Combination of troponin markers for early discrimination of type 2 versus type 1 acute myocardial infarction

Abstract

The present invention relates to a method for assessing myocardial infarction comprising the steps of: determining the amount of a first biomarker in a sample of a subject, wherein said first biomarker is a cardiac troponin; determining the amount of a second biomarker in a sample of a subject, wherein said second biomarker is selected from the group consisting of BMP10 type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP type peptide, cardiac myosin binding protein C (cMyBPC) and ANG2 (angiopoietin 2); comparing the amount of the biomarker with a standard for said biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker; and assessing the subject based on the comparison and / or calculation. The present invention also relates to the use of a first biomarker, which is a cardiac troponin, and a second biomarker selected from the group consisting of BMP10 type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP type peptide, cardiac myosin binding protein C (cMyBPC) and ANG2 (angiopoietin 2), or at least one detection agent for said first biomarker and at least one detection agent for said second biomarker, for assessing myocardial infarction. Furthermore, the present invention further relates to a computer-implemented method for assessing myocardial infarction, as well as a device and a kit for assessing myocardial infarction.

Description

[Technical field]

[0001] The present invention relates to a method for assessing myocardial infarction comprising the steps of: determining the amount of a first biomarker in a sample from a subject, wherein the first biomarker is a cardiac troponin; determining the amount of a second biomarker in the sample from the subject, wherein the second biomarker is selected from the group consisting of BMP10 type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP type peptide, cardiac myosin binding protein C (cMyBPC) and ANG2 (angiopoietin 2); comparing the amount of the biomarker with a standard for the biomarker and / or calculating a score for assessing myocardial infarction based on the amount of the biomarker; and assessing the subject based on the comparison and / or calculation. The present invention also relates to the use of a first biomarker which is a cardiac troponin and a second biomarker selected from the group consisting of BMP10 type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP type peptide, cMyBPC and ANG2 (angiopoietin 2), or at least one detection agent for said first biomarker and at least one detection agent for said second biomarker for assessing myocardial infarction. Furthermore, the present invention further relates to a computer-implemented method for assessing myocardial infarction, as well as a device and a kit for assessing myocardial infarction. [Background technology]

[0002] The aim of modern medicine is to provide personalized or individualized treatment regimens. These are treatment regimens that take into account the individual needs or risks of the patient. In cases where a possible treatment regimen needs to be determined within a short period of time, personalized or individualized treatment regimens shall also be taken into account. In such situations, the measurement of circulating biomarkers by blood tests can help to diagnose disease states (such as cardiomyocyte injury by troponin tests), mechanistically understand disease risk (e.g., elevated lipids and atherosclerosis), identify biological pathways involved (e.g., protective effect of BMP10 in atherosclerotic plaques; Upton PD et al., J Cell Sci 2020;133:jcs239715;doi:10.1242 / jcs.239715), and improve therapeutic outcomes.

[0003] In patients with suspected AMI (acute myocardial infarction), diagnostic workup requires admission to the ED, registration of a 12-lead electrocardiogram (ECG), blood tests to diagnose or exclude myocardial injury, evaluation of clinical signs and medical history, physical examination, and other diagnostic tests to diagnose AMI or differential diagnosis.

[0004] According to the Universal Definition of AMI (Thygesen K et al., Circulation 2018;138:e618-e651.doi:10.1161 / CIR.0000000000000617.), five different types of AMI are defined based on different mechanistic pathways underlying AMI, with type 1 and type 2 myocardial infarction being described in more detail below. According to the guidelines, types 3 to 5 of AMI are clinically well distinguished by the skilled artisan for three specific clinical situations, such as type 3 (defined as "death before it is possible to obtain blood for cardiac biomarker measurement, or the patient may die shortly after the onset of symptoms before an elevation in biomarker values ​​occurs"), type 4 (PCI and stent-associated AMI) and type 5 (CABG-associated AMI). Thus, the present invention is aimed at the most common situations of diagnosing suspected AMI (types 1 and 2), the distinction (type 1 vs. type 2) being highly relevant for the choice of treatment:

[0005] Type 1 myocardial infarction (T1MI): Coronary atherothrombosis caused by plaque rupture and plaque erosion in one or more coronary arteries and / or distal embolism leads to intraluminal thrombosis and subsequent reduction in myocardial perfusion and necrosis.

[0006] Type 2 myocardial infarction (T2MI): secondary to an acute imbalance in myocardial oxygen supply and demand. Decreased myocardial perfusion can result from stable coronary atherosclerosis (without plaque rupture), coronary spasm, microvascular dysfunction, coronary embolism or dissection, as well as impaired systemic hemodynamics, including hypotension, hypertension, tachycardia, or hypoxemia (DeFilippis et al, Circulation 2019:140;1661-1678).

[0007] Type 2 MI tends to be more frequent in women, as it is often associated with the presence of other underlying comorbidities (Thygesen et al,Eur Heart J 2018:40(3);246-247), and has been described as exhibiting higher short-term and long-term mortality than type 1 MI. The 5-year survival rate of patients with type 2 MI has been described as low, at 40% or less (McCarthy et al,JAMA 2018:320(5);433).

[0008] Treatment of type 1 and type 2 myocardial infarction is substantially different because the myocardial damage in type 2 MI is not due to atherosclerosis and unstable coronary artery disease (CAD), so treatment should address the root cause and underlying cause (e.g., oxygen therapy in case of hypoxemia or volume replacement in case of hypotension). This is in contrast to type 1 myocardial infarction, which requires immediate invasive procedures or fibrinolysis depending on the extent of infarction and the availability of percutaneous coronary intervention (PCI). In case of type 2 MI, coronary artery examination may be useful to determine whether underlying CAD is present (Thygesen et al,Eur Heart J 2018:40(3);246-247). Default treatment of type 2 MI with specific therapy for type 1 MI is currently not supported by clinical evidence and may even have an unfavorable impact on outcome (Collinson et al,ACC May 18,2016,Diagnosing Type 2 Myocardial Infarction), thus making this distinction even more important. Troponin peak levels tend to be higher in T1 vs. T2 MI, but discriminatory power in clinical practice remains inadequate (Smilowitz et al,Coronary Artery Disease 2018;29(1);46-52). Thus, there is an important unmet need to identify and differentiate patients with type 2 vs. type 1 MI (Nagele 2020 Circulation.2020;141:1431-1433.DOI:10.1161 / CIRCULATIONAHA.119.044996).

[0009] Myocardial necrosis biomarkers, including troponin, were observed at different concentrations in patients with type 1 and type 2 AMI (see, e.g., Nestelberger et al., 2021 JAMA Cardiol. doi:10.1001 / jamacardio.2021.0669).

