Prognosis prediction method

Abstract

To provide a method for treatment of ACS in a subject, and a device, a kit, and a cardiac biomarker related to a method for prognosis prediction of ACS.SOLUTION: The present invention is a method for prognosis prediction of ACS in a subject includes: measuring the concentration of blood plasma MIF and Nt-proBNP (or BNP) in a sample from the subject; diagnosing ACS when the concentration of blood plasma of the subject is higher than reference MIF and Nt-proBNP (or BNP); and predicting the degree of ACS from the concentration of blood plasma MIF and Nt-proBNP (or BNP) of the subject.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from PCT / CN2017 / 104752, the entire contents of which are incorporated herein by reference. FIELD OF THE INVENTION The present invention relates to a method for predicting the prognosis of acute coronary syndromes and cardiac biomarkers for use in said method. The present invention also relates to devices and kits for use in accordance with said method. [Background technology]

[0002] The use of plasma biomarkers has become central to the diagnosis and prognosis of cardiovascular events. For example, the prognostic impact of elevated myoglobin in patients with coronary artery disease (CAD) is well established.

[0003] Current therapies and timely primary percutaneous coronary intervention (PCI) have significantly improved the prognosis of patients with ST-segment elevation acute myocardial infarction (STEMI) over the past few decades. However, recurrent major adverse cardiovascular events (MACE) after STEMI remain. Early risk stratification of patients at high risk for long-term MACE is crucial for the aggressiveness of treatment and intensity of care to improve their prognosis. Current plasma biomarkers that can be used to diagnose and / or predict the prognosis of STEMI or acute coronary syndrome include myoglobin, creatine kinase-MB (CK-MB), and troponin. However, each of these plasma biomarkers has its own set of limitations. For example, myoglobin in plasma peaks approximately 2 hours after a cardiac event, but has poor cardiac specificity. CK also peaks approximately 10 hours after a cardiac event, but cumulative plasma CK concentrations are not available until at least 48 hours after the cardiac event.

[0004] Troponin has become the primary plasma biomarker for the early detection of acute coronary syndromes, such as myocardial necrosis, and has largely replaced CK measurements. A single measurement of plasma troponin is currently one of the most sensitive and specific tests for myocardial necrosis. Current evidence suggests that a single low admission troponin level can be used to rule out (exclude) the diagnosis of ACS in subjects with a low probability of ACS, although most patients require serial measurements over 6 hours or more to safely rule out such a diagnosis. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need for new or improved methods for predicting the prognosis of acute coronary syndromes. The reference herein to any prior art is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would be understood by, considered relevant, and / or reasonably expected to be combined with other pieces of prior art by those skilled in the art. [Means for solving the problem]

[0006] The present invention provides a method for providing a prognosis of acute coronary syndrome (ACS) in a subject, comprising: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof and Predict ACS when the subject's plasma MIF and Nt-proBNP concentrations are higher than the reference plasma NIF and Nt-proBNP concentrations. The present invention provides a method comprising:

[0007] The present invention provides a method for providing a prognostic factor for acute coronary syndrome (ACS) in a subject, comprising: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) B-type natriuretic peptide (BNP) or a fragment thereof and Predict ACS when a subject's plasma MIF and BNP concentrations are higher than the reference plasma NIF and BNP concentrations The present invention provides a method comprising:

[0008] The present invention also provides a method for providing a prognosis for a subject with ACS, comprising: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof Measure both concentrations; The concentration of MIF is compared with a reference MIF concentration. The Nt-proBNP concentration was compared with the reference Nt-proBNP concentration, The respective concentrations of MIF and Nt-proBNP compared to their respective reference concentrations are indicative of the subject's prognosis. The present invention provides a method comprising:

[0009] The present invention also provides a method for providing a prognosis for a subject with ACS, comprising: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) B-type natriuretic peptide (BNP) or a fragment thereof Measure both concentrations; The concentration of MIF is compared with a reference MIF concentration. The BNP concentration is compared with the reference BNP concentration. The levels of MIF and BNP compared to their respective reference levels are indicative of the subject's prognosis. The present invention provides a method comprising:

[0010] The present invention also provides a method for providing a prognosis for a subject having ACS, comprising detecting in a sample from said subject: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof Measure both concentrations; The concentration of MIF is compared with a reference MIF concentration. The Nt-proBNP concentration was compared with the reference Nt-proBNP concentration, the reference concentrations of MIF and Nt-proBNP are concentrations below which, at a later time point, are correlated with increased survival and a decreased probability of non-fatal cardiac events, and concentrations above which, at a later time point, are correlated with decreased survival and an increased probability of non-fatal cardiac events, thereby providing a prognostic prediction for subjects with ACS.

[0011] The present invention also provides a method for providing a prognosis for a subject having ACS, comprising detecting in a sample from said subject: (c) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (d) B-type natriuretic peptide (BNP) or a fragment thereof Measure both concentrations; The concentration of MIF is compared with a reference MIF concentration. The BNP concentration is compared with the reference BNP concentration. wherein the reference concentrations of MIF and BNP are concentrations below which increased survival and decreased probability of non-fatal cardiac events at a later time point are correlated, and concentrations above which decreased survival and increased probability of non-fatal cardiac events at a later time point are correlated, thereby providing a prognostic prediction for subjects with ACS. The present invention provides a method comprising:

[0012] The present invention also provides a method for providing a prognosis for a subject having ACS, comprising analyzing in a sample from the subject the levels of both: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof; determining the concentration of MIF or a fragment thereof and the concentration of Nt-proBNP or a fragment thereof in a sample from said subject; comparing the concentration of said MIF or fragment thereof to a reference MIF concentration; comparing said concentration of Nt-proBNP or a fragment thereof to a reference Nt-proBNP concentration; assigning the subject to a risk group based on whether the concentration of MIF or a fragment thereof is higher or lower than a reference concentration and based on whether the concentration of Nt-proBNP or a fragment thereof is higher or lower than a reference concentration; a concentration of MIF or a fragment thereof greater than the reference MIF concentration indicates a decreased likelihood of survival and / or an increased likelihood of a non-fatal cardiac event; a concentration of Nt-proBNP or a fragment thereof higher than the reference Nt-proBNP concentration indicates a lower likelihood of survival and / or a higher likelihood of a non-fatal cardiac event; thereby providing a prognostic prediction for subjects with ACS. The present invention provides a method comprising:

[0013] The present invention also provides a method for providing a prognosis for a subject having ACS, comprising analyzing in a sample from the subject the levels of both: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) B-type natriuretic peptide (BNP) or a fragment thereof; determining a concentration of macrophage migration inhibitory factor (MIF) or a fragment thereof and a concentration of B-type natriuretic peptide (BNP) or a fragment thereof in a sample from the subject; comparing the concentration of said MIF or fragment thereof to a reference MIF concentration; comparing the concentration of said BNP or fragment thereof to a reference BNP concentration; assigning the subject to a risk group based on whether the concentration of MIF or a fragment thereof is higher or lower than a reference concentration and based on whether the concentration of BNP or a fragment thereof is higher or lower than a reference concentration; a concentration of MIF or a fragment thereof greater than the reference MIF concentration indicates a decreased likelihood of survival and / or an increased likelihood of a non-fatal cardiac event; a concentration of BNP or a fragment thereof higher than the reference BNP concentration indicates a lower likelihood of survival and / or a higher likelihood of a non-fatal cardiac event; thereby providing a prognostic prediction for subjects with ACS. The present invention provides a method comprising:

[0014] In one aspect of the invention, the reference concentrations of MIF, Nt-proBNP (or BNP) and / or troponin are determined from reference concentrations in a plasma, blood or serum sample obtained from at least one individual previously identified as suffering from ACS.

[0015] The present invention also provides a method for providing a prognosis for a subject having ACS, comprising detecting in a sample from said subject: determining the concentration of macrophage migration inhibitory factor (MIF) or a fragment thereof; wherein if the MIF concentration in the sample from the subject is greater than or equal to about 70 ng / mL, the subject is determined to have a decreased survival rate and an increased probability of a non-fatal cardiac event at a later time point; determining that the subject has an increased survival rate and an increased probability of a non-fatal cardiac event at a later time point if the MIF concentration in the sample from the subject is less than about 70 ng / mL; thereby providing a prognosis for subjects with ACS A method is provided.

[0016] In one aspect of the invention, a subject is determined to have a decreased survival rate and an increased probability of a non-fatal cardiac event at a later time point if the concentration of MIF in a sample from the subject is greater than or equal to about 73 ng / mL, and further, a subject is determined to have an increased survival rate and a decreased probability of a non-fatal cardiac event at a later time point if the concentration of MIF in a sample from the subject is less than about 73 ng / mL.

[0017] The present invention also provides a method for providing a prognosis for a subject with ACS, comprising determining the concentration of macrophage migration inhibitory factor (MIF) or a fragment thereof in a sample from said subject; comparing the concentration of MIF to reference MIF concentrations of about 40 ng / mL and about 70 ng / mL; wherein if the MIF concentration in the sample from the subject is about 40 ng / mL or less, the subject is determined to have a high probability of survival and a low probability of a non-fatal cardiac event at a later time point; wherein if the MIF concentration in the sample from the subject is greater than or equal to about 70 ng / mL, the subject is determined to have a low survival rate and a high probability of a non-fatal cardiac event at a later time point; thereby providing a prognostic prediction for subjects with ACS. The present invention provides a method comprising:

[0018] In one aspect of the invention, the method compares the MIF concentration to reference MIF concentrations of about 40 ng / mL and about 70 ng / mL, where if the MIF concentration in a sample from a subject is about 73 ng / mL or greater, the subject is determined to have a reduced survival rate and a higher probability of a non-fatal cardiac event at a later time point.

[0019] In any aspect of the invention, the prognosis is survival, preferably long-term survival, or non-fatal cardiac events. Survival may be selected from MACE-free survival, all-cause mortality-free survival, cardiac death-free survival, or heart failure (HF) rehospitalization-free survival, or any other survival described herein. Non-fatal cardiac events include MACE and adverse improvement in LVEF.

[0020] In any aspect of the invention, prognosis can be an indication of survival for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 28, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80 months or more after diagnosis of ACS.

[0021] In any aspect of the invention, the method further comprises determining the concentration of troponin or a fragment thereof. Preferably, the troponin is high-sensitivity troponin T (hs-TnT). The method further comprises comparing the concentration of troponin or a fragment thereof to a reference troponin concentration. The reference troponin concentration is a concentration lower than that correlated with increased survival and decreased probability of non-fatal cardiac events at a later time point, and higher than that correlated with decreased survival and increased probability of non-fatal cardiac events.

[0022] In any aspect of the present invention, the concentration of either BNP or a fragment thereof, or the N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof, is measured, analyzed, or determined. BNP is synthesized as a 134-amino acid preprohormone (preproBNP) encoded by the human NPPB gene. Removal of the 25-residue N-terminal signal peptide generates the prohormone proBNP, which is stored intracellularly as an O-linked glycoprotein. ProBNP is then cleaved by a specific convertase between arginine 102 and serine 103 into Nt-proBNP and the biologically active 32-amino acid polypeptide BNP, which are secreted into the blood in equimolar amounts.

[0023] In any aspect of the invention, the method comprises determining the concentration of MIF, Nt-proBNP (or BNP) and / or troponin from plasma, blood or serum. Preferably, the method comprises determining the concentration of MIF, Nt-proBNP and / or troponin from plasma.

[0024] In any aspect of the invention, the method may not include the step of collecting plasma, blood, or serum from the subject. In other words, the method may involve determining the concentration of MIF, Nt-proBNP (or BNP) and / or troponin from plasma, blood, or serum previously collected from the subject, i.e., obtained before performing the method of the invention. Furthermore, the plasma, blood, or serum sample may be an in vitro sample of plasma, blood, or serum.

[0025] In any aspect of the invention, the acute coronary syndrome is acute myocardial infarction (AMI). The AMI can be ST-elevation myocardial infarction (STEMI) or non-ST-elevation myocardial infarction (non-STEMI). Preferably, the AMI is STEMI. In some embodiments, the subject with STEMI may have been treated with primary percutaneous coronary intervention (PCI).

[0026] In any aspect of the invention, the method further comprises administering percutaneous coronary intervention (PCI) and / or thrombolysis to the subject. Preferably, the administering of percutaneous coronary intervention (PCI) and / or thrombolysis is administered only to subjects identified as having a poor prognosis, in other words, identified as having a reduced or low likelihood (probability) of survival and an increased or high likelihood of a non-fatal cardiac event.

[0027] In any aspect of the invention, the method involves determining the MIF concentration in a sample obtained within 4 hours after the onset of symptoms or admission to hospital. Alternatively, the MIF sample can be obtained from the subject within 210, 180, 150, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 minutes after the onset of symptoms or admission to hospital.

[0028] In any aspect of the invention, the method comprises determining the Nt-proBNP or BNP concentration in a sample taken around or between any of the following days: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or more days after the onset of symptoms or hospital admission. Preferably, the Nt-proBNP or BNP concentration is measured in a sample obtained from the patient approximately 3 days after the onset of symptoms or hospital admission.

