat blood draws after set intervals. This area of active research has no consensus regarding resampling interval, magnitude of concentration change, use of absolute or relative change in concentration, or effect of assay vendor. 4,5,21,[38][39][40] Third, as a prospective diagnostic study, we cannot exactly quantify the clinical benefit associated with the use of cMyC as an alternative or addition to hs-cTn. Further cluster-randomized studies will be required to address this issue. Fourth, we cannot comment on the accuracy of cMyC among patients with terminal kidney failure on renal replacement therapy or ST elevation myocardial infarction because such patients were excluded from this study. Currently, biomarkers have no role in the assessment of patients with ST elevation myocardial infarction, and hence this group was not analyzed. Fifth, of 3029 patients recruited, 875 had no baseline cMyC measured. However, a comparison between the analyzed cohort and the excluded patient sample has not demonstrated substantial differences in baseline characteristics (Table III in the online-only  Data Supplement). Sixth, in patients with low levels of cMyC (eg, the noncardiac chest pain group), we observed a significant difference in biomarker values dependent on certain underlying conditions (such as prior coronary artery disease) (Tables VII and VIII in the online-only Data Supplement). However, this effect is muted in patients with AMI and indeed did not negatively influence specificity. Finally, cMyC was measured using a research platform, whereas hs-cTnI and hs-cTnT were measured using widely available clinical laboratory analyzers. The sandwich immunoassay is comparable to the setup used to test for hs-cTn, but cMyC is not yet available on a random-access laboratory analyzer for routine clinical use.
In summary, cMyC is a promising new biomarker of myocardial necrosis, with overall discriminatory power comparable with the leading troponin assays in AMI diagnosis. A potential advantage of cMyC is its ability to more effectively rule out AMI at presentation, particularly among those presenting early after symptom onset.