[0010] However, although troponin elevation is specific for myocardial injury, not all troponin elevations are MI, and additional information is still needed to enable reliable and early assessment of myocardial infarction (Collet JP et al, Eur Heart J 2021;42:1289-1367. doi:10.1093 / eurheartj / ehaa575) and in particular to better distinguish type 1 from type 2 MI. Summary of the Invention

[0011] Thus, the present invention provides means and methods that meet these needs.

[0012] Advantageously, in the study underlying the present invention, it was found that a combination of a first biomarker with a second biomarker, preferably a third biomarker, allows a reliable early assessment of myocardial infarction. In the study, patients presenting to the emergency department were investigated. For this purpose, patients with confirmed myocardial infarction were subdivided into those suffering from type 1 and type 2 myocardial infarction. The amounts of the various biomarkers were determined, and the biomarkers were analyzed and mathematically combined via logistic regression analysis. The area under the receiver operating characteristic curve (AUC) was used to evaluate biomarker performance. The AUC value is the mathematical integral of the function f(x) within the interval [a][b]. The AUC was also examined for biomarker pairs and triplets. Combined, biomarker combinations were identified that showed an improved AUC over the best single biomarker AUC. The results are described in the attached examples below.

[0013] In particular, when a patient suspected of having a myocardial infarction is in, for example, an emergency unit, early assessment of the patient dictates the initiation of therapeutic measures, including drug administration, physical or other therapeutic interventions. In particular, it is important to distinguish between type 1 and type 2 myocardial infarction, since their treatment differs significantly (see above). Thanks to the present invention, the identification of AMI patients can be assessed by early biomarker determination, which is important for preventing myocardial damage (Collet JP et al, Eur Heart J 2021;42:1289-1367.doi:10.1093 / eurheartj / ehaa575), thus preventing life-threatening developments. The biomarker pairs and triplets identified in the research underlying the present invention are a reliable basis for medical decisions, and the assessment can be performed in a time- and cost-effective manner.

[0014] The present invention provides a method for assessing myocardial infarction in a subject, comprising: (a) determining an amount of a first biomarker in a sample from the subject, wherein the first biomarker is a cardiac troponin, such as cardiac troponin T or I; (b) determining the amount of a second biomarker in the subject's sample, wherein the second biomarker is a BMP10-type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), a BNP-type peptide, such as NT-proBNP or BNP, cardiac myosin-binding protein C (cMyBPC) or ANG2 (angiopoietin-2); (c) comparing the amount of said biomarker with a standard for said biomarker and / or calculating a score for assessing myocardial infarction based on the amount of said biomarker; (d) assessing myocardial infarction based on the comparison and / or calculation performed in step (c).

[0015] In one embodiment of the method of the present invention, the method further comprises determining the amount of a third biomarker. In particular, in step (b) of the method of the present invention, (i) if the amount of a BMP10-type peptide is determined as the second biomarker, the method may further comprise determining the amount of cMyBPC (cardiac myosin binding protein C), at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6, as a third biomarker, or (ii) if the amount of FGF23 is determined as the second biomarker, the method may further comprise determining the amount of at least one lipid biomarker, such as cMyBPC, CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7, as a third biomarker; or (iii) if the amount of a BNP-type peptide is determined as the second biomarker, the method may further comprise determining the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2, as a third biomarker; or (iv) If the amount of ANG2 is determined as the second biomarker, the method may further comprise determining the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7, as a third biomarker.

[0016] Accordingly, the present invention provides a method for assessing myocardial infarction in a subject, comprising: (a) determining the amount of a first biomarker in the subject's sample, wherein the first biomarker is a cardiac troponin; (b) determining the amount of said second and third biomarkers in said sample of said subject; (c) comparing the amount of said biomarker with a standard for said biomarker and / or calculating a score for assessing myocardial infarction based on the amount of said biomarker; (d) assessing myocardial infarction based on the comparison and / or calculation performed in step (c). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] It should be understood that as used in the specification and claims, "a" or "an" can mean one or more, depending on the context in which it is used. Thus, for example, reference to "an" item can mean that at least one item can be utilized.

[0018] As used below, the terms "have", "comprise" or "include" or any grammatical variants thereof are used in a non-exclusive manner. Thus, these terms may refer both to the situation where no further features are present in the entity described in this context, in addition to the features introduced by these terms, and to the situation where one or more additional features are present. As an example, the expressions "A has B", "A comprises B" and "A includes B" may both refer to the situation where no other elements are present in A, other than B (i.e., A consists solely and exclusively of B), and to the situation where, other than B, one or more further elements are present in the entity A, such as element C, elements C and D, and even further elements. The term "comprising" has a restrictive meaning in the sense of "consisting of", also encompassing embodiments where only the mentioned items are present.

[0019] Furthermore, when used below, the terms "particularly", "more particularly", "typically" and "more typically" or similar terms are used in conjunction with additional / alternative features without limiting the possibilities of substitution. Thus, features introduced by these terms are additional / alternative features and are not intended to limit the scope of the claims in any way. The invention may be implemented by using alternative features, as would be understood by a person skilled in the art. Similarly, features introduced by "in one embodiment of the invention" or similar expressions are intended to be additional / alternative features, without any limitations on alternative aspects of the invention, without any limitations on the scope of the invention, and without any limitations on the possibilities of combining the features so introduced with other additional / alternative or non-additional / alternative features of the invention.

[0020] Furthermore, as used herein, the term "at least one" means that one or more of the items followed by this term can be used according to the present invention. For example, when this term indicates that at least one sampling unit should be used, this can be understood as one sampling unit or two or more sampling units, i.e., two, three, four, five or any other number. Depending on the item to which this term refers, those skilled in the art will understand which upper limit, if any, this term can refer to.

[0021] As used herein, the term "about" means that for any number listed after the term, there is an interval of precision within which the technical effect can be achieved. Thus, "about" as referred to herein preferably refers to the exact numerical value or a range around that exact numerical value of ±20%, preferably ±15%, more preferably ±10%, or even more preferably ±5%.

[0022] Additionally, the terms "first," "second," "third," etc. in the specification and claims are used to distinguish between similar elements and not necessarily to describe a chronological or chronological order. For example, the amount of a second biomarker may be determined before the amount of a first biomarker.

[0023] The method of the present invention may consist of the above steps or may include additional steps, such as a step for further evaluation of the assessment obtained in step (d), a step for recommending or initiating a therapeutic measure, such as a treatment. Furthermore, the method of the present invention may include steps prior to step (a), such as a step relating to pretreatment of the sample. However, it is preferably envisaged that the above-mentioned method is an ex vivo method that does not require any steps to be performed on the human or animal body. Thus, the method is an in vitro method. Furthermore, the method may be assisted by automation. Typically, the determination of biomarkers may be assisted by a robotic device, and the comparison and assessment may be assisted by a data processing device, such as a computer.