[0029] In any aspect of the invention, the method includes determining the troponin concentration in a sample taken around or between any of the following days: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11, 11.5, 12 or more. Preferably, the troponin is high-sensitivity troponin T (hs-TnT).

[0030] In any aspect of the invention, the concentrations of MIF, Nt-proBNP or BNP, and troponin are determined in the same sample, or Nt-proBNP or BNP, troponin, and MIF may be determined from different samples.

[0031] The present invention provides a method for providing a prognosis for a subject after diagnosis of acute coronary syndrome (ACS), comprising measuring the concentrations of both macrophage migration inhibitory factor (MIF) and N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof in a sample from the subject, and predicting ACS when the subject's MIF and Nt-proBNP concentrations are greater than reference MIF and Nt-proBNP concentrations.

[0032] In any aspect of the invention, the method includes determining whether the MIF concentration falls within a concentration range of about 40 ng / mL to 70 ng / mL, is less than about 40 ng / mL, or is greater than about 70 ng / mL. In any aspect of the invention, a MIF concentration equal to or greater than about 70 ng / mL is associated with a poor prognosis. In another aspect, a MIF concentration of about 73 ng / mL or greater is associated with a poor prognosis (malignant prognosis). In any aspect of the invention, the reference concentration can be 40 ng / mL, 70 ng / mL, 73 ng / mL, or any one of the concentrations described herein, particularly those listed in Table 2. Determination of the MIF concentration can be determined by any assay known in the art, including those described herein.

[0033] In any aspect of the invention, the method includes determining whether the hs-TnT concentration falls within the range of about 2.5 ng / mL to about 4.5 ng / mL, is less than or equal to about 2.5 ng / mL, or is greater than or equal to about 4.5 ng / mL. Preferably, an hs-TnT concentration of about 4.5 ng / mL or greater is associated with a poor prognosis. In any aspect of the invention, the reference concentration can be 2.5 ng / mL, 4.5 ng / mL, or any one of the concentrations described herein, particularly those listed in Table 2. Determining the concentration of hs-TnT can be determined by any assay known in the art, including those described herein.

[0034] In any aspect of the invention, the method comprises determining whether the Nt-proBNP concentration falls within the range of about 700 pg / mL to about 1200 pg / mL, is less than or equal to about 700 pg / mL, or is greater than or equal to about 1200 pg / mL. Preferably, Nt-proBNP concentrations greater than about 1200 pg / mL are associated with a poor prognosis. In any aspect of the invention, the reference concentration can be 700 pg / mL, 1200 pg / mL, or any one of the concentrations described herein, particularly those listed in Table 2. Determining the concentration of Nt-proBNP can be by any known assay, including those described herein.

[0035] The present invention provides a method of treating acute coronary syndrome (ACS) in a subject, comprising: measuring the concentrations of both macrophage migration inhibitory factor (MIF) or a fragment thereof and N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof in a sample collected from the subject, and predicting ACS if the subject's MIF and Nt-proBNP concentrations are greater than reference MIF and Nt-proBNP concentrations, and performing percutaneous coronary intervention (PCI) and / or thrombolysis in the subject.

[0036] The present invention provides a method of treating acute coronary syndrome (ACS) in a subject, comprising: providing an individual determined to have a low likelihood of survival and / or a high likelihood of a non-fatal cardiac event by any of the methods of the invention described herein; The subject undergoes percutaneous coronary intervention (PCI) and / or thrombolysis; thereby treating the subject for ACS. The present invention provides a method comprising:

[0037] In any embodiment of the invention, the method further comprises measuring the concentration of troponin. Preferably, the concentration of MIF, Nt-proBNP and / or troponin is measured from plasma.

[0038] The present invention provides a device comprising means for measuring the concentration of macrophage migration inhibitory factor (MIF) and the concentration of B-type natriuretic peptide (BNP) or the N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) in a sample from a subject, for use in any of the methods of the invention described herein.

[0039] In any aspect of the invention, the device further comprises means for measuring troponin concentration. Preferably, the device is a point-of-care detection-enabled device. Preferably, the concentrations of MIF, Nt-proBNP and / or troponin are measured from plasma.

[0040] In any aspect of the invention, the concentration of MIF, Nt-proBNP and / or troponin may be determined by immunoassay.

[0041] In any aspect of the present invention, a kit is provided, comprising reagents for measuring macrophage migration inhibitory factor (MIF) concentration and N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) concentration in a sample from a subject, and / or comprising a device as defined above.Preferably, the kit is for use in any of the methods described herein.

[0042] In any aspect of the present invention, there is provided a kit comprising reagents and / or a device as defined above for measuring macrophage migration inhibitory factor (MIF) and brain natriuretic peptide (BNP) concentrations in a sample from a subject, preferably for use in any of the methods described herein.

[0043] In any aspect of the invention, the kit further comprises means for measuring the concentration of troponin. Preferably, the troponin is high-sensitivity troponin T (hs-TnT). Preferably, the concentrations of MIF, Nt-proBNP (or BNP) and / or troponin are determined from plasma.

[0044] In any aspect of the invention, the reagent may comprise an anti-MIF antibody, an anti-Nt-proBNP (or BNP) antibody and / or an anti-troponin antibody.

[0045] In an optional aspect of the present invention, a cardiac biomarker panel is provided that includes plasma MIF and Nt-proBNP (or BNP) in a sample from a subject, wherein a plasma MIF and Nt-proBNP (or BNP) concentration greater than a reference plasma MIF and Nt-proBNP (or BNP) concentration is prognostic of the magnitude of ACS in the subject. The cardiac biomarker panel may further include plasma troponin in the sample from the subject.

[0046] The invention also provides the use of a thrombolytic agent in the manufacture of a medicament for the treatment of ACS for a subject determined to have a low likelihood of survival and / or a high likelihood of a non-fatal cardiac event by any of the methods of the invention described herein.

[0047] The present invention provides definitive lytic agents for use in treating ACS for subjects determined to have a low likelihood of survival and / or a high likelihood of a non-fatal cardiac event by any of the methods of the invention described herein.

[0048] The present invention also provides the use of a means for detecting MIF, Nt-proBNP and / or troponin in the manufacture of or when using a reagent or kit for use in prognosing ACS. Preferably, the prognosis of ACS is by the method of the present invention described herein.

[0049] As used herein, unless the context requires otherwise, the term "comprises" and variations of that term, such as "comprising," "including," and "included," are not intended to exclude further additives, components, integers, or steps.

[0050] Further aspects of the invention set out in the preceding paragraphs and further embodiments of said aspects will become apparent from the following description, which is given by way of example only, and by reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0051] [Figure 1] Study flow chart. A total of 658 patients with a confirmed diagnosis of STEMI were recruited into this prospective study. Of these, 42 patients were excluded based on the exclusion criteria, and another 50 patients were excluded due to missing MIF measurements at admission (n = 14) or discontinuity during follow-up, resulting in a final cohort of 566 patients. Echocardiography was performed on day 3 and then at 12 months during follow-up. Biochemical assays included MIF (at admission), hs-TnT and CK-MB (within 48 hours), Nt-proBNP, and Hs-CRP (both on day 3). CAG, coronary angiography; PPCI, primary percutaneous coronary intervention; hs-TnT, high-sensitivity troponin T; CK-MB, creatine kinase-MB; Nt-proBNP, N-terminal prohormone of brain natriuretic peptide; CRP, C-reactive protein. [Figure 2] Admission MIF correlated with LVEF and improvement at 3 days and 12 months. (AB) Admission MIF was negatively correlated with LVEF by echocardiography performed at 3 days and 12 months (F12) after STEMI. (C) Patients were divided into three groups according to the tertiles of migration inhibitory factor. By calculating the difference in LVEF between the two time points (ΔLVEF), patients with high tertile MIF values ​​failed to improve LVEF compared with the other two tertiles (P<0.001). [Figure 3] Kaplan-Meier event-free survival curves for (A) all-cause mortality, (B) cardiovascular mortality, (C) MACE, and (D) HF readmission according to tertiles of MIF concentration at admission. Patients with high tertile MIF levels (red line, ≥73.0 ng / mL; n=188) were compared with those in the middle tertile (black line, 40.2–73.0 ng / mL; n=189) and the lower tertile (dashed black line, <40.2 ng / mL; n=189). [Figure 4]Risk stratification of MACE in STEMI patients according to tertiles of plasma MIF and Nt-proBNP concentrations. The combination of admission MIF and Nt-proBNP (day 3) identified a subgroup of patients with an increased risk of MACE during the follow-up period. Patients were divided into tertile groups based on MIF and Nt-proBNP levels separately. Patients with both biomarkers in the higher tertile group had a significantly increased risk of MACE compared with patients with both biomarkers in the lower tertile group (*P<0.001). [Figure 5] All-cause mortality and MACE in patients according to whether they had MIF, Nt-proBNP, and / or hs-TnT in the high tertile groups. Comparison of all-cause mortality and major adverse cardiovascular events in patients with ST-segment elevation myocardial infarction based on tertiles of macrophage migration inhibitory factor (MIF) and Nt-proBNP. Kaplan-Meier event-free survival curves for (A) all-cause mortality and (B) major adverse cardiovascular events (MACE) in patients based on migration inhibitory factor and Nt-proBNP levels. Patients were separately classified into tertile groups, defined as a positive (+) group with the high tertile and a negative (-) group with the middle or low tertile. Four groups were generated: Nt-proBNP(+) MIF(+) (red line; n=77), Nt-proBNP (-) MIF(+) (black line; n=111), Nt-proBNP (+) MIF (-) (red dashed line; n=111), and Nt-proBNP (-) MIF (-) (black dashed line; n=267). P values ​​in the inset indicate the difference relative to the Nt-proBNP (-) MIF (-) group. [Figure 6] Frequency distribution of MIF in STEMI patients, healthy subjects, and patients with non-ischemic chest pain. Patients with non-ischemic chest pain were those who presented to the emergency department with chest pain without evidence of cardiac ischemia, infection, or malignancy, ultimately followed up through medical records or direct telephone contact with the patient. DETAILED DESCRIPTION OF THE INVENTION

[0052] It will be understood that the invention disclosed or defined herein extends to any and all alternative combinations of two or more of the individual features mentioned or apparent in the text or drawings, all of which different combinations constitute various alternative embodiments of the invention.

[0053] Several embodiments of the invention will now be described in detail. While the invention will be described in conjunction with these embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the invention as defined by the claims.

[0054] Those skilled in the art will be aware of many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to any and all alternative combinations of two or more of the individual features mentioned or apparent in the text or drawings. All of these different combinations constitute various alternative embodiments of the present invention.

[0055] All patents and publications referenced herein are incorporated by reference in their entirety.

[0056] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0057] Long-term mortality and morbidity after acute myocardial infarction (AMI) are largely determined by myocardial infarction (MI) size and the degree of left ventricular (LV) dysfunction. Primary percutaneous coronary intervention (PPCI) has now become the established standard of care in patients with ST-segment elevation MI (STEMI) to limit infarct size and mortality. The present inventors have surprisingly discovered that measurement of plasma MIF concentration alone or measurement of MIF and Nt-proBNP concentrations greater than normal (i.e., greater than reference concentrations) can be prognostic for ACS, particularly STEMI, and can also be prognostic for survival and non-fatal cardiac events. The present inventors have also advantageously discovered that plasma concentrations of MIF, Nt-proBNP, and troponin greater than normal (i.e., greater than reference concentrations) at admission can be prognostic for ACS, particularly STEMI, or can be prognostic for survival and non-fatal cardiac events.

[0058] Most patients diagnosed with ACS, such as AMI, are treated by PCI. In hospitals that do not have PCI facilities, either permanently or temporarily, the measurement of plasma concentrations of only MIF at admission that are higher than normal (i.e., higher than the reference concentration); MIF and Nt-proBNP; or MIF, Nt-proBNP, and troponin can determine whether a given patient should be transferred to a hospital with PCI facilities. Furthermore, the inventors have discovered that the above-defined combination has a prognostic effect, and therefore, early and accurate estimation of MI size in patients with AMI is advantageous, especially in complex patients, or in places where local medical resources are limited.

[0059] The inventors have unexpectedly discovered that the above plasma biomarkers are prognostic factors for survival or non-fatal cardiac events. The inventors demonstrate that measuring a certain concentration of MIF alone; the concentrations of both MIF and Nt-proBNP; or the concentrations of MIF, Nt-proBNP, and troponin is an accurate approach to help predict the prognosis of ACS. The concentrations of MIF and Nt-proBNP; or MIF, Nt-proBNP, and troponin are better indicators of prognostic outcome than measuring MIF alone. The inventors have validated their findings and, in several respects, offer at least the following advantages: (1) High plasma MIF concentrations after ACS diagnosis correlate with more severe short- and long-term prognosis after diagnosis: (2) subjects who experience high plasma MIF concentrations after ACS diagnosis are more likely to experience MACE, cardiac death, heart failure, or death from any cause; (3) high plasma MIF and Nt-proBNP concentrations are associated with a higher risk of events such as MACE and death and are more accurate prognostic predictors when compared with their individual components; and / or (4) High plasma MIF, NT-proBNP, and troponin concentrations are associated with a higher risk of events such as MACE and death, and are more accurate prognostic tools when compared with their individual components.

[0060] In other words, high plasma concentrations of MIF and Nt-proBNP; or high plasma concentrations of MIF, Nt-proBNP, and troponin can serve as independent indicators of adverse ACS outcomes (poor prognosis). This approach may facilitate the identification of high-risk groups that may be associated with poor prognosis after ACS. Individuals with high levels of either plasma MIF and Nt-proBNP; or MIF, Nt-proBNP, and troponin can be identified as having a poor prognosis after ACS. Thus, elevated concentrations of MIF and Nt-proBNP; or MIF, Nt-proBNP, and troponin have implications for prognosis and patient management.

[0061] The present invention provides information about the likelihood of non-fatal cardiac events and survival to physicians or clinicians caring for patients. Based on the results of the methods of the present invention, the clinician or physician can, among other things: (i) enroll the patient in a clinical trial for a new therapy for ACS, (ii) treat the subject with an alternative therapy, e.g., one that targets a biomarker, (iii) discuss potential treatments and outcome status with the subject, (iv) provide regular or extensive post-treatment surveillance for subjects identified as having a low likelihood of survival and / or a high likelihood of a non-fatal cardiac event, and / or (v) proceed with treatment of subjects identified as having a high risk with added confidence that the treatment will likely benefit the subject.

[0062] In any embodiment of the present invention, the method can include further therapeutic steps such as PCI and / or thrombolysis. Thrombolysis and PCI can be important in reducing morbidity and mortality in STEMI. Early prognosis during patient management decision-making offers numerous advantages. First, clinicians assessing patients with an unclear or uncertain diagnosis of STEMI can benefit from the knowledge that elevated biomarkers are predictive of patient outcome, which will facilitate the decision-making process regarding treatment, reperfusion, and the timeliness of post-reperfusion supportive cardiac care required in a coronary care unit or intensive care unit. Second, in resource-limited areas, early prognostic findings can influence whether a patient should be transferred to a PCI-capable hospital or whether thrombolysis should be attempted first, especially in cases with significant comorbidities. When used in combination with Nt-proBNP, or alternatively in combination with Nt-proBNP and troponin, MIF serves as a valid prognostic indicator in clinical settings, particularly in the emergency room setting.

[0063] The present invention also provides for the prognosis prediction of poor myocardial reperfusion recovery and adverse improvement of LVEF. In particular, the inventors have demonstrated that MIF level is an independent predictor of poor myocardial reperfusion recovery. Individuals identified as being at risk for poor myocardial reperfusion recovery can undergo additional or more intensive intervention or monitoring. Furthermore, individuals at risk for poor or impaired LVEF recovery can be administered heart failure medications (e.g., ACE inhibitors, beta-blockers, etc.) and heart failure preventive therapy can be administered.

[0064] Finally, the information provided by the methods of the present invention is useful in clinical trials, allowing subjects at high risk for certain adverse outcomes to be included in clinical trials. Post-ACS clinical trials of novel therapies are typically constrained by the requirement to use proven therapies, resulting in low clinical endpoint event rates. This creates the need to use very large test cohorts to demonstrate further improvements with new drugs at a statistically significant level. Being able to predictively identify patients at high risk for clinical events potentially results in substantial cost savings by allowing the use of smaller patient cohorts. Furthermore, patients who are unlikely to benefit are not exposed to unproven drugs. Thus, the methods of the present disclosure benefit patients and can also optimize clinical trials by reducing the number of participants by selecting those with high event rates.

[0065] Measurement of a combination of MIF and Nt-proBNP; or a combination of MIF, Nt-proBNP and troponin, would therefore be highly beneficial in the ongoing management, including the use of adjuvant therapy and in patients after PPCI, since it would provide additional prognostic information regarding MI size in addition to the advantages outlined above.

[0066] Those skilled in the art will recognize that the magnitude of plasma MIF, Nt-proBNP and / or troponin concentrations may vary depending on the characteristics of the assay used to measure MIF, Nt-proBNP and / or troponin (e.g., various antibodies). Nevertheless, those skilled in the art will also understand that suitable reference plasma MIF concentrations can be determined, provided that appropriate control samples are analyzed.

[0067] Measurement of MIF, Nt-proBNP (BNP) or troponin levels relative to a reference value (e.g., reference concentration) can be used to predict the prognosis of ACS, particularly STEMI, or to predict the prognosis of survival and non-fatal cardiac events according to the methods described herein.Preferably, the reference concentration is predetermined or determined from a cohort of patients with known ACS (preferably ST-segment elevation myocardial infarction) outcomes, preferably survival and non-fatal cardiac events, as described herein.

[0068] In some embodiments, the reference concentration classifies subjects into one of two subgroups using the following rule: if the test or target concentration is lower than the reference concentration, the subject is assigned to a group with increased survival and / or decreased probability of a non-fatal cardiac event; if the test or target concentration is higher than or equal to the reference concentration, the subject is assigned to a group with decreased survival and / or increased probability of a non-fatal cardiac event.

[0069] Alternatively, there may be multiple reference concentrations that classify (stratify) subjects into low, intermediate, or high likelihood of survival or non-fatal cardiac event.

[0070] The reference concentration can be selected according to any method known in the art. In certain embodiments, the reference concentration can be a predetermined value. Alternatively, the reference concentration can be determined during the assay process. For example, samples from known non-diseased and / or diseased subjects can be tested simultaneously with the test sample and the reference value determined therefrom. As a further alternative, test samples from a mixed population can be analyzed, and a reference value can be determined based on the distribution of results, for example, using statistical methods known in the art.

[0071] It is expected that, over time, additional testing will generate new and additional information regarding the MIF, Nt-proBNP (or BNP) or troponin profiles of subjects suffering from ACS, particularly STEMI. The additional information can increase the accuracy, reliability, and certainty of the reference profile, and thus the accuracy, reliability, and certainty of the decisions and recommendations embodied by implementing the method. Thus, newly generated or revised reference concentrations and reference profiles can be utilized in accordance with the methods described herein. Thus, those skilled in the art will recognize that reference concentrations may change over time, and appropriate reference MIF, Nt-proBNP (or BNP) concentrations can be determined once appropriate control samples are analyzed.

[0072] A plasma MIF, Nt-proBNP (or BNP) or troponin concentration is greater than a reference plasma MIF concentration when it exceeds the reference plasma MIF, Nt-proBNP (or BNP) or troponin concentration by 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more. A plasma MIF, Nt-proBNP (or BNP) or troponin concentration that is 50% greater than a reference plasma MIF, Nt-proBNP (or BNP) or troponin concentration is equivalent to a plasma MIF, Nt-proBNP (or BNP) or troponin concentration that is 1.5 times greater, and a plasma MIF, Nt-proBNP (or BNP) or troponin concentration that is 100% greater than a reference plasma MIF, Nt-proBNP (or BNP) or troponin concentration is equivalent to a plasma MIF, Nt-proBNP (or BNP) or troponin concentration that is 2 times greater, etc. Thus, a plasma MIF, Nt-proBNP (or BNP) or troponin concentration is greater than a reference plasma MIF, Nt-proBNP (or BNP) or troponin concentration when it is 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold or more greater than the reference plasma MIF, Nt-proBNP (or BNP) or troponin concentration. In another embodiment, a plasma MIF, Nt-proBNP (or BNP) or troponin concentration is greater than a reference plasma MIF, Nt-proBNP (or BNP) or troponin concentration when it exceeds the reference MIF, Nt-proBNP (or BNP) or troponin concentration, and the difference is statistically significant as determined by methods known to those skilled in the art.Alternatively, subjects with MIF, Nt-proBNP (or BNP) or troponin values ​​higher than about the 50th, 60th, 70th, 80th, 90th, 95th, 96th, 97th, 98th, 99th percentile or higher when compared to an appropriate matched control population can be identified as affected (i.e., as those with a moderate or high (malignant) prognosis).

[0073] Those skilled in the art will understand that MIF concentrations of about 40 ng / mL to 70 ng / mL are associated with a moderate prognosis, concentrations higher than about 70 ng / mL MIF are associated with a poor prognosis (malignant prognosis), while concentrations lower than about 40 ng / mL are associated with a benign prognosis. Subjects with MIF levels higher than about 70 ng / mL exhibit a prognosis of about 35% to 40%, preferably about 40%, 5-year MACE rate and about 20% mortality rate. In another aspect, MIF concentrations of about 40 ng / mL to 73 ng / mL are associated with a moderate prognosis, concentrations higher than about 73 ng / mL MIF are associated with a moderate prognosis, and MIF concentrations higher than about 73 ng / mL are associated with a poor prognosis, while MIF concentrations lower than about 40 ng / mL are associated with the best prognosis. In this regard, subjects with MIF levels of about 73 ng / mL or greater have a prognosis of about 35-40%, preferably about 40%, 5-year MACE rate and about 20% mortality rate.

[0074] Those skilled in the art will appreciate that troponin concentrations of about 2.5 ng / mL to about 4.5 ng / mL are associated with a moderate prognosis, concentrations greater than about 4.5 ng / mL are associated with a poor prognosis, while concentrations less than about 2.5 ng / mL are associated with a benign prognosis, and in combination with MIF levels less than 40 ng / mL or Nt-proBNP (or BNP) levels less than 700 pg / mL are associated with the best prognosis.

[0075] Subjects with troponin concentrations greater than about 4.5 ng / mL, combined with MIF greater than about 70 ng / mL or about 73 ng / mL and BNP greater than about 1200 pg / mL, exhibit a 5-year MACE predictive rate (prognostic value) of about 50% and a mortality predictive rate (prognostic value) of about 25%.

[0076] Subjects with MIF levels greater than about 70 or 73 ng / mL and Nt-proBNP (or BNP) greater than about 1200 pg / mL show a 5-year predictive rate of MACE (prognostic value) of about 50% and a 5-year predictive rate of death (prognostic value) of about 25%.

[0077] Those skilled in the art will appreciate that Nt-proBNP (or BNP) concentrations of about 700 pg / mL to about 1200 pg / mL are associated with a moderately severe prognosis, concentrations greater than about 1200 pg / mL are associated with a poor prognosis, and in combination with MIF concentrations less than 40 ng / mL or troponin concentrations less than 2.5 ng / mL are associated with the best prognosis.

[0078] As shown herein, MIF is a significant early indicator of prognosis of cardiovascular or acute myocardial ischemic events, while the combination of MIF and Nt-proBNP (or BNP); or the combination of MIF, Nt-proBNP (or BNP) and troponin is the most clinically relevant measure of prognosis of ACS compared to each individual component alone. Thus, in some embodiments, the present invention relates to methods for treating and prognosing ACS by measuring the concentrations of MIF and Nt-proBNP (or BNP); or the concentrations of MIF, Nt-proBNP (or BNP) and troponin.

[0079] As used herein, a "method" for prognosing or treating ACS in a subject, comprising measuring plasma MIF and Nt-proBNP (or BNP); or MIF, Nt-proBNP (or BNP) and troponin concentrations, may be provided in alternative formats. As one example, the method may be in the form of "use" of plasma MIF concentration for diagnosing, prognosing, or treating ACS in a subject. As a second example, the method may be in the form of "use" of plasma MIF concentration for prognosing or treating ACS in a subject. In yet another form, the method may be in the Swiss format of "use of plasma MIF concentration in the manufacture" of a prognostic factor or drug.

[0080] In a preferred embodiment, the method for predicting the prognosis of ACS in a subject is performed "in vitro" on a plasma (or serum or blood) sample. In other words, any method of the invention is an in vitro method. For example, the determining, measuring, or analyzing step in any method of the invention described herein is performed in vitro.

[0081] In one embodiment, the method of the present invention does not include the step of obtaining a sample from a subject.

[0082] Following prognosis of ACS in a subject, the method may further comprise treating the subject with percutaneous coronary intervention (PCI) and / or thrombolysis.

[0083] The currently recommended treatment for STEMI, if available and performed in a timely manner, is primary PCI (i.e., PCI performed as soon as possible after diagnosis). PCI involves the placement of a lumen-containing catheter into the femoral, radial, or occasionally branchial artery, which is then introduced, under x-ray imaging, into the coronary artery containing the stenosis / thrombosis causing the STEMI. The stenosis is then dilated using a fluid-filled balloon. In some cases, this is followed by the placement of a stent (a cylindrical metallic scaffolding material) at the site of the dilated area. The stent may or may not be impregnated with a drug solution to prevent the stenosis from recurring (this depends on the clinical situation and angiographic findings). If primary PCI is not feasible, STEMI patients are generally treated with fibrinolytic agents to dissolve any thrombus present in the culprit area. Fibrinolytic agents are delivered via peripheral venous cannulation. In some cases, residual symptoms and physical signs persist (or recur) despite fibrinolytic treatment, and in such cases, patients can subsequently undergo "rescue" PCI.

[0084] The treatment may further include the administration of an agent capable of dissolving thrombi. Such agents may also be referred to as thrombolytic agents. Examples of thrombolytic agents include urokinase, recombinant tissue plasminogen activator (TPA), pro-urokinase, anisoylated purified streptokinase activator complex (APSAC), and streptokinase. The treatment may further include the administration of an agent capable of preventing or reducing thrombosis or rethrombosis. Such agents may also be referred to as antithrombotic agents. Examples of antithrombotic agents include antiplatelet agents, such as glycoprotein IIB / IIIA inhibitors (e.g., abciximab, eptifibatide, or tirofiban), or adenosine diphosphate (ADP) receptor inhibitors (e.g., clopidogrel, prasugrel, ticagrelor, or ticlopidine).