[0024] According to the present invention, myocardial infarction shall be assessed. The term "myocardial infarction" is well known in the art. As used herein, the term refers to acute myocardial infarction (abbreviated as "AMI"). Clinical diagnostic criteria for acute myocardial infarction indicate the presence of acute myocardial damage (i.e., cardiac troponin (cTn) values ​​above the 99th percentile upper reference limit (URL), including rising and / or falling cTn values) in the context of evidence of acute myocardial ischemia. Evidence of acute myocardial ischemia includes clinical signs and symptoms, ischemic ECG changes, occurrence of pathological Q waves, and imaging evidence of new loss of viable myocardium or new regional wall motion abnormalities in a pattern consistent with an ischemic etiology (Thygesen et al, Eur J Heart 2019:40(3);237-269, DOI:10.1093 / eurheartj / ehy462).

[0025] In one embodiment of the present invention, the term "assessing myocardial infarction" relates to distinguishing between type 1 and type 2 myocardial infarction. Thus, it is determined whether a subject suffers from type 1 or type 2 myocardial infarction.

[0026] Thus, the present invention provides a method for distinguishing between type 1 and type 2 myocardial infarction, comprising: (a) determining the amount of a first biomarker in the subject's sample, wherein the first biomarker is a cardiac troponin; (b) determining the amount of a second biomarker and optionally a third biomarker in the sample of said subject; (c) comparing the amount of said biomarker to a standard for said biomarker and / or calculating a score for distinguishing between type 1 and type 2 myocardial infarction based on the amount of said biomarker; (d) distinguishing between type 1 and type 2 myocardial infarction based on the comparison and / or calculation performed in step (c).

[0027] The term "type 1 myocardial infarction" is well known in the art. As used herein, this term refers to AMI with an underlying ischemic etiology of atherosclerotic plaque rupture / erosion with occlusive or non-occlusive thrombus formation in one or more coronary arteries. Thus, the diagnostic criteria for type 1 AMI include the aforementioned criteria for AMI, including, for example, the identification of coronary thrombus by angiography, including intracoronary imaging or autopsy, as evidence of acute myocardial ischemia (Thygesen et al, Eur J Heart 2019:40(3);237-269, DOI:10.1093 / eurheartj / ehy462).

[0028] The term "type 2 myocardial infarction" is well known in the art. As used herein, the term refers to AMI with an underlying ischemic etiology due to an oxygen supply-demand balance disorder resulting from a) increased oxygen demand (e.g., sustained tachyarrhythmia or severe hypertension) or b) decreased oxygen supply (e.g., hypotension, bradyarrhythmia, respiratory failure, severe anemia, coronary vasospasm, coronary artery dissection or microvascular dysfunction). Thus, the diagnostic criteria for type 2 AMI include the aforementioned diagnostic criteria for AMI, including evidence of an imbalance between myocardial oxygen supply and demand unrelated to acute coronary atherothrombosis (Thygesen et al, Eur J Heart 2019:40(3);237-269, DOI:10.1093 / eurheartj / ehy462).

[0029] In another embodiment of the invention, the term "assessing myocardial infarction" relates to the diagnosis of type 2 myocardial infarction.

[0030] Thus, the present invention provides a method for diagnosing type 2 myocardial infarction, comprising: (a) determining the amount of a first biomarker in the subject's sample, wherein the first biomarker is a cardiac troponin; (b) determining the amount of a second biomarker and optionally a third biomarker in the sample of said subject; (c) comparing the amount of said biomarker with a standard for said biomarker and / or calculating a score for diagnosing type 2 myocardial infarction based on the amount of said biomarker; (d) diagnosing type 2 myocardial infarction based on the comparison and / or calculation performed in step (c).

[0031] As used herein, the term "diagnosing" means assessing whether a subject referred to in accordance with the method of the present invention is suffering from type 2 myocardial infarction or not.

[0032] In another embodiment of the present invention, the term "assess myocardial infarction" preferably relates to the assessment of the treatment for myocardial infarction or the treatment of myocardial infarction in the affected subject.According to the present invention, treatment decision can be made and subject can be treated accordingly.For example, it can be determined whether patient undergoes treatment aimed at treating type 2 myocardial infarction or treatment aimed at treating type 1 myocardial infarction.

[0033] As used herein, the term "diagnosing" means assessing whether a subject referred to in accordance with the method of the present invention is suffering from type 2 myocardial infarction or not.

[0034] As understood by those skilled in the art, the assessment such as discrimination or diagnosis made according to the present invention is preferably correct for 100% of the subjects examined, but may not be 100% correct. This term typically requires that the statistically significant portion of the subjects can be accurately assessed. Whether a portion is statistically significant can be further easily determined by those skilled in the art using various well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley&Sons, New York 1983. Typically, the assumed confidence interval is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is typically 0.2, 0.1, 0.05.

[0035] According to the present invention, the assessment of myocardial infarction is typically understood as an aid in the assessment of myocardial infarction, for example, as an aid in distinguishing between type 2 and type 1 myocardial infarction, or as an aid in diagnosing type 2 myocardial infarction.The method and use of the present invention can therefore be part of a complete assessment.A complete assessment can include the assessment of further markers, clinical parameters and / or ECG (which can, for example, contribute to the score).The final assessment (discrimination, etc.) is generally performed by a physician.

[0036] As used herein, the term "subject" refers to an animal, preferably a mammal, more typically a human. The subject investigated by the method of the present invention is suspected to have myocardial infarction or has myocardial infarction. Preferably, the subject has myocardial infarction, such as non-ST elevation myocardial infarction (NSTEMI). In one embodiment, the subject is male. In another embodiment, the subject is female.

[0037] However, the subject to be tested is preferably not suffering from type 3, type 4 or type 5 myocardial infarction. An overview of these types of AMI is given in Thygesen K et al.Circulation 2018;138:e618-e651.doi:10.1161 / CIR.0000000000000617.), which is incorporated herein by reference. Type 3 (defined as "death before blood can be obtained for cardiac biomarker measurement, or the patient may die shortly after the onset of symptoms before an elevation in biomarker value occurs"), type 4 (PCI and stent-related AMI) and type 5 (CABG-related AMI). Thus, the subject shall suffer from type 1 or type 2 myocardial infarction. Alternatively, the subject shall be suspected of suffering from type 1 or type 2 myocardial infarction.

[0038] In a preferred embodiment, the subject is a diabetic patient, i.e. suffers from diabetes.For example, the subject may suffer from type 1 or type 2 diabetes.Typically, the diabetic subject suffers from type 2 diabetes.Table 5 in the example shows the preferred marker and marker combination for the patient suffering from diabetes.Preferably, the second marker in diabetic patients is ANG2.

[0039] The term "sample" as used herein refers to any sample that contains the first, second and / or third biomarkers referred to herein under physiological conditions. More typically, the sample is a body fluid sample, such as a blood sample or a sample derived therefrom (e.g., serum or plasma), a urine sample, a saliva sample, interstitial fluid, lymph sample, etc. Most typically, the sample is a blood, serum or plasma sample.