[0085] As used herein, performing thrombolysis includes administering any one or more thrombolytic agents as described herein.

[0086] Preferably, the sample for measuring MIF, Nt-proBNP (or BNP) and troponin is plasma. Plasma can be obtained by suppressing blood coagulation with EDTA, sodium heparin, lithium heparin, sodium citrate or sodium oxalate. Alternatively, the sample for measuring MIF, Nt-proBNP (or BNP) and troponin is serum or blood. In one embodiment, the sample can be whole blood.

[0087] "Acute coronary syndrome" or "ACS" refers to a spectrum of symptoms including chest discomfort or other symptoms caused by a lack of oxygen supply to the heart. Symptoms occur as a result of erosion, cord fissure, or rupture of pre-existing atherosclerotic plaque and occur spontaneously. In the absence of evidence of myocardial necrosis, unstable angina is diagnosed, whereas in the presence of evidence of myocardial necrosis (e.g., plasma biomarkers), AMI is diagnosed. Thus, ACS may include unstable angina or AMI. "ACS" does not include stable angina.

[0088] Patients with ACS present with a variety of physical symptoms, including chest pain that often radiates to the neck, jaw, or shoulders, or travels down the inside of the left arm or arms, and may have accompanying symptoms of dyspnea, sweating, palpitations, mild headache, and nausea. Patients experiencing ACS present to their physician with clinical symptoms including unstable angina, non-ST elevation non-Q wave myocardial infarction ("NST-MI"), ST elevation non-Q wave MI, and transmural (Q wave) MI.

[0089] "Acute myocardial infarction" or "AMI" refers to the interruption of blood supply to a portion of the heart, resulting in restricted blood supply ("ischemia"), oxygen deprivation, and cell death ("necrosis"), which is a type of ACS. This can lead to damage or death of heart muscle tissue (myocardium). Thus, "myocardial necrosis" refers to the death of heart cells. AMI can be classified as ST-segment elevation myocardial infarction (STEMI), diagnosed by elevation of the ST segment on an electrocardiogram, and non-ST-segment elevation myocardial infarction (non-STEMI), diagnosed by the absence of such electrocardiographic changes. STEMI can be treated with thrombolysis or PCI. Non-STEMI is managed with medical therapy, but PCI is often performed during hospitalization.

[0090] As used herein, the term MACE (major adverse cardiac event) refers to cardiac death and other non-fatal cardiovascular outcomes. Non-exclusive examples of MACE include myocardial infarction, unstable angina, heart failure, percutaneous cardiac intervention, coronary artery bypass graft, malignant dysrhythmia, cardiac shock, implantable defibrillator, and malignant dysrhythmia.

[0091] As used herein, "HF-excluded readmission survival" refers to the prognosis of patients who are not readmitted to the hospital due to heart failure after a diagnosis of ACS. In other words, HF readmission can be defined as a readmission in which HF was the primary reason.

[0092] As used herein, the term "survival excluding all-cause mortality" refers to the prognosis of patients who do not die from any underlying disease.

[0093] As used herein, the term "survival excluding cardiac death" refers to the prognosis of patients who do not die from any heart-related disease.

[0094] In any aspect of the invention, the prognosis can indicate survival or non-fatal cardiac events for 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 28, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80 or more months after diagnosis of ACS.

[0095] "Coronary event" refers to any severe or acute cardiovascular condition, including AMI, unstable angina, or cardiac mortality.

[0096] "Left ventricular hypertrophy" or "LVH" refers to an enlargement of the myocardium (muscle) in the left ventricle of the heart.

[0097] "Left ventricular end-diastolic volume" or "LVEDV" is defined as the volume of blood in the left ventricle just before contraction.

[0098] "Left ventricular end-systolic volume" or "LVESV" is defined as the volume of blood remaining in the left ventricle at the end of systole.

[0099] "Stroke volume" refers to the amount of blood ejected from the left ventricle with each contraction (heart beat).

[0100] "Left ventricular ejection fraction" or "LVEF" is defined as the fraction of the LVEDV that is ejected with each contraction (heart beat); i.e., the "stroke volume" divided by the LVEDV. LVEF can be expressed as a percentage.

[0101] As used herein, "infarct size" is measured by cardiovascular resonance (CMR), integrated biomarker levels, or echocardiography and is defined as the area of ​​the enhancing myocardium on each short-axis slice (bounded by manually traced endocardial and epicardial contours) multiplied by the slice thickness and a myocardial density of 1.05 g / mL to obtain the infarct mass, which is expressed as a percentage (%) of the left ventricular mass.

[0102] As used herein, "indexed left ventricular mass" refers to the left ventricular mass (g) divided by the square of the subject's height (m), which is expressed as g / m 2 It is expressed in units of .

[0103] As used herein, a "biomarker" refers to a measurable substance, the detection of which typically indicates a particular cardiac disease. A "biomarker" can indicate a change in the expression or state of the measurable substance that correlates with disease prognosis. A "biomarker" can be a protein or peptide. A "biomarker" can be measured in a body fluid such as plasma, blood, or serum. As used herein, "biomarker" includes plasma macrophage migration inhibitory factor (MIF), B-type natriuretic peptide (BNP), and troponin, but may further include myoglobin, C-reactive protein, or creatine kinase (CK).

[0104] In one embodiment, MIF, Nt-proBNP (or BNP) and troponin are full-length. In another embodiment, MIF, BNP and troponin comprise fragments thereof. Preferably, MIF, Nt-proBNP (or BNP) and troponin are human.

[0105] The troponin can be troponin I, e.g., cardiac troponin I (cTnI), troponin T, or high-sensitivity troponin T (hs-TnT). Those skilled in the art will appreciate that hs-TnT is a form of troponin that allows very low concentrations of troponin to be measured early and accurately after ACS.

[0106] Preferably, for clinical prognosis, the MIF is human MIF and is represented by the NCBI reference sequence: NP 002406.1 (SEQ ID NO: 1): MPMFIVNTNVPRASVPDGFLSELTQQLAQATGKPPQYIAVHVVPDQLMAFGGSSEPCALCSLHSIGKIGGQNRSYSKLLCGLLAERLRISPDRVYINYYDMNAANVGWNNSTFA The amino acid sequence provided herein is

[0107] Alternatively, for veterinary prognosis, the MIF can be from another mammal, such as a primate, mouse, cow, sheep, goat, pig, dog, or cat.

[0108] As used herein, "prognosis" and related terms refer to the description of the estimated outcome of ACS. This includes risk of MACE, survival excluding MACE, survival excluding HF-rehospitalization, survival excluding all-cause mortality, and survival excluding cardiac death. Prognosis may also include prediction of a favorable response to ACS treatment, such as thrombolysis. Because measurements of plasma biomarker concentrations correlate with the magnitude of AMI (e.g., quantification of infarct size), the plasma concentrations of the biomarkers defined above allow for the assessment of estimated morbidity and mortality due to infarction (prognosis). As will be understood by those skilled in the art, predictions may not necessarily be accurate for 100% of the subjects evaluated. However, the term requires that a statistically significant population of subjects can be identified as having an increased probability of having a given outcome.

[0109] Furthermore, measurement of plasma MIF, BNP and / or troponin concentrations can quantify ACS, thereby enabling prognosis of ACS.

[0110] As used herein, "onset" or "onset" is the point at which a subject begins to experience deviations from normal physiology.

[0111] As used herein, "admission" refers to the formal admission of a subject by a medical institution or health care facility to which medical care should be provided. In particular, "admission" will be associated with the exact time at which a subject is admitted for medical care.

[0112] As used herein, admission plasma MIF concentration refers to the MIF concentration measured in plasma derived from a blood sample obtained as soon as practicable after admission to hospital, but typically less than 4 hours after symptom onset. Alternatively, admission plasma MIF concentration is understood to mean 210, 180, 150, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 minutes or less after symptom onset.

[0113] If the subject is not receiving medical care, e.g., at home or at work, admission plasma MIF is understood to mean 240 minutes, or 210 minutes, 180 minutes, 150 minutes, 120 minutes, 110 minutes, 100 minutes, 90 minutes, 80 minutes, 70 minutes, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 5 minutes, or less after the onset of symptoms.

[0114] As used herein, Nt-proBNP (or BNP), preferably plasma Nt-proBNP (or BNP) concentration, refers to the Nt-proBNP (or BNP) concentration measured in a blood sample obtained from a patient after onset of symptoms or after hospitalization, preferably the concentration measured in plasma derived from the blood sample. In particular, the sample can be plasma derived from a blood sample obtained within or between approximately the following number of days after onset of symptoms: about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or more days after onset of symptoms. Preferably, the Nt-proBNP (or BNP) concentration is measured in plasma derived from a blood sample obtained from a patient on day 3 after onset of symptoms or hospitalization.

[0115] As used herein, plasma troponin concentration refers to troponin measured in plasma derived from a blood sample obtained from a patient after the onset of symptoms (onset) or hospitalization. In particular, the sample can be plasma derived from a blood sample obtained within or between about the following number of days: about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11, 11.5, 12 or more days after onset of symptoms or hospitalization.

[0116] The time at which a sample can be taken from a subject is applicable to all aspects of the present invention.

[0117] As used herein, "means for measuring" plasma MIF, Nt-proBNP (or BNP), or troponin refers to any mechanism capable of measuring (assaying or quantifying) MIF, Nt-proBNP (or BNP), or troponin. For example, plasma MIF, Nt-proBNP (or BNP), or troponin can be measured in a sample using any method known to those skilled in the art for measuring proteins, including, but not limited to, ELISA, enzyme immunoassay (EIA), Western blot, slot blot, dot blot, or immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), chromatography, etc. Dendrimer-enhanced radial partitioning immunoassays and immunofluorescence assays are known in the art and commercially available. Troponin can also be measured using a highly sensitive troponin assay.

[0118] As used herein, "assay" and variations thereof refer to the measurement or quantification of the concentration of plasma MIF, Nt-proBNP (or BNP) or troponin, or other biomarkers as defined herein.

[0119] The term "antibody(s)" refers to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, grafted antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies, as well as antigen-binding fragments of any of the foregoing. In particular, antibodies encompass immunoglobulin molecules, immunologically active fragments of immunoglobulins, i.e., molecules that contain an antigen-binding site. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. The heavy-chain constant domains (Fc) that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The terms "antibody" and "immunoglobulin" are used interchangeably in the broadest sense. In some embodiments, an antibody is part of a larger molecule formed by covalent or noncovalent association of the antibody with one or more additional proteins or peptides.

[0120] The terms "binding fragment," "functional fragment," "antibody fragment," or "antigen-binding fragment" are used for the purposes of this specification and claims to mean a portion or fragment of an intact antibody molecule, preferably where the fragment retains antigen-binding function. Examples of antibody fragments include Fab, Fab', F(ab'), Fd (VH and CH1 domains), Fd', and Fv (VH and CH1 domains of the single-chain arms of an antibody). L and V HThese include variable domain (Vl) fragments, diabodies, linear antibodies, variable light chains (Vl), variable heavy chains (VH), single-chain antibody molecules, single-chain binding polypeptides, scFv, scFv2 (tandem linkage of two scFv molecules head to tail in one chain), bivalent scFv, tetravalent scFv, half antibodies, dAb fragments, variable NAR domains, and bispecific and multispecific antibodies formed from antibody fragments.

[0121] Methods for producing antibodies are known in the art and / or are described in Antibodies: A Laboratory Manual, edited by Harlow & Lane, Cold Spring Harbor Laboratory (1988). Generally, such methods involve administering an antigen or an immunogenic fragment or epitope thereof, or a cell expressing and presenting it (i.e., immunogen), optionally formulated with any suitable or desirable carrier, adjuvant, or pharmaceutically acceptable excipient, to a non-human animal, such as a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig. Such immunogens can be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0122] Polyclonal antibody production can be monitored by sampling the blood of the immunized animal at various points after immunization. One or more additional immunizations may be given if necessary to achieve the desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. Once the desired level of immunogenicity is obtained, the immunized animal is bled, and the serum is isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0123] Monoclonal antibodies are one typical form of antibody contemplated by the present invention. The term "monoclonal antibody" or "mAb" refers to a homogeneous antibody population capable of binding to the same antigen, e.g., the same epitope within the antigen. This term is not intended to be limiting with respect to the source of the antibody or the method by which it is produced.

[0124] mAbs can be produced using any one of a number of known techniques, such as those procedures exemplified in US Pat. No. 4,196,265 or Harlow & Lane (1988) supra.

[0125] For example, a suitable animal is immunized with an immunogen under conditions sufficient to identify antibody-producing cells. Rodents such as rabbits, mice, and rats are representative animals. Mice genetically engineered to express human antibodies, e.g., mice that do not express mouse antibodies, can also be used to produce the antibodies of the present invention (e.g., as described in International Publication WO 2002 / 066630).

[0126] After immunization, somatic cells with the potential to produce antibodies, particularly B lymphocytes (B cells), are selected for use in the mAb production protocol. These cells can be obtained from biopsies of the spleen, tonsils, or lymph nodes, or from a peripheral blood sample. The B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell line, usually from the same species as the animal immunized with the immunogen.

[0127] The hybrids are amplified by culture in selective media containing agents that block de novo synthesis of nucleotides in tissue culture media, typical agents being aminopterin, methotrexate and azaserine.

[0128] The amplified hybridomas are subjected to functional selection for antibody specificity and / or titer, for example, by flow cytometry and / or immunohistochemistry and / or immunoassay (e.g., radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, etc.).

[0129] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to generate mAb-secreting cell lines (e.g., as described in Largaespada et al., J. Immunol. Methods. 197: 85-95, 1996).

[0130] Antibodies can be produced or isolated by screening display libraries, eg, phage display libraries, as described, eg, in US Pat. Nos. 6,300,064 and / or 5,885,793.

[0131] Any antibody used in accordance with the present invention may be a synthetic antibody, for example, the antibody is a chimeric antibody, a humanized antibody, an antibody synthetically humanized with a human antibody, a primatized antibody, or a deimmunized antibody.

[0132] One exemplary agent for detecting a protein of interest is an antibody or fragment thereof capable of specifically binding to plasma MIF, Nt-proBNP (or BNP) or troponin. The antibody may be detectably labeled, either directly or indirectly.

[0133] Anti-MIF antibodies are commercially available from suppliers such as Abcam and include: chicken polyclonal anti-MIF antibody (ab34644); goat polyclonal anti-MIF antibody (ab36146, ab14574); rabbit polyclonal anti-MIF (C-terminus) antibody (ab65869); rabbit polyclonal anti-MIF antibody (ab86670); mouse polyclonal anti-MIF antibody (ab55445); and mouse anti-MIF monoclonal antibody [2Ar3] (ab14575).

[0134] Troponin and anti-hsTnT antibodies are commercially available from suppliers such as Roche (07007302190) and Abcam (ab47003 or EP1106Y). An approach for measuring hs-TnT involves fragment-antigen binding of two hs-TnT-specific monoclonal antibodies, detectable in a sandwich format. The antibodies recognize epitopes corresponding to amino acids 125-131 and 135-147 of hs-TnT. Detection can be achieved by chemiluminescence using Tris(bipyrididol)-ruthenium(II).

[0135] Anti-Nt-proBNP (or BNP) antibodies are available from commercial suppliers, including Abcam (15F11 or 5B6) or ThrmoFischer Scientific (MA1-20631). Polyclonal antibodies bind to epitopes on residues 1-21 and 29-50, and their expression can be detected through means routine in the art, including labeling with biotin followed by ruthenium. The complex binds to Nt-proBNP, which is detected by streptavidin-labeled microparticles.

[0136] Immunoassays for plasma MIF, Nt-proBNP (or BNP), or troponin involve incubating a sample with a detectably labeled antibody or antibody fragment capable of specifically binding plasma MIF, Nt-proBNP (or BNP), or troponin, and detecting the bound antibody by any of a number of techniques well known in the art. As described in more detail below, the term "labeled" refers to direct labeling of an antibody, for example, by coupling (i.e., physically binding) a detectable substance to the antibody, and can also refer to indirect labeling of an antibody through reactivity with another directly labeled reagent. An example of indirect labeling is the detection of a primary antibody using a fluorescently labeled secondary antibody.

[0137] The sample can be brought into contact with or immobilized on a solid support or carrier or other solid support capable of immobilizing soluble proteins. The support can then be washed with an appropriate buffer and subsequently treated with a detectably labeled antibody. The solid support can then be washed a second time with buffer to remove unbound antibody. The amount of label bound to the solid support can then be detected by conventional methods.

[0138] By "solid support or carrier" is intended any support capable of binding an antigen or antibody. Well-known supports or carriers include nitrocellulose, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, and magnetite. The nature of the solid support or carrier can be either soluble to some extent or insoluble.

[0139] A solid support can have virtually any possible structural configuration so long as the molecule attached to it is capable of binding to an antigen or antibody. Thus, the shape of the support can be spherical, such as a bead, or cylindrical, such as the inside surface of a test tube, or the exterior surface of a rod. Alternatively, the surface can be flat, such as a sheet, test strip, etc. Those of skill in the art will know many other suitable carriers for binding antibodies or antigens, or will be able to ascertain such using routine experimentation.

[0140] One way to directly label an antibody specific for plasma MIF, Nt-proBNP (or BNP), or troponin is to link the antibody to an enzyme for enzyme immunoassay. The enzyme bound to the antibody will react with an appropriate substrate, preferably a chromatographic support, in a manner that produces a chemical moiety that can be detected, for example, by spectroscopic, fluorometric, or visual means. Enzymes that can be used to detectably label an antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, Δ5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Detection and measurement can be achieved by colorimetric methods using chromogenic substrates for the enzymes. Detection and measurement can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

[0141] Detection and measurement can also be performed using any of a variety of other immunoassays. For example, by radioactively labeling an antibody or functional antibody fragment, it is possible to detect plasma levels of the biomarker using a radioimmunoassay (RIA). Radioisotopes (e.g., 125 I, 131 I,35 S, 32 P or 3 H) can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

[0142] Antibodies can also be labeled with fluorescent or chemiluminescent compounds. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected due to fluorescence. The most commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluosescamine.

[0143] The antibody is 152 Detectable labeling can also be achieved using fluorescent-emitting metals, such as Eu or others of the lanthanide series. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Fluorescent energy transfer compounds can also be used.

[0144] An antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged (tagged) antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, luminescent acridinium esters, imidazole, acridinium salts, and oxalate esters. Similarly, a bioluminescent compound can be used to label an antibody. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of the bioluminescent protein is detected by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase, and aequorin.

[0145] In another embodiment, specific binding molecules other than antibodies, such as aptamers, may be used to bind plasma MIF, Nt-proBNP (or BNP) or troponin.

[0146] "Means for measuring" plasma MIF, Nt-proBNP (or BNP) or troponin include proteomic approaches using chromatography or electrophoresis with dye-based detection, spectroscopic methods such as mass spectrometry.

[0147] Spectroscopic methods can be used to measure dye-based assays that include visible dyes, fluorescent or luminescent reagents.

[0148] Protein chip assays can also be used to measure plasma MIF, Nt-proBNP (or BNP) or troponin.

[0149] Plasma MIF, Nt-proBNP (or BNP), or troponin can also be measured or assayed using one or more of the following methods, including, for example, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, such as electrospray ionization mass spectrometry (ESl-MS), Esl-MS / MS, Esl-MS / (MS)n (where n is an integer greater than zero), matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time of flight mass spectrometry (SELDl-TOF-MS), desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS) quadrupole time of flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS). Other mass spectrometry methods include quadrupole Fourier transform mass spectrometry (FTMS) and ion trap mass spectrometry. Other suitable methods include chemical extraction partitioning, column chromatography, ion exchange chromatography, hydrophobic (reverse phase) liquid chromatography, isoelectric focusing, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) or other chromatography such as thin layer, gas or liquid chromatography, or a combination thereof.

[0150] In one embodiment, LDI-TOF-MS allows for the generation of a large amount of information in a relatively short time. A biological sample is applied to one of several types of supports that bind MIF, BNP, or troponin in the sample. The sample is applied to these surfaces in a volume as small as 0.5 μL, with or without prior purification or fractionation. The sample can be concentrated or diluted before application to the support. Using laser desorption / ionization, a mass spectrum of the sample can be generated in as little as 3 hours.

[0151] Bead assays can also be used to measure plasma MIF, Nt-proBNP (or BNP) or troponin concentrations.

[0152] As used herein, a "device" refers to the physical arrangement of components for performing an assay to measure plasma MIF, Nt-proBNP (or BNP), or troponin. The device can be a point-of-care device used by a healthcare professional to measure plasma MIF, Nt-proBNP (or BNP), or troponin without the need for laboratory measurements. Alternatively, a point-of-care device may be used in the home, for example, in subjects at risk for a first or subsequent coronary event. Alternatively, the device may be in a laboratory located remotely from the subject in whom plasma MIF, Nt-proBNP (or BNP), or troponin is to be measured.

[0153] The device may use an electrochemical cell. The electrochemical cell may use electrodes placed within the cell in a side-by-side or "coplanar" configuration to minimize electrical interference between the electrodes. Alternatively, non-coplanar electrodes may be used that take advantage of electrical interference between the electrodes to provide additional information about the sample. Such information includes information that can correct for patient-to-patient variations in hematocrit and for interfering chemicals that may be present in the sample.

[0154] The device can provide a quantitative output (e.g., Yes / No, present / absent, high / low), a numerical or numeric output (e.g., concentration), or an output for visual inspection (e.g., color for comparison to a reference scale).

[0155] As used herein, "kit" refers to a physical arrangement of components, one of which can be a device for measuring plasma MIF, Nt-proBNP (or BNP), or troponin. The kit can include reagents such as anti-MIF, anti-Nt-proBNP (or BNP), or anti-troponin immunogenic components, secondary detection reagents for detecting the immunogenic components, or reagents for sample preparation and / or processing, such as buffers. The kit can also include means, such as reagents, for performing a highly sensitive assay, such as for detecting hs-TnT.

[0156] The device or kit may be accompanied by instructions or directions for using the device or kit in any of the methods described herein.

[0157] As used herein, a device or kit may be in any form. One form specifies either suitability for or limitation to a particular use and is indicated by the phrase "for." Another form is limited to only a particular use and is indicated by the phrase "for use" or similar phrases. In one embodiment of a method for treating ACS in a subject, plasma MIF, Nt-proBNP (or BNP), or troponin is measured using a device disclosed herein.

[0158] Survival analysis can be performed using the Kaplan-Meier method (described in the Examples herein and shown in Figures 3 and 5). The Kaplan-Meier method estimates a survival function (cumulative survival rate) from lifespan data. In medical research, it can be used to measure the fraction of patients who survive a certain amount of time after treatment. A Kaplan-Meier plot of the survival function is a series of horizontal step plots of decreasing magnitude, which, given a sufficiently large sample size, approximates the true survival function for the population. The value of the survival function between successive independently sampled observations ("clicks") is assumed to be constant.

[0159] A key advantage of the Kaplan-Meier curve is that it can account for data loss that is "censored" from the sample before the final outcome is observed (e.g., when a patient drops out of the study). On the plot, a small vertical tick mark indicates loss when patient data are censored. If no rejection or censoring occurs, the Kaplan-Meier curve is equivalent to the empirical distribution.

[0160] In statistics, the log-rank test (also known as the Mantel-Cox test) is a hypothesis test for comparing the survival distributions of two patient groups. It is a nonparametric test and is suitable for use when data are right-censored. It is widely used in clinical trials to validate the efficacy of new drugs compared to a control group when the measured value is time to an event. The log-rank test statistic compares the predicted values ​​of the hazard functions of two groups at each observed event time. It is constructed by computing the number of observed and expected events in one group at each observed event time and then adding them to obtain an overall summary across all time points where an event exists. The log-rank statistic can be derived as a score test of the Cox proportional hazards model comparing two groups. Therefore, it is asymptotically equivalent to the likelihood ratio test statistic based on that model.

[0161] It will be understood that the invention disclosed and defined herein extends to any and all alternative combinations of two or more of the individual features mentioned or apparent in the text or drawings, all of which various combinations constitute various alternative embodiments of the invention.

[0162] These examples are intended to demonstrate these and other aspects of the present invention. While the examples describe some embodiments of the present invention, it will be understood that the examples do not limit the embodiments thereto. Various changes can be made, various equivalents can be substituted, and various modifications can be implemented without departing from the spirit and / or theory of the present invention as described above. All such changes, equivalents, and modifications are intended to be within the scope of the claims set forth herein. [Example]

[0163] Example 1 This study was conducted to determine whether a single measurement of MIF on admission, alone or in combination with BNP and / or troponin, can provide predictive information for long-term survival and non-fatal cardiovascular events in patients with STEMI. method

[0164] Study population and study design We consecutively recruited patients with STEMI who underwent PCI treatment at the Department of Cardiology, Peking University Third Hospital, between June 2010 and April 2015. Patient inclusion criteria were as follows: (1) presentation of STEMI (typical symptoms for >30 minutes and <12 hours plus persistent ST-segment elevation of ≥2 mV in at least two consecutive precordial ECG leads or persistent ST-segment elevation of ≥1 mV in at least two consecutive limb ECG leads, or new-onset left bundle branch block); (2) invasive treatment with PCI; (3) availability of MIF measurement from a blood sample at admission. Patients with one or more of the following criteria were excluded: (1) previous ACS within 1 month; (2) emergency angioplasty; (3) current infectious disease, known malignancy, inflammatory, or autoimmune disease; (4) end-stage renal disease (estimated glomerular filtration rate <30 mL / min / kg); and (5) unwillingness to undergo surgery. The recruitment process and study protocol are depicted in Figure 1 .