[0040] It is further envisaged that the blood sample is a dried blood spot sample. A dried blood spot sample can be obtained by applying a drop of blood to an absorbent filter. The blood is allowed to completely saturate the paper and allowed to air dry for several hours. The blood may have been taken from the subject to be tested, for example from a finger, by a lancet.

[0041] In a preferred embodiment, the sample is a blood (i.e., whole blood), serum or plasma sample. Serum is the liquid fraction of whole blood obtained after the blood is allowed to clot. To obtain serum, the clot is removed by centrifugation and the supernatant is then collected. Plasma is the cell-free fluid portion of blood. To obtain a plasma sample, whole blood is collected in an anticoagulant-treated tube (e.g., a citrate-treated or EDTA-treated tube). Cells are removed from the sample by centrifugation to obtain the supernatant (i.e., the plasma sample).

[0042] Blood samples include capillary blood samples, which may be obtained, for example, from a finger prick.

[0043] Preferably, the subject suffers from myocardial infarction at the time the sample is obtained, although the subject may not have been diagnosed as suffering from myocardial infarction at that time. The diagnosis may be made later (e.g., based on the determination of cardiac troponin in a second sample obtained from the subject 1-3 hours after the first sample). Methods for diagnosing myocardial infarction are well known in the art and are described, for example, in Collet JP et al. (ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. Eur Heart J. 2020 Aug 29: ehaa575. doi: 10.1093 / eurheartj / ehaa575. Epub ahead of print. PMID: 32860058). Currently available protocols for the diagnosis of non-ST elevation ACS combine biomarker concentrations at the time of presentation and at later time points such as 1 hour, 2 hours or 3 hours.

[0044] Advantageously, the method of the present invention allows early assessment of myocardial infarction.The assessment referred to herein can be reliably performed based on the amount of the first, second and third biomarkers in a sample (preferably a single sample) obtained from the subject at the time of presentation.Thus, in one embodiment, the sample is obtained from the subject at the time of presentation in the emergency department.

[0045] According to the present invention, the amounts of at least three biomarkers, namely a first biomarker, a second biomarker, and optionally a third biomarker, shall be determined in a sample (preferably a single sample) from the subject obtained at the time of presentation.

[0046] The first biomarker is cardiac troponin, a well-known marker in the diagnostic field. The term "cardiac troponin" typically refers to human cardiac troponin T or cardiac troponin I.

[0047] In one embodiment, the cardiac troponin is troponin T (measured in the Examples section).

[0048] In an alternative embodiment, the cardiac troponin is cardiac troponin I.

[0049] The second marker is at least one biomarker selected from the group consisting of BMP10 type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), BNP type peptide, such as NT-proBNP or BNP; cardiac myosin-binding protein C (cMyBPC) and ANG2 (angiopoietin-2).Preferred second biomarkers are BMP10 type peptide, FGF23 and ANG2 in order of preference.

[0050] In one embodiment, the second biomarker is a BMP10 type peptide (bone morphogenetic protein type 10 peptide). The BMP10 type peptide is preferably NT-proBMP10, proBMP10 or BMP, more preferably NT-proBMP10 or proBMP10, and most preferably NT-proBMP10.

[0051] BMP10 type peptide is well known in the art.Preferred BMP10 type peptide is disclosed, for example, in Susan-Resiga et al. (J Biol Chem.2011 Jul 1;286(26):22785-94), which is incorporated herein by reference in its entirety (see, for example, Figure 3A of Susan-Resiga et al., or US Patent Application Publication No. 2012 / 0213782).

[0052] In one embodiment, the BMP10-type peptide is unprocessed preproBMP10. In another embodiment, the BMP10-type peptide is the propeptide proBMP10. This marker comprises an N-terminal prosegment and BMP10. In another embodiment, the BMP10-type peptide is the N-terminal prosegment of BMP10 (the N-terminus is proBMP10). In another embodiment, the BMP10-type peptide is BMP10.

[0053] In one embodiment, the BMP10-type peptide is part of a homo- or heterodimeric complex.

[0054] Human preproBMP10 (i.e., unprocessed preproBMP10) has a length of 424 amino acids. The amino acid sequence of human preproBMP10 is shown, for example, in SEQ ID NO: 1 of WO2020 / 035605A1 or in FIG. 3 of US2012 / 0213782, which are incorporated herein by reference in their entirety. SEQ ID NO: 1 of WO2020 / 035605A1 is the same sequence as SEQ ID NO: 2 in the sequence listing of the present application.

[0055] The amino acid sequence of preproBMP10 can also be assessed by Uniprot (see sequence under accession number O95393-1). Human preproBMP10 contains a short signal peptide (amino acids 1-21) that is enzymatically cleaved to release proBMP10. Thus, human proBMP10 contains amino acids 22-424 of human preproBMP10 (i.e., a polypeptide having the sequence shown in SEQ ID NO: 1 of WO 2020 / 035605 A1). Human proBMP10 is further cleaved into an N-terminal prosegment of BMP10 and an active (non-glycosylated) form of BMP10. The N-terminal prosegment of BMP10 contains amino acids 22-316 of a polypeptide having the sequence shown in SEQ ID NO: 1 of WO 2020 / 035605 A1 (i.e., human preproBMP10). BMP10 comprises amino acids 317 to 424 of a polypeptide having the sequence set forth in SEQ ID NO:1 of WO 2020 / 035605 A1.

[0056] The preferred BMP10-type peptides are BMP10 and N-terminal proBMP10. After cleavage of proBMP10, BMP10 and N-terminal proBMP10 remain structurally close to form homodimers or heterodimers of BMP10 or remain combined with other BMP family proteins (Yadin et al., CYTOGFR 2016, 27 (2016) 13-34). Dimerization occurs by the formation of Cys-Cys bridges or strong adhesion in the C-terminal peptides of both binding partners. Thus, a structure consisting of two subunits is formed.

[0057] Since proBMP10 is cleaved into BMP10 and the N-terminal prosegment in equimolar proportions, the amount of BMP10 reflects the amount of the N-terminal prosegment, and therefore the amount of BMP10 can be determined by determining the amount of the N-terminal prosegment, and vice versa.

[0058] Preferably, the amount of BMP10-type peptide is determined using one or more antibodies (or antigen-binding fragments thereof) that specifically bind to the BMP10-type peptide.

[0059] For example, one or more antibodies that specifically bind to the N-terminal prosegment of BMP10 can be used. Since such antibodies (or fragments) also bind to proBMP10 and preproBMP10, the total amount of the N-terminal prosegment of BMP10, proBMP10 and preproBMP10 is determined in step a) of the method of the present invention. Therefore, the expression "determine the amount of the N-terminal prosegment of BMP10" also means "determine the total amount of the N-terminal prosegment of BMP10, proBMP10 and preproBMP10".