[0165] Basic clinical data, including medical and medication history, were collected from medical records. Hypertension was diagnosed as a diastolic blood pressure of ≥140 mmHg and / or a systolic blood pressure of ≥90 mmHg on at least two separate occasions, in the presence of active treatment with antihypertensive drugs or by other means. Hypercholesterolemia was diagnosed as a total cholesterol level of ≥6.22 mmol / L or a low-density lipoprotein cholesterol level of ≥4.14 mmol / L, in the presence of active treatment with lipid-lowering drugs. Current smokers were defined as those who currently smoke any cigarettes. Diabetes was confirmed by active treatment with antidiabetic drugs or by a fasting plasma glucose level of ≥7 mmol / L or a non-fasting plasma glucose level of ≥11.1 mmol / L. Patients were prospectively classified according to the Maximum Killip classification system by three clinicians at or during hospitalization. This prospective cohort study was approved by the Ethics Review Board of the Peking University Health Science Center and conducted in accordance with the requirements of the Declaration of Helsinki. Informed consent was obtained from all participants.

[0166] PPCI and drug therapy Coronary angiography was performed after a loading dose of 300 mg aspirin and 600 mg clopidogrel. Quantitative coronary angiography was performed before and after intervention. Culprit lesions, the number of significantly narrowed vessels, and TIMI reclassification before and after PCI were recorded. Interventions were performed according to the latest guidelines. Thrombus aspiration, use of glycoprotein IIb / IIIa inhibitors (tirofiban), and intra-arterial balloon pump (IABP) implantation were performed at the operator's discretion. Two blinded observers were included in the calculation of ST-segment resolution (ST-level elevation / depression) according to predefined criteria 60 minutes after revascularization. A cutoff value of <50% was defined as incomplete ST-segment resolution.

[0167] After PCI, patients were prescribed enoxaparin sodium (100 U / kg / q12h for 3 days) and received other secondary prevention therapies such as aspirin (100 mg / day), clopidogrel (75 mg / day for 12 months), cholesterol-lowering medications (statins), beta-receptor blockers, and angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (ACEI / ARB). All patients received standard and personalized medical care and management at the discretion of their cardiologist.

[0168] Study endpoints and tracking The short-term outcome of this study was incomplete ST-segment resolution (ST-level elevation / depression) after primary PCI as a surrogate for inefficient myocardial reperfusion. Long-term follow-up was performed by reviewing medical records and by individual telephone contact with patients or their relatives. Information was collected on the occurrence of major adverse cardiac events (MACE), consisting of cardiovascular death (CVD), all-cause mortality, recurrent MI, and readmission due to heart failure (HF). The long-term outcome was a composite of all-cause mortality and MACE. Recurrent MI was defined according to the universal definition proposed in 2012. HF readmission was defined as readmission to the hospital with HF as the primary reason.

[0169] Echocardiography Echocardiography was performed using a Vivid 7 (Vingmed, GE, Horten, Norway) with a 3.3-MHz multiphasic array transducer on day 3 post-MI and at approximately 12-month follow-up. Standard echocardiograms were obtained under the supervision of an experienced cardiologist. Left ventricular diastolic dimensions and ejection fraction (LVEF) were obtained using a modified biplane Simpson technique.

[0170] Routine laboratory measurements Venous blood samples were collected for CK-MB and Hs-TnT assays at admission and every 6 hours for the first 2 days. Peak concentrations were identified to estimate occlusion size. Because the prognostic value of post-MI day 3 was superior to other times during the acute phase, median Nt-proBNP and hs-CRP concentrations were measured at this time point.

[0171] All routine biochemistry assays were performed immediately after blood sample collection using commercially available automated platforms. CK-MB, hs-CRP, blood lipids, and plasma creatine concentrations were analyzed using an AU5400 automated chemistry analyzer (Beckman Coulter, California, USA). Hs-TnT and NT-pro-BNP were measured using an E601 immunoassay analyzer (Roche Diagnostics, Mannheim, Germany). Estimated glomerular filtration rate (eGFR) was calculated according to the Cockcroft-Gault equation. All test results were obtained at the Department of Clinical Biochemistry, Peking University Third Hospital, based on manufacturer recommendations or literature.

[0172] Measurement of plasma MIF concentration Immediately after admission, before antiplatelet medication and primary PCI procedures, blood samples were collected by venipuncture into collection tubes containing lithium heparin. Plasma was isolated from whole blood by centrifugation at 3000 rpm for 10 minutes at 4°C, then aliquoted and stored at -80°C until analysis. Repeated freeze-thaw cycles were avoided. Plasma MIF was measured in duplicate using the Quantikine MIF ELISA kit (DMF00B, R&D Systems) according to the manufacturer's specifications. The inter- and intra-assay coefficients of variation were 2.8 ± 1.6% and 5.8 ± 1.3%, respectively. For comparison, MIF levels were also measured in healthy subjects (n = 65) and in patients admitted to the emergency department with chest pain not due to cardiac ischemia (n = 600). All assays were performed by personnel blinded to patient identity and outcomes.

[0173] statistical analysis Aortas were primarily analyzed by identifying the three (1st to 3rd) tertiles of initial MIF measurement. Categorical variables were summarized as percentages (%) and chi-squared (χ 2 Tertiles of MIF were compared between groups using the variance test. Continuous variables are presented as mean ± SD or median and interquartile range (IQR), and their association with tertiles of MIF was tested by one-way ANOVA or Kruskal-Wallis rank-sum test. Associations between MIF concentrations and other continuous variables (e.g., biomarkers, LVEF) were tested by Spearman's rank-order correlation. Due to non-normal distributions, all biomarkers were log- or log2-transformed before entering the statistical model. The primary endpoint (complete ST resolution) was analyzed using a logistic regression model.

[0174] Kaplan-Meier curves were constructed to visualize the correlation between tertiles of MIF concentration and long-term prognosis. Univariate and multivariate analyses were performed using Cox proportional hazards models. Four models were used to adjust for covariates: Model 1, adjusted for age, sex, eGFR, and log2MIF; Model 2, adjusted for all factors in Model 1 plus body mass index (BMI), hemoglobin, previous MI, diabetes, hypertension, current smoking, hypocholesterolemia, time from onset to admission <6 h, 3-vessel disease, Killip classification >1, culprit left anterior descending artery (LAD), ST-segment resolution (ST-level elevation / depression), thrombus aspiration, glycoprotein IIb / IIIa inhibitors during PCI, and TIMI reclassification before and after PCI; Model 3, adjusted for all factors in Model 2 plus traditional biomarkers including hs-TnT peak, Nt-proBNP, and hs-CRP; and Model 4, adjusted for all factors in Model 3 plus LVEF on day 3.

[0175] Patients were separately defined as those with each biomarker in the upper tertile as the positive group (+), whereas those with each biomarker in the middle and lower tertiles as the negative group (-). The prognostic value of different combinations, including Nt-proBNP / MIF, hs-TnT peak / MIF, Nt-proBNP / hs-TnT peak, and triple groups, was investigated. Discrimination was assessed using the C statistic. Sequential net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were also calculated to quantify the reclassification accuracy as a result of adding admission MIF to the clinical risk model. All probability values ​​were two-sided, and a value of <0.05 was considered statistically significant. C-statistics, NRI, and IDI were implemented using the “surviC1” and “survIDINRI” packages ( R Development Core Team, 2016 ) in the R programming language 3.4.0 for Windows, and other data analyses were implemented using SPRR (version 22.0; SPSS, Inc. Chicago, IL).

[0176] result Clinical characteristics A total of 658 patients with a confirmed diagnosis of STEMI were initially recruited into this prospective study. Of these, 56 patients were excluded based on the exclusion criteria, another 36 patients were lost to follow-up (n = 25), and eight patients had unavailable blood samples or missing MIF measurements on admission (n = 8), resulting in a final study cohort of 421 patients (Figure 1). The median age of patients was 61 years, and 79.9% were male. The median MIF level on admission (interquartile range) was 55.1 (35.3-83.6) ng / mL, significantly higher than the other two reference groups: healthy controls (16.9 (12.8-22.9) ng / mL) and patients with chest pain not of ischemic etiology who presented to the emergency department (26.8 (21.7-34.6) ng / mL) (Figure 6).

[0177] The characteristics of this patient cohort are summarized according to MIF tertiles in Tables 1 and 2. MIF levels were not associated with age, sex, or eGFR, BMI, diastolic blood pressure, or heart rate. STEMI patients in the high MIF tertile group tended to have a higher prevalence of hypertension (P = 0.029) and were more likely to have culprit vessel disease in the LAD (P = 0.001). Other conditions regarding pre-existing risk factors for coronary heart disease and CAG results were similar in the three groups (Table 1). No significant differences were observed between the three groups in the use of secondary prevention treatment with aspirin, clopidogrel, statins, ACEIs or ARBs, and β-blockers at admission (not shown) or discharge (Table 1).

[0178] A modest but highly statistically significant correlation was observed between the concentrations of MIF and necrosis markers at admission, and peak levels of hsTnT (r=0.486, P<0.001) and CK-MB (r=0.343, P<0.001). Meanwhile, MIF levels were also associated with inflammatory markers such as white blood cell count (r=0.210, P<0.001), non-fasting glucose level at first presentation (r=0.126, P<0.001), and CRP on day 3 (r=0.154, P<0.001), but not with hemoglobin, serum cholesterol, or HbA1c%.

[0179] [Table 1]

[0180] [Table 2]

[0181] Data are given as mean ± SD, percentage, or median (25th percentile; 75th percentile). Categorical variables are shown as percentage (%) of patients. eGFR: estimated glomerular filtration rate. LAD = left anterior descending artery; IABP = intra-aortic balloon pump; LDL = low-density lipoprotein; LVEF = left ventricular ejection fraction; LVEDD = left ventricular end-diastolic diameter; PCI = percutaneous coronary intervention. P values ​​are based on the Mann-Whitney U statistical method, one-way ANOVA test, or chi-square (χ ) for comparisons between MIF tertile groups. 2 ) test.

[0182] [Table 3]

[0183] Data are either mean ± SD, percentage, or median (25th percentile; 75th percentile). NT-proBNP indicates the N-terminal prohormone of brain natriuretic peptide; LDL-c = low-density lipoprotein cholesterol; HDL-c = high-density lipoprotein cholesterol; CK-MB = creatine kinase MB fraction; CRP = C-reactive protein; hs-TnT = high-sensitivity troponin T. P values ​​were derived from the Mann-Whitney U test or one-way ANOVA test for comparisons between MIF tertile groups.

[0184] MIF and cardiac function at admission during the acute and 12-month periods Patients in the higher tertile of admission MIF had a higher proportion of highest Killip class >1 during hospitalization compared with patients in the lower tertile (23.4% vs 11.1%, p=0.006).Admission MIF levels were associated with elevated Nt-proBNP levels (r=0.190, P<0.001), reduced LVEF [r=-0.298, 95% CI (-0.382,-0.215), P<0.001], and enlarged LVDD (r=0.115, P<0.006) on day 3 post-MI. Repeat echocardiography was performed at 12 months during the follow-up period, and MIF was strongly correlated with F12 LVEF [r=-0.474, 95% CI (-0.550,-0.384), P<0.001] and LVDD (r=-0.261, P<0.001, n=414). Importantly, after calculating the change in LVEF, our data revealed that higher tertiles of MIF were associated with a lack of improvement in LVEF% (P<0.001) at 12 months post-MI compared with day 3 values ​​(Figure 2).

[0185] MIF and incomplete ST-segment deviation In the subgroup of patients with MIF in the upper tertile, the incidence of ST resolution <50% at 60 minutes after PCI was 3.3-fold and 1.9-fold higher, respectively, than in the lower and middle tertiles (p<0.001, Table 1). In contrast, admission hs-TnT and CK-MB were not associated with incomplete ST resolution (P=0.263 and P=0.486, respectively). In multivariate logistic regression analysis, when continuous variables were log-2 transformed, admission MIF was an independent predictor of incomplete ST-segment elevation resolution with an odds ratio (OR) of 1.75 (95% CI 1.35-2.18; P<0.001) for twice the MIF concentration after adjusting for age, sex, eGFR, time from symptom onset to admission <6 hours, location of obstruction, history of diabetes, current smoking, and WBC level at initial presentation. Another remaining significant predictor was anterior infarct location, with an OR of 2.02 (95% CI 1.35-3.01; P=0.001).

[0186] MIF and long-term adverse outcomes A total of 160 patients developed MACE during a median follow-up period of 64 months (ranging from 0.03 to 83 months). Among them, 62 patients died, 46 of which died of cardiovascular causes. 56 patients were re-hospitalized due to HF, and 42 experienced recurrent MI. MIF levels at admission were found to be closely associated with long-term adverse outcomes. As shown in Figure 3, Kaplan-Meier survival curves and rank correlation analysis demonstrated the distribution of all-cause mortality, cardiovascular death, HF-rehospitalization, and MACE according to MIF tertiles (all P<0.001). To further explore the independence of MIF in prognostic prediction, we applied univariate and multivariate Cox regression analyses using different models (Table 3). In all four clinical risk models tested, including clinical characteristics and existing biomarkers such as Nt-proBNP, peak hs-TnT, hs-CRP, and day 3 LVEF, MIF remained an independent predictor of all-cause mortality, cardiovascular death, and MACE.

[0187] [Table 4]

[0188] Model 1: adjusted for age, sex, eGFR and log2MIF; Model 2: In addition to Model 1, adjustments were made for body mass index (BMI), hemoglobin, previous MI, diabetes, hypertension, current smoking, hypocholesterolemia, time from onset to admission <6 h, 3-vessel disease, Killip class >1, culprit lesion in the LAD, ST-segment resolution (ST level elevation / depression), use of glycoprotein IIb / IIIa inhibitors during PCI, and TIMI reclassification before and after PCI. Model 3: Model 2 plus adjustment for log Nt-proBNP, log TnT peak, and logh s-CRP; Model 4: In addition to Model 3, adjusted for LVEF.

[0189] We used a clinical risk model consisting of age, sex, eGFR, hemoglobin, previous MI, diabetes, hypertension, current smoking, time from symptom onset to admission <6 h, culprit LAD lesion, 3-vessel disease, Killip class >1, culprit LAD lesion, ST-segment resolution (ST-level elevation / depression), TIMI class before and after PCI, hs-TnT peak, and LVEF on day 3. Our data showed that including MIF significantly improved the predictive ability estimated by the C-statistic for all-cause mortality [0.84 (0.77-0.90) vs. 0.89 (0.83-0.94), P = 0.020 0.006] and MACE [0.72 (0.67-0.77) vs. 0.74 (0.70-0.79), P = 0.047]. Meanwhile, we calculated how many patients were reclassified after adding continuous log2MIF using a continuous NRI of 0.34 (95% CI: 0.04-0.47) for all-cause mortality and 0.24 (95% CI: 0.11-0.34) for MACE (Table 4). The calculated IDIs showed similar improvements of 0.06 (95% CI: 0.00-0.144) for all-cause mortality and 0.05 (95% CI: 0.01-0.09) for MACE (Table 4).

[0190] [Table 5]

[0191] Clinical model included age, sex, eGFR, BMI, hemoglobin, previous MI, diabetes, hypertension, current smoking, hypocholesterolemia, time from onset to admission <6 h, 3-vessel disease, Killip class >1, culprit lesion in the LAD, ST-segment resolution (ST-level elevation / depression), use of glycoprotein IIb / IIIa inhibitor (tirofiban) during PCI, Timi class before and after PPCI, hs-TnT peak, and LVEF. NIR = Net Reclassification Index; IDI = Integrated Discrimination Improvement.

[0192] Combined prognostic value of MIF and Nt-proBNP The prognostic ability of MIF compared with peak hs-TnT, CRP, and Nt-proB was compared using C-statistics. MIF (C-statistic: 0.71, 95% CI: 0.64-0.78) was found to provide better prognostic information than peak hs-TnT (C-statistic: 0.63, 95% CI: 0.56-0.71, P<0.03) and hs-CRP (C-statistic: 0.53, 95% CI: 0.62-0.75, P<0.001), but was comparable to Nt-proBNP (C-statistic: 0.70, 95% CI: 0.62-0.75, P=0.33) for all-cause mortality. Cox regression analysis revealed that after adjustment for model 3 (including gold-standard biomarkers such as MIF and Nt-proBNP, peak hs-TnT, and hs-CRP), only admission MIF and Nt-proBNP were independent predictors of adverse outcomes in STEMI patients. However, after adjustment for model 4, in which day 3 LVEF was added, Nt-proBNP remained a statistically significant factor only for cardiovascular death (Table 5).

[0193] [Table 6]

[0194] Model 1: adjusted for age, sex, eGFR and log Nt-proBNP; Model 2: In addition to Model 1, adjustment was made for body mass index (BMI), hemoglobin, previous MI, diabetes, hypertension, current smoking, hypocholesterolemia, time from onset to admission <6h, 3-vessel disease, Killip class >1, culprit lesion in the LAD, ST-segment resolution (ST level elevation / depression), use of glycoprotein IIb / IIIa inhibitor (tirofiban) during PCI, and TIMI classification before and after PCI; Model 3: Model 2 plus adjustment for log Nt-proBNP, log TnT peak, and log hs-CRP; Model 4: In addition to Model 3, adjusted for LVEF.

[0195] To investigate the additive prognostic value of the combination of MIF and Nt-proBNP, we risk-stratified STEMI patients for the endpoints according to tertiles of MIF and Nt-proBNP levels. The risk of all-cause mortality (26% vs. 0.0%, P<0.001) and MACE (57.1% vs. 7.4%, P<0.001; Figure 4) was significantly increased in patients with both biomarkers in the higher tertile compared with their counterparts with both biomarkers in the lower tertile. To further explore the prognostic value of different combinations, STEMI patients were divided into two subgroups: those with each biomarker in the higher tertile (positive group) and those with each biomarker in the middle or lower tertile (negative group).

[0196] Compared with patients in the double-negative group, patients in the Nt-proBNP(+) MIF(+) group had an 11-fold increased risk of all-cause mortality [hazard ratio (HR) 11.28; 95% CI: 4.82-26.94; P<0.001, Figure 5], which was comparable to that of the triple-positive group [HR 11.39; 95% CI: 4.29-29.68; P<0.001]. However, the hazard ratios for patients in the hs-TnT(+) MIF(+) or Nt-proBNP(+) peak hs-TnT(+) groups were 4.12 (95% CI: 2.16-7.85) and 6.60 (95% CI: 3.32-13.10), respectively. Similar results were observed in the Nt-proBNP (+) MIF (+) group regarding the risk of MACE (Figure 5 and Table 6).

[0197] [Table 7]

[0198] Patients were classified separately according to the high tertile of MIF, Nt-proBNP, or hs-TnT peak.

[0199] These findings demonstrated that in STEMI patients, admission MIF has prognostic value for the adverse progression of LV systolic dysfunction, long-term mortality, and MACE, independently of clinically established risk factors, acute LVEF, and routinely measured biomarkers. Additionally, admission MIF, combined with Nt-proBNP and / or hs-TnT, facilitates better prognosis prediction. This study established admission MIF as a useful marker for predicting short- and long-term outcomes in STEMI patients. Thus, admission MIF allows risk stratification of high-risk STEMI patients, who could potentially benefit from more intensive therapy for secondary prevention. These findings support the utility of biomarker-guided management strategies in patients with potentially poor long-term outcomes.

[0200] First, we demonstrated that admission MIF levels are predictors of subsequent changes in necrosis markers (peak CK-MB and peak hs-TnT) and inflammatory parameters (hs-CRP, white blood cell count). Furthermore, admission MIF is an independent risk factor for poor myocardial reperfusion recovery (ST resolution <50% by 60 minutes after PCI). Second, a subgroup with high MIF levels exhibited impaired LVEF recovery up to 12 months after STEMI. Third, multivariate analysis demonstrated that admission MIF, as a continuous variable, remained an independent predictor of long-term all-cause mortality, cardiovascular death, and MACE, even after adjusting for established risk factors and biomarkers. Finally, we demonstrated that the combination of MIF and day 3 Nt-proBNP improved risk stratification for all-cause mortality and MACE in STEMI patients in this study. These findings suggest that admission MIF levels provide useful information in STEMI patients beyond what is currently available from clinical and angiographic features.

[0201] Myocardial ischemia / reperfusion injury remains a common event in STEMI patients. Despite optimal angiographic revascularization, microvascular injury, manifesting as the no-reflow phenomenon, is detected in 30–60% of STEMI patients. We first demonstrated that patients with initial MIF in the higher tertile had a 2.5-fold higher prevalence of incomplete ST resolution (ST-level elevation / depression) compared with those in the lower tertile, and that MIF was an independent predictive risk factor for incomplete ST resolution. This is the first evidence that plasma MIF levels before primary PCI are predictive of reperfusion success and microvascular obstruction. The ability of admission MIF levels to predict reperfusion injury may be partly due to its predictive properties for obstruction size. [1] 1. A method for providing a prognosis for acute coronary syndrome (ACS) in a subject, comprising: measuring the concentration of both (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof in a sample from the subject; and Predict ACS when a subject's plasma MIF and Nt-proBNP concentrations are higher than the reference plasma NIF and Nt-proBNP concentrations The method includes: [2] 1. A method for providing a prognosis for a subject having an acute coronary syndrome (ACS), comprising measuring in a sample from the subject the concentrations of both: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) B-type natriuretic peptide (BNP) or a fragment thereof; comparing the concentration of MIF or a fragment thereof to a reference MIF concentration; comparing the concentration of Nt-proBNP or a fragment thereof to a reference Nt-proBNP concentration; The concentration of each of MIF or a fragment thereof and Nt-proBNP or a fragment thereof compared to the respective reference concentrations is an indicator of the subject's prognosis. The method includes: [3] 1. A method for providing a prognosis for a subject having ACS, comprising measuring in a sample from the subject the concentrations of both: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof; comparing the concentration of MIF or a fragment thereof to a reference MIF concentration; comparing the concentration of Nt-proBNP or a fragment thereof to a reference Nt-proBNP concentration; the reference concentrations of MIF and Nt-proBNP are concentrations below which, at a later time point, are correlated with increased survival and a decreased probability of non-fatal cardiac events, and concentrations above which, at a later time point, are correlated with decreased survival and an increased probability of non-fatal cardiac events; thereby providing a prediction of subjects with ACS The method includes: [4] 1. A method for providing a prognosis for a subject having ACS, comprising measuring in a sample from the subject the concentrations of both: (a) macrophage migration inhibitory factor (MIF) or a fragment thereof; and (b) B-type natriuretic peptide (BNP) or a fragment thereof; comparing the concentration of MIF or a fragment thereof to a reference MIF concentration; comparing the concentration of BNP or a fragment thereof to a reference BNP concentration; assigning the patient to a risk group based on whether the concentration of said MIF or fragment thereof is higher or lower than a reference concentration and whether the concentration of Nt-proBNP or fragment thereof is higher or lower than a reference concentration; wherein a concentration of MIF or a fragment thereof greater than said reference MIF concentration indicates a decreased likelihood of survival and an increased likelihood of a non-fatal cardiac event; wherein a concentration of Nt-proBNP or a fragment thereof higher than said reference Nt-proBNP concentration indicates a lower likelihood of survival and a higher likelihood of a non-fatal cardiac event; thereby providing a prognostic prediction for subjects with ACS. The method includes: [5] 1. A method for providing a prognosis for a subject having an ACS, comprising: determining the concentration of macrophage migration inhibitory factor (MIF) or a fragment thereof in a sample from the subject; wherein if the concentration of MIF or a fragment thereof in the sample from the subject is greater than or equal to about 70 ng / mL, the subject is determined to have a decreased survival rate and an increased probability of a non-fatal cardiac event at a later time point; wherein if the concentration of MIF or a fragment thereof in the sample from the subject is less than about 70 ng / mL, the subject is determined to have an increased survival rate and a decreased probability of a non-fatal cardiac event at a later time point; thereby providing a prognostic prediction for subjects with ACS. The method includes: [6] 1. A method for providing a prognosis for a subject having an ACS, comprising: determining the concentration of macrophage migration inhibitory factor (MIF) or a fragment thereof in a sample from the subject; comparing the concentration of MIF or a fragment thereof to reference MIF concentrations of about 40 ng / mL and about 70 ng / mL; wherein if the concentration of MIF or a fragment thereof in the sample from the subject is about 40 ng / mL or less, the subject is determined to have a high probability of survival and a low probability of a non-fatal cardiac event at a later time point; wherein if the concentration of MIF or a fragment thereof in the sample from the subject is greater than or equal to about 70 ng / mL, the subject is determined to have a reduced survival rate and a high probability of a non-fatal cardiac event at a later time point; thereby providing a prognostic prediction for subjects with ACS. The method includes: [7] 7. The method according to any one of items 1 to 6, further comprising measuring the concentration of troponin or a fragment thereof. [8] 8. The method according to any one of items 1 to 7, wherein the concentration of MIF, Nt-proBNP and / or troponin, or fragments thereof, is determined from plasma. [9] 9. The method according to any one of items 1 to 8, wherein the ACS is acute myocardial infarction (AMI).

[10] 10. The method of item 9, wherein the AMI is ST-elevation myocardial infarction (STEMI).

[11] 11. The method of any one of items 1 to 10, comprising measuring the subject's MIF concentration in a sample taken within 4 hours after the onset of symptoms.

[12] 12. The method of claim 11, wherein the MIF sample is collected within 3 hours, 2 hours, 1 hour, or 30 minutes after the onset of symptoms.

[13] 13. The method according to any one of items 1 to 12, wherein the concentration of brain natriuretic peptide (BNP) is measured instead of the concentration of the N-terminal prohormone of brain natriuretic peptide (Nt-proBNP).

[14] 14. The method according to any one of items 7 to 13, wherein the troponin is high-sensitivity troponin T (hs-TnT).

[15] 15. The method of any one of items 1 to 14, wherein the prognosis is determined by assessing survival excluding MACE, survival excluding all-cause mortality, survival excluding cardiac death, or survival excluding HF readmissions.

[16] 15. The method of any one of items 1 to 14, wherein the non-fatal cardiac event is a MACE.

[17] 15. The method of any one of items 1 to 14, wherein the non-fatal cardiac event is an impairment of myocardial reperfusion recovery.

[18] 15. The method of any one of items 1 to 14, wherein the non-fatal cardiac event is impairment of LVEF improvement.