[0060] Structural predictions based on knowledge from other BMP-type proteins, such as BMP9, indicate that BMP10 remains in a complex with proBMP10, and therefore detection of the N-terminal prosegment also reflects the amount of BMP10.

[0061] For example, one or more antibodies that specifically bind to BMP10 can be used. Since such antibodies (or fragments) also bind to proBMP10 and preproBMP10, the total amount of BMP10, proBMP10 and preproBMP10 is determined in step a) of the method of the present invention. Therefore, the expression "determine the amount of BMP10" also means "determine the total amount of BMP10, proBMP10 and preproBMP10".

[0062] Structural predictions based on knowledge from other BMP-type proteins, such as BMP9, indicate that BMP10 remains in a complex with proBMP10, and thus detection of BMP10 also reflects the amount of the N-terminal prosegment.

[0063] It is further envisaged to determine the sum of the amounts of all four BMP10 type peptides mentioned above, namely BMP10, the N-terminal prosegment of BMP10, proBMP10 and preproBMP10.

[0064] Thus, according to the invention, the following amounts of BMP10 type peptides can be determined: Amount of BMP10 Amount of N-terminal prosegment of BMP10 Amount of proBMP10 Amount of preproBMP10 The sum of BMP10, proBMP10 and preproBMP10 levels the sum of the amounts of the N-terminal prosegment of BMP10, proBMP10 and preproBMP10, or The sum of the amounts of BMP10, the N-terminal prosegment of BMP10, proBMP10 and preproBMP10

[0065] In one embodiment, the amount of BMP10-type peptide is determined as described in WO 2020 / 035605 A1, which is incorporated herein by reference in its entirety. In another embodiment, the amount of BMP10-type peptide is determined as described in WO 2021 / 165465 A1, which is incorporated herein by reference in its entirety. In the Examples section of this application, the amount of BMP10-type peptide was determined by using an antibody that binds to NT-proBMP10.

[0066] In another embodiment, the second biomarker is FGF23. The biomarker fibroblast growth factor-23 (abbreviated as "FGF-23") is well known in the art. FGF-23 plays an important role in regulating calcium phosphate and vitamin D metabolism, and plays a causal role in the pathogenesis of LV hypertrophy and renal disease, which are major determinants of cardiovascular events. Preferably, the FGF-23 is human FGF-23. The sequence of human FGF-23 is well known in the art, for example, the amino sequence can be assessed by GenBank accession number NM_020638.1 GI:10190673. Furthermore, the sequence is also disclosed in Shimada et al., 2001, PNAS, vol.98(11), pages 6500-6505.

[0067] In another embodiment, the second biomarker is a BNP-type peptide.Preferably, the BNP-type peptide is NT-proBNP, proBNP or BNP, more preferably NT-proBNP or BNP, most preferably NT-proBNP.The BNP-type peptide may be glycosylated (as described elsewhere herein) or non-glycosylated.

[0068] Brain natriuretic peptide-type peptides (also referred to herein as BNP-type peptides) are preferably selected from the group consisting of pre-proBNP, proBNP, NT-proBNP, and BNP. The pre-propeptide (134 amino acids for pre-proBNP) contains a short signal peptide, which is enzymatically cleaved to release the propeptide (108 amino acids for proBNP). The propeptide is further cleaved into the N-terminal propeptide (NT-propeptide, 76 amino acids for NT-proBNP) and the active hormone (32 amino acids for BNP). Preferably, the brain natriuretic peptides according to the invention are NT-proBNP, BNP and variants thereof. BNP is the active hormone and has a shorter half-life than its respective inactive counterpart NT-proBNP. Preferably, the brain natriuretic peptide-type peptide is BNP (brain natriuretic peptide), more preferably NT-proBNP (N-terminal of the prohormone brain natriuretic peptide).

[0069] In one embodiment, the BNP-type peptide is BNP.

[0070] In another embodiment, the BNP-type peptide is NT-proBNP.

[0071] NT-proBNP may be "total NT-proBNP" (also referred to herein as tNT-proBNP) or "non-glycosylated NT-proBNP", e.g. NT-proBNP that is non-glycosylated at position S44.

[0072] The sequence of human NT-proBNP is well known in the art and has already been described in detail in the prior art, for example in WO 02 / 089657, WO 02 / 083913, Bonow 1996, New Insights into the cardiac natriuretic peptides. Circulation 93:1946-1950. Preferably, human NT-proBNP has the amino acid sequence shown in SEQ ID NO:1.

[0073] NT-proBNP as well as its precursor proBNP can be O-glycosylated. Overviews of O-glycosylation of NT-proBNP and proBNP are provided, for example, by Schellenberger et al. and Halflinger et al., both of which are incorporated by reference in their entirety (Schellenberger et al., Arch Biochem Biophys 2006;451; Halfinger et al., Clinical Chemistry 63:1,359-368(2017)).

[0074] The term "O-linked glycosylation" is well known in the art (also referred to herein as "glycosylation"). O-linked glycosylation is the attachment of a sugar molecule, i.e., a carbohydrate, to a serine or threonine residue. It is a post-translational modification that occurs after a protein is synthesized. How NT-proBNP and proBNP are glycosylated is described, for example, in Schellenberger et al. and Halflinger et al. (cited above).

[0075] An overview regarding the O-glycosylation sites of NT-proBNP is provided herein below. The sequence below is the human NT-proBNP sequence (here serving as the reference sequence SEQ ID NO: 1). The O-glycosylation sites are underlined. [ka]

[0076] Thus, human NT-proBNP (as shown in SEQ ID NO:1) contains at least the following glycosylation sites: T36, S37, S44, T48, S53, T58 and T71.

[0077] Preferably, as used herein, the term "non-glycosylated NT-proBNP" refers to NT-proBNP that is not glycosylated (i.e., not O-glycosylated) at one or more positions selected from the group consisting of T36, S37, S44, T48, S53, T58 and T71 of human NT-proBNP. Thus, at least one of the following amino acid residues of human NT-proBNP is not glycosylated, i.e., does not contain O-glycosylation: T36, S37, S44, T48, S53, T58 and T71.

[0078] In some embodiments, the term "unglycosylated NT-proBNP" refers to NTproBNP in which at least two of the aforementioned amino acid residues (i.e., T36, S37, S44, T48, S53, T58 and T71) are unglycosylated. In some embodiments, the term "unglycosylated NT-proBNP" refers to NTproBNP in which at least three of the aforementioned amino acid residues are unglycosylated. In some embodiments, the term "unglycosylated NT-proBNP" refers to NTproBNP in which at least three, at least four, at least five, at least six, or all of the aforementioned amino acid residues are unglycosylated. In some embodiments, the term "unglycosylated NT-proBNP" refers to NTproBNP in which at least the serine residue at position 44 (i.e., S44) is unglycosylated. Thus, unglycosylated NT-proBNP may be unglycosylated at position S44 (i.e., Ser44).

[0079] In a preferred embodiment, determining the amount of unglycosylated NT-proBNP in a sample from a subject comprises contacting the sample with an antibody or antigen-binding fragment thereof that specifically detects unglycosylated NT-proBNP. Preferably, the antibody or antigen-binding fragment thereof that specifically detects unglycosylated NT-proBNP specifically binds to an epitope of NT-proBNP, the epitope including the glycosylation site, but not glycosylated at the glycosylation site. The complex formed between the antibody (or fragment) and the biomarker shall be proportional to the amount of unglycosylated NT-proBNP.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof that specifically detects non-glycosylated NT-proBNP specifically binds to an epitope of NT-proBNP, said epitope comprising the T36 amino acid residue, said T36 amino acid residue being unglycosylated. Preferably, said antibody or fragment does not essentially bind to NT-proBNP comprising a glycosylated T36 amino acid residue.

[0081] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, which epitope comprises the S37 amino acid residue, and said S37 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP that comprises a glycosylated S37 amino acid residue.

[0082] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, the epitope comprising the S44 amino acid residue, and the S44 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP comprising a glycosylated S44 amino acid residue. In some embodiments, the epitope of the antibody or antigen-binding fragment thereof comprises amino acid residues 42-46 of human NT-proBNP (as set forth in SEQ ID NO:1).

[0083] In a preferred embodiment, the antibody that specifically detects non-glycosylated NT-proBNP is the monoclonal antibody MAB 1.21.3 disclosed in WO2004099253A1, or an antibody comprising the six CDRs of said antibody. Furthermore, it is envisaged to use an antigen-binding fragment of said antibody.

[0084] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, which epitope comprises the T48 amino acid residue, and the T48 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP that comprises the glycosylated T48 amino acid residue.

[0085] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, which epitope comprises the S53 amino acid residue, and said S53 amino acid residue is unglycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP that comprises a glycosylated S53 amino acid residue.

[0086] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, which epitope comprises the T58 amino acid residue, and the T58 amino acid residue is not glycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP that comprises a glycosylated T58 amino acid residue.

[0087] In some embodiments, the antibody or fragment thereof specifically binds to an epitope of NT-proBNP, which epitope comprises the T71 amino acid residue, and said T71 amino acid residue is unglycosylated. Preferably, the antibody or fragment does not essentially bind to NT-proBNP that comprises a glycosylated T71 amino acid residue.

[0088] Preferably, as used herein, the term "glycosylated NT-proBNP" refers to NT-proBNP that is glycosylated (i.e., O-glycosylated), preferably at one or more positions (i.e., amino acid residues) selected from the group consisting of T36, S37, S44, T48, S53, T58 and T71 of human NT-proBNP. Thus, at least one of the following amino acid residues of human NT-proBNP is glycosylated, i.e., includes O-glycosylation: T36, S37, S44, T48, S53, T58, and T71.

[0089] In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least two of the aforementioned amino acid residues (i.e., T36, S37, S44, T48, S53, T58 and T71) are glycosylated. In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least three of the aforementioned amino acid residues are glycosylated. In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least three, at least four, at least five, at least six, or all of the aforementioned amino acid residues are glycosylated. In some embodiments, the term "glycosylated NT-proBNP" refers to NTproBNP in which at least the serine residue at position 44 (i.e., S44) is glycosylated.

[0090] "Total amount of NT-proBNP" is preferably the amount of glycosylated and non-glycosylated NT-proBNP. Thus, the term refers to the sum of the amount of glycosylated NT-proBNP and non-glycosylated NT-proBNP.

[0091] Preferably, determining the amount of total NT-proBNP comprises contacting the sample with an antibody or antigen-binding fragment thereof that specifically detects total NT-proBNP. More preferably, said antibody or antigen-binding fragment thereof that specifically detects total NT-proBNP binds to a region of human NT-proBNP that cannot be glycosylated. Thus, said antibody (or fragment thereof) shall specifically bind to a region of NT-proBNP, in particular a region of human NT-proBNP, that does not carry a glycosylation site, i.e. an O-glycosylation site. The complex formed between the antibody (or fragment) and the biomarker shall be proportional to the amount of total NT-proBNP.

[0092] In particular, the antibody (or fragment thereof) shall specifically bind to a region of NT-proBNP that does not carry the T36, S37, S44, T48, S53, T58 or T71 glycosylation sites (of human NT-proBNP). For example, the first 35 amino acid residues, i.e., the N-terminal amino acid residues 1 to 35, are known to have no O-glycosylation sites. Preferably, the antibody or antigen-binding fragment thereof that specifically detects total NT-proBNP binds to an epitope present within the first 35 amino acids of NT-proBNP, more preferably, to an epitope present within the first 20 amino acids of NT-proBNP, and most preferably, to an epitope present within amino acid residues 10 to 20 of human NT-proBNP. In a preferred embodiment, the epitope of the antibody or antigen-binding fragment thereof comprises amino acid residues 13 to 16 of human NT-proBNP. The sequence of human NT-proBNP is shown above (see SEQ ID NO: 1).

[0093] In a preferred embodiment, the antibody that specifically detects total NT-proBNP is the monoclonal antibody MAB 17.3.1 disclosed in WO2004099253A1, or an antibody comprising the six CDRs of said antibody.Furthermore, it is envisaged to use an antigen-binding fragment of said antibody.

[0094] In another embodiment, the second biomarker is ANG2. The biomarker angiopoietin-2 (abbreviated as "ANG2" and often referred to as ANGPT2) is well known in the art. It is an antagonist of both naturally occurring Ang-1 and TIE2 (see, for example, Maisonpierre et al., Science 277 (1997) 55-60). The protein can induce tyrosine phosphorylation of TEK / TIE2 in the absence of ANG-1. In the absence of angiogenic inducers such as VEGF, ANG2-mediated loosening of cell-matrix contacts can induce endothelial cell apoptosis with resulting vascular regression. In cooperation with VEGF, it can promote endothelial cell migration and proliferation, thus functioning as a permissive angiogenic signal. The sequences of human angiopoietins are well known in the art. Uniprot lists three isoforms of angiopoietin-2: isoform 1 (Uniprot identifier: O15123-1), isoform 2 (identifier: O15123-2) and isoform 3 (O15123-3). In a preferred embodiment, the total amount of angiopoietin-2 is determined. This total amount is preferably the sum of the amount of complexed and free angiopoietin-2.

[0095] In another embodiment, the second biomarker is cMyBPC (cardiac myosin binding protein C). Myosin binding protein C is a myosin-associated protein found in the cross-linked region (C region) of the A-band of striated muscle.

[0096] According to the invention, the amount of the cardiac isoform, namely cMyBPC (cardiac myosin-binding protein C), shall be determined (also called MYBPC3, CMD1MM, CMH4, FHC, LVNC10, MYBP-C, myosin-binding protein C, cardiac, cMyBP-C, myosin-binding protein C3). cMyBPC is produced in cardiac muscle. It is encoded by the MYBPC3 gene. Further information on human cMyBPC can be found in the UniProtKB database under the accession number Q14896 (MYPC3_HUMAN).

[0097] As noted above, the amount of a third biomarker can be determined in accordance with the present invention.

[0098] For example, when the amount of BMP10-type peptide is determined as the second biomarker, the method may further comprise, in one embodiment, determining the amount of cMyBPC (cardiac myosin binding protein C), at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6, as a third biomarker.Preferred third biomarkers are cMyBPC, at least one lipid biomarker and hsCRP.

[0099] When the amount of FGF23 is determined as the second biomarker, the method may in one embodiment further comprise determining the amount of at least one lipid biomarker, such as cMyBPC, CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7, as a third biomarker. Preferred third biomarkers are cMyBPC and at least one lipid biomarker.

[0100] When the amount of a BNP-type peptide is determined as the second biomarker, the method further comprises determining the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2, as a third biomarker.

[0101] When the amount of ANG2 is determined as the second biomarker, the method may in one embodiment comprise determining the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7, a third biomarker, particularly cMyBPC, LDL or CHOL. Alternatively, the third biomarker may be IGFBP7, apolipoprotein A-1, or LDL. The use of IGFBP7, APOAT or LDL, particularly LDL, as the third biomarker is advantageous in diabetic patients, for example in combination with cardiac troponin, such as cardiac troponin T and ANG2.

[0102] The term "growth differentiation factor-15" or "GDF-15" refers to a polypeptide that is a member of the transforming growth factor (TGF)-cytokine superfamily. The terms polypeptide, peptide and protein are used interchangeably throughout this specification. GDF-15 was initially cloned as macrophage inhibitory cytokine 1, and was subsequently identified as placental transforming growth factor-15, placental bone morphogenetic protein, nonsteroidal anti-inflammatory drug-activated gene 1, and prostate-derived factor (Bootcov loc cit; Hromas, 1997 Biochim Biophys Acta 1354:40-44; Lawton 1997, Gene 203:17-26; Yokoyama-Kobayashi 1997, J Biochem (Tokyo), 122:622-626; Paralkar 1998, J Biol Chem 273:13760-13767). The amino acid sequence of GDF-15 is disclosed in WO 99 / 06445, WO 00 / 70051, WO 2005 / 113585, etc., Bottner 1999, Gene 237:105-111, Bootcov loc.cit, Tan loc.cit., Baek 2001, Mol Pharmacol 59:901-908, Hromas loc cit, Paralkar loc cit, Morrish 1996, Placenta 17:431-441.

[0103] As used herein, the term "sFlt-1" refers to a polypeptide that is a soluble form of fms-like tyrosine kinase 1. The polypeptide is also referred to in the art as soluble VEGF receptor 1 (sVEGF R1) (see, e.g., Sunderji 2010, Am J Obstet Gynecol 202:40e1-7). Soluble Flt-1 was identified in the conditioned culture medium of human umbilical vein endothelial cells. Endogenous sFlt1 receptor is chromatographically and immunologically similar to recombinant human sFlt1 and binds [125I]VEGF with comparable high affinity. Human sFlt1 has been shown to form a VEGF-stabilized complex with the extracellular domain of KDR / Flk-1 in vitro. Preferably, sFlt1 refers to human sFlt1 as described in Kendall 1996, Biochem Biophs Res Commun 226(2):324-328, with respect to the amino acid sequence, e.g., Genebank accession numbers P17948, GI:125361 for human and BAA24499.1, GI:2809071 for mouse sFlt-1 (Genbank is available from NCBI, USA, under www.ncbi.nlm.nih.gov / entrez).

[0104] Interleukin-6 (abbreviated IL-6), secreted by T cells and macrophages, is an interleukin that stimulates immune responses, for example during infection and after trauma, especially during burns or other tissue damage that leads to inflammation. It acts as both a pro-inflammatory and an anti-inflammatory cytokine. In humans, it is encoded by the IL6 gene. The sequence of human IL-6 can be assessed by GenBank (see NM_000600.3 for the polynucleotide sequence and NP_000591.1 for the amino acid sequence). IL-6 signals through a cell surface type I cytokine receptor complex consisting of the ligand-binding IL-6Rα chain (CD126) and the signaling component gp130 (also called CD130). CD130 is a common signaling agent for several cytokines, including leukemia inhibitory factor (LIF), ciliary neurotropic factor, oncostatin M, IL-11 and cardiotrophin-1, and is expressed almost ubiquitously in most tissues. In contrast, the expression of CD126 is restricted to certain tissues. When IL-6 interacts with its receptor, it triggers gp130 and IL-6R proteins to form a complex, thus activating the receptor. These complexes bring together the intracellular regions of gp130 to initiate a signaling cascade via specific transcription factors, Janus kinases (JAKs) and signal transduction and activator of transcription.

[0105] CRP (C-reactive protein) is an acute phase protein that was discovered over 75 years ago to be a blood protein that binds to the C-polysaccharide of Streptococcus pneumoniae. CRP is known as a reactive inflammatory marker and is produced by distal organs (i.e., the liver) in response to or in reaction to chemokines or interleukins derived from the primary lesion site. CRP is known to consist of five single subunits that are non-covalently linked and assembled as a cyclic pentamer with a molecular weight of about 110-140 kDa. Preferably, CRP as used herein relates to human CRP. The sequence of human CRP is well known and has been disclosed, for example, by Woo et al. (J.Biol.Chem.1985.260(24),13384-13388). Levels of CRP are usually low in normal individuals, but can be elevated 100-200 fold or more due to inflammation, infection or injury (Yeh(2004)Circulation.2004;109:11-11-11-14). CRP is known to be an independent factor for predicting cardiovascular risk.CRP can be measured by immunoassays, such as ELISA, which are well known in the art and are commercially available.In a preferred embodiment, CRP is hsCRP (high sensitivity CRP).

[0106] IGFBP-7 (insulin-like growth factor binding protein 7) is a modular glycoprotein of 30 kDa known to be secreted by endothelial cells, vascular smooth muscle cells, fibroblasts, and epithelial cells (Ono, Y., et al, Biochem Biophys Res Comm 202 (1994) 1490-1496). Preferably, the term "IGFBP-7" refers to human IGFBP-7. The protein sequence is well known in the art and can be accessed, for example, through UniProt (Q16270, IBP7_HUMAN), or GenBank (NP_001240764.1). A detailed definition of the biomarker IGFBP-7 is provided, for example, in WO 2008 / 089994, which is incorporated herein by reference in its entirety. IGFBP-7 has two isoforms, isoform 1 and 2, which are generated by alternative splicing. In one embodiment of the invention, the total amount of both isoforms is measured (for sequences see UniProt database entries (Q16270-1 and Q16270-2)).

[0107] The biomarker endothelial cell specific molecule 1 (abbreviated ESM-1) is well known in the art. The biomarker is also often called endocan. ESM-1 is a secreted protein and is expressed mainly in endothelial cells of human lung and kidney tissues. Public domain data suggests that it is expressed in thyroid, lung, kidney, but also in cardiac tissue. See, for example, the entry for ESM-1 in the Protein Atlas database (Uhlen M.et al.,Science 2015;347(6220):1260419). Expression of this gene is regulated by cytokines. ESM-1 is a proteoglycan composed of a 20 kDa mature polypeptide and a 30 kDa O-linked glycan chain (Bechard D et al.,J Biol Chem 2001;276(51):48341-48349). In a preferred embodiment of the invention, the amount of human ESM-1 polypeptide is determined in a sample from a subject. The sequence of the human ESM-1 polypeptide is well known in the art (see, e.g., Lassale P. et al., J. Biol. Chem. 1996;271:20458-20464, and can be assessed, e.g., via the Uniprot database, see entry Q9NQ30 (ESM1_HUMAN). Two isoforms of ESM-1 are generated by alternative splicing, isoform 1 (having the Uniprot identifier Q9NQ30-1) and isoform 2 (having the Uniprot identifier Q9NQ30-2). Isoform 1 is 184 amino acids in length. In isoform 2, amino acids 101 to 150 of isoform 1 are missing. Amino acids 1 to 19 form the signal peptide (which can be cleaved).

[0108] In a preferred embodiment, the amount of isoform 1 of the ESM-1 polypeptide is determined, ie isoform 1 has the sequence as shown under UniProt accession number Q9NQ30-1.

[0109] In another preferred embodiment, the amount of isoform 2 of the ESM-1 polypeptide is determined, ie isoform 2 has the sequence as shown under UniProt accession number Q9NQ30-2.

[0110] In another preferred embodiment, the amount of isoform-1 and isoform-2 of the ESM-1 polypeptide, ie total ESM-1, is determined.

[0111] According to the present invention, the amount of one or more lipid biomarkers can be determined.

[0112] The lipid biomarkers referred to herein are preferably lipid biomarkers selected from the group consisting of cholesterol (also referred to herein as "CHOL"), TAG (triglyceride, also referred to herein as "TRIGLY"), LDL (low density lipoprotein), HDL (high density lipoprotein), and apolipoprotein A-1 (also referred to herein as "APOAT"). Thus, one or more markers from the aforementioned marker groups can be determined.

[0113] In one embodiment, the lipid biomarker is cholesterol.Cholesterol is a steroid with a secondary hydroxyl group at C3 position.It is synthesized in many types of tissues, but especially in the liver and intestinal wall.About 3 / 4 of cholesterol is newly synthesized, and 1 / 4 comes from dietary intake.Cholesterol assay is used to screen the risk of atherosclerosis, and to diagnose and treat disorders associated with elevated cholesterol levels and lipid and lipoprotein metabolism disorders.

[0114] In another embodiment, the lipid biomarker is apolipoprotein A-1. Apolipoproteins are the protein components of lipoproteins. Lipoproteins are classified according to their ultracentrifugal buoyant density. Apolipoprotein A-1 is the major protein component of high density lipoprotein (HDL). Apolipoprotein A-1 activates the enzyme lecithin-cholesterol-acyltransferase (LCAT), which catalyzes the esterification of cholesterol, thereby enhancing the lipid-carrying capacity of lipoproteins. Further information is apolipoprotein A-1 under UniProt-Accession-Number UniProtKB-P02647 (APOA1_HUMAN).

[0115] In yet another embodiment, the lipid biomarker is triglyceride.Triglycerides are esters of the trihydric alcohol glycerol and three long-chain fatty acids.They are partially synthesized in the liver and partially ingested in food.The determination of triglyceride is utilized in the diagnosis and treatment of patients with diabetes mellitus, nephropathy, hepatic obstruction, lipid metabolism disorders and many other endocrine diseases.

[0116] In yet another embodiment, the lipid biomarker is LDL. Thus, the LDL content is determined. Low density lipoprotein (LDL) plays an important role in causing and influencing the progression of atherosclerosis, especially coronary atherosclerosis. LDL is one of the five major groups of lipoproteins that transport all fat molecules around the body in extracellular water. Blood tests generally report the amount of cholesterol that is estimated to be contained in LDL particles on average by using the LDL-cholesterol: Friedewald formula. Furthermore, LDL content can be assessed via the cholesterol content in LDL.

[0117] In yet another embodiment, the lipid biomarker is HDL.HDL is synthesized by intestine and liver.High density lipoprotein (HDL) is responsible for the reverse transport of cholesterol from peripheral cells to liver.HDL-cholesterol concentration is important in assessing atherosclerosis risk, so monitoring HDL-cholesterol in serum or plasma is clinically important.

[0118] Preferably, the at least one vascular biomarker referred to herein is selected from the group consisting of FGF23 (fibroblast growth factor 23), sFlt-1 (soluble fms-like tyrosine kinase-1), GDF15 (growth differentiation factor 15), ESM-1 (endothelial cell-specific molecule 1), ANG2 (angiopoietin 2) and IGFBP7 (insulin-like growth factor binding protein 7).

[0119] In one embodiment, the vascular biomarker is FGF23.

[0120] In another embodiment, the vascular biomarker is sFlt1.

[0121] In yet another embodiment, the vascular biomarker is GDF15.

[0122] In yet another embodiment, the vascular biomarker is ESM1.

[0123] In yet another embodiment, the vascular biomarker is ANG2.

[0124] In yet another embodiment, the vascular biomarker is IGFBP7.

[0125] Preferably, the at least one inflammatory biomarker referred to herein is CRP, such as hsCRP (high sensitivity C-reactive protein) and / or IL6 (Interleukin 6).

[0126] In one embodiment, the inflammatory biomarker is CRP. In an alternative embodiment, the inflammatory biomarker is IL6.

[0127] In one embodiment, the first marker is a cardiac troponin and the second marker is a BMP10-type peptide.

[0128] In one embodiment, the first marker is a cardiac troponin and the second marker is FGF23.

[0129] In one embodiment, the first marker is a cardiac troponin and the second marker is cMyBPC.

[0130] In one embodiment, the first marker is a cardiac troponin and the second marker is ANG2.

[0131] In one embodiment, the first marker is a cardiac troponin and the second marker is a BNP-type peptide, such as NT-proBNP or total NT-proBNP. Preferred marker combinations of