[19] 19. The method according to any one of items 1 to 18, wherein Nt-proBNP (or BNP) and MIF are measured in the same sample.

[20] 20. The method of any one of items 1 to 19, further comprising performing percutaneous coronary intervention (PCI) and / or serolysis on the subject. [twenty one] 1. A method of treating acute coronary syndrome (ACS) in a subject, comprising: (a) measuring the concentrations of both macrophage migration inhibitory factor (MIF) or a fragment thereof and N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof in a sample obtained from the subject, and prognosing ACS if the subject's MIF and Nt-proBNP concentrations are greater than reference MIF and Nt-proBNP concentrations; and (b) performing percutaneous coronary intervention (PCI) and / or thrombolysis on said subject. [twenty two] 22. A device for use in the method according to any one of items 1 to 21, comprising means for measuring the concentrations of both macrophage migration inhibitory factor (MIF) or a fragment thereof and the N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) in a sample from a subject. [twenty three] 23. The device according to item 22, comprising means for performing immunoassays for measuring the concentrations of both MIF and Nt-proBNP (or BNP). [twenty four] 24. The device of any one of items 22 or 23, wherein the device is a point-of-care enabled device. [twenty five] 25. The method of item 21 or the device of any one of items 22 to 24, further comprising measuring the concentration of troponin or a fragment thereof.

[26] 26. The method or device according to item 25, wherein the troponin is high-sensitivity troponin T (hs-TnT).

[27] if the concentration of troponin in the sample from the subject is greater than or equal to about 4.5 ng / mL, the subject is determined to have a decreased survival rate and an increased probability of a non-fatal cardiac event at a later time point; wherein if the concentration of troponin in the sample from the subject is less than about 4.5 ng / mL, the subject is determined to have an increased survival rate and a decreased probability of a non-fatal cardiac event at a later time point; thereby providing a prognostic prediction for subjects with ACS. 21. The method according to any one of items 7 to 20, comprising:

[28] 27. The method or device according to any one of items 22 to 26, wherein a BNP concentration is measured instead of the concentration of N-terminal prohormone of brain natriuretic peptide (Nt-proBNP), or the device comprises means for measuring a BNP concentration.

[29] determining that the subject has a decreased survival rate and an increased probability of a non-fatal cardiac event at a later time point if the concentration of Nt-proBNP (or BNP) in the sample from the subject is greater than or equal to about 1200 pg / mL; determining that the subject has an increased survival rate and a decreased probability of a non-fatal cardiac event at a later time point if the concentration of Nt-proBNP (or BNP) in the sample from the subject is less than about 1200 pg / mL; thereby providing a prognostic prediction for subjects with ACS. 22. The method according to any one of items 1 to 21, comprising:

[30] 30. The method or device according to any one of items 21 to 29, wherein the concentrations of MIF, Nt-proBNP (or BNP) and / or troponin are measured from plasma.

[31] A kit comprising reagents for measuring the concentrations of both macrophage migration inhibitory factor (MIF) and Nt-proBNP (or BNP) in a sample from a subject, for use in a method for prognosing ACS in a subject, the method comprising measuring concentrations of both MIF and Nt-proBNP (or BNP) in a sample, and predicting ACS if the subject's MIF and Nt-proBNP (or BNP) concentrations are greater than reference MIF and Nt-proBNP (or BNP) concentrations; and / or The device according to any one of items 22 to 24 is included. A kit characterized by:

[32] 32. The kit of item 31, further comprising measuring the concentration of troponin or a fragment thereof.

[33] 33. The kit according to item 31 or 32, wherein the reagent comprises an anti-MIF antibody, an anti-Nt-proBNP (or BNP) antibody and / or an anti-troponin antibody.

[34] 34. The kit of item 32 or 33, wherein the troponin is high-sensitivity troponin T (hs-TnT).

[35] 35. The kit according to any one of items 31 to 34, wherein instead of the N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) concentration, a BNP concentration is measured, or the kit comprises a reagent for measuring BNP.

[36] A cardiac biomarker panel comprising plasma macrophage migration inhibitory factor (MIF) and the N-terminal prohormone of brain natriuretic peptide, Nt-proBNP, in a sample from a subject, wherein MIF and Nt-proBNP concentrations greater than reference MIF and Nt-proBNP concentrations are prognostic of the extent of ACS in the subject.

[37] 37. The cardiac biomarker panel of item 36, further comprising troponin in a sample from the subject, wherein MIF, Nt-proBNP, and troponin concentrations greater than reference MIF, Nt-proBNP, and troponin concentrations are prognostic factors for the severity of ACS in the subject.

[38] 38. The cardiac biomarker panel of item 36 or 37, wherein the ACS is AMI.

[39] 39. The cardiac biomarker panel according to any one of items 36 to 38, wherein the AMI is ST-elevation myocardial infarction (STEMI).

[40] 31. The method of any one of items 1 to 21 or items 25 to 30, wherein the BNP concentration is measured in plasma obtained from a blood sample obtained from the patient on day 3 after the onset of symptoms.

[41] 41. The method of any one of paragraphs 1 to 21, 25 to 30 or 40, further comprising measuring the concentration of another biomarker selected from the group consisting of myoglobin, creatine kinase (CK) or C-reactive protein (CRP).

[42] 7. The method according to item 5 or 6, wherein a MIF level higher than about 70 ng / mL is indicative of a 5-year MACE prognostic rate of about 35% and a mortality prognostic rate of about 20%.

[43] 30. The method according to item 29, wherein a MIF level higher than about 70 ng / mL and a Nt-proBNP level higher than about 1200 pg / mL are indicators of a 5-year MACE prognosis predictive rate of about 50% and a mortality prognosis predictive rate of about 25%.

[44] 44. The method according to item 43, wherein a troponin level higher than about 4.5 ng / mL is an indicator of a 5-year MACE prognosis predictive rate of about 35 to 40% and a mortality prognosis predictive rate of about 20%.

[45] Item 45. The method according to item 44, wherein the MACE prognostic prediction rate is 40%.

[46] if the concentration of MIF or a fragment thereof in a sample from said subject is greater than or equal to about 73 ng / mL, then said subject is determined to have a decreased survival rate and an increased probability of a non-fatal cardiac event at a later time point; and / or If the concentration of MIF or a fragment thereof in the sample from the subject is less than about 73 ng / mL, the subject is determined to have an increased survival rate and a decreased probability of a non-fatal cardiac event at a later time point. 7. The method according to any one of items 5 or 6.

[47] 31. The method according to item 8 or 30, wherein the method comprises measuring the concentration of MIF, Nt-proBNP (or BNP) and / or troponin in plasma previously obtained from the subject.

[48] 31. The method of item 8 or 30, wherein the plasma sample is an in vitro sample of plasma.

[49] 1. A method of treating acute coronary syndrome (ACS) in a subject, comprising: providing an individual determined to have a low likelihood of survival and / or a high likelihood of a non-fatal cardiac event according to any one of items 3 to 6; The subject undergoes percutaneous coronary intervention (PCI) and / or thrombolysis; thereby treating the subject for ACS. The method includes:

[50] 10. Use of a thrombolytic agent in the manufacture of a medicament for treating ACS in a subject who has been determined to have a low likelihood of survival and / or a high likelihood of a non-fatal cardiac event according to any one of items 3 to 6.

[51] A thrombolytic agent for use in the treatment of ACS in subjects who have been determined to have a low likelihood of survival and / or a high likelihood of non-fatal cardiac events according to items 3-6.

[52] 46. ​​Use of a means for detecting MIF, Nt-proBNP and troponin in the manufacture of a reagent or kit for or when used in prognosing ACS according to any one of items 1 to 20, 27, 29 or 42 to 45.

[53] 53. The method of any one of items 1 to 4, 7 to 21, 27, 29, 30, 40 to 41, 43 to 45, 47 to 52, wherein the MIF, Nt-proBNP and / or troponin reference concentrations are determined from reference MIF, Nt-proBNP and / or troponin concentrations of a sample obtained from at least one individual previously identified as suffering from ACS.

Claims

1. 1. A method for providing an index for predicting the prognosis of a subject with ACS, comprising the steps of: determining the concentration of macrophage migration inhibitory factor (MIF) or a fragment thereof in a sample from a subject, wherein the sample is selected from blood, plasma, or serum; comparing the concentration of MIF or a fragment thereof with reference MIF concentrations of 40 ng / ml and 73 ng / ml; Including, an indication is determined that the subject has a higher probability of survival and a lower probability of a non-fatal cardiac event at a later time point if the concentration of MIF or a fragment thereof from the sample from the subject is less than or equal to 40 ng / ml; determining an indication that the subject has a reduced probability of survival and a higher probability of a non-fatal cardiac event at a later time point if the concentration of MIF or a fragment thereof from the sample from the subject is equal to or greater than 73 ng / ml; Thereby providing a prognosis for a subject with ACS.

2. determining the concentration of N-terminal prohormone of brain natriuretic peptide (Nt-proBNP) or a fragment thereof, or brain natriuretic peptide (BNP) or a fragment thereof in a sample from the subject, wherein the sample is selected from blood, plasma, or serum; determining an index indicating that if a concentration of NT-proBNP or BNP from a sample from the subject is equal to or greater than 700 pg / ml, the subject will have a decreased probability of survival and an increased probability of a non-fatal cardiac event at a later time point; 2. The method of claim 1, wherein an indication is determined that the subject has an increased probability of survival and a decreased probability of a non-fatal cardiac event at a later time if the concentration of Nt-proBNP or BNP from a sample from the subject is lower than 700 pg / ml.

3. The method of claim 2, wherein Nt-proBNP or BNP and MIF are measured in the same sample.

4. 4. The method of claim 2 or 3, wherein the concentration of BNP is measured in plasma from a blood sample obtained from the subject three days after the onset of symptoms.

5. The method of any one of claims 2 to 4, further comprising measuring the concentration of troponin or a fragment thereof.

6. The method of claim 5, wherein the troponin is high-sensitivity troponin T (hs-TnT).

7. determining an indication that if a concentration of troponin (or hs-TnT) from a sample from the subject is equal to or greater than 4.5 ng / ml, the subject will have a decreased probability of survival and an increased probability of a non-fatal cardiac event at a later time point; 7. The method of claim 5 or 6, wherein an indication is determined that indicates that the subject has an increased probability of survival and a decreased probability of a non-fatal cardiac event at a later time point if the concentration of troponin (or hs-TnT) from a sample from the subject is lower than 4.5 ng / ml.

8. The method according to any one of claims 5 to 7, wherein the concentration of MIF, Nt-proBNP or BNP and / or troponin, or fragments thereof, is determined from plasma.

9. The method of any one of claims 1 to 8, wherein the ACS is acute myocardial infarction (AMI).

10. 10. The method of claim 9, wherein the AMI is ST-elevation myocardial infarction (STEMI).

11. 11. The method of any one of claims 1 to 10, comprising determining the subject's MIF concentration in a sample taken less than 4 hours after symptom onset.

12. 12. The method of claim 11, wherein the sample taken less than 4 hours after symptom onset is taken 3 hours or less, 2 hours or less, 1 hour or less, or 30 minutes or less after symptom onset.

13. The method of any one of claims 1 to 12, wherein the prognosis is determined by assessing MACE-free survival, all-cause mortality-free survival, cardiac death-free survival or HF-rehospitalization-free survival.

14. The method of any one of claims 1 to 13, wherein the non-fatal cardiac event is MACE.

15. The method of any one of claims 1 to 13, wherein the non-fatal cardiac event is impaired recovery of myocardial reperfusion.

16. The method of any one of claims 1 to 13, wherein the non-fatal cardiac event is failure to improve LVEF.

17. The method of any one of claims 1 to 16, wherein an MIF level above 73 ng / ml indicates a 5-year MACE prognosis of 35% and a mortality prognosis of 20%.

18. 18. The method of any one of claims 1 to 17, further comprising determining the concentration of another biomarker selected from the group consisting of myoglobin, creatine kinase (CK) or C-reactive protein (CRP).

19. 19. A method of providing an indication for percutaneous coronary intervention (PCI) and / or thrombolytic therapy for use in treating acute coronary syndrome (ACS) in a subject, wherein the subject has been identified as having or at risk of having ACS by the method of any one of claims 1 to 18.

20. 1. A device comprising: means for determining the concentration of macrophage migration inhibitory factor (MIF) or a fragment thereof in a sample from a subject, wherein the sample is selected from blood, plasma, or serum; A means for determining a prognostic indicator for subjects with acute coronary syndrome (ACS) wherein an indication is determined that if a concentration of MIF or a fragment thereof from a sample from the subject is equal to or greater than 73 ng / ml, the subject will have a decreased probability of survival and an increased probability of a non-fatal cardiac event at a later time point; The device determines an indication that if the concentration of MIF or a fragment thereof from a sample from the subject is equal to or lower than 40 ng / ml, the subject has an increased probability of survival and a decreased probability of a non-fatal cardiac event at a later time point, thereby providing a prognosis for the subject with ACS.

21. 21. The device of claim 20, wherein the means for determining the concentration of MIF or a fragment thereof is a means for performing an immunoassay.

22. 22. The device of claim 20 or 21, wherein the device is a point-of-care device.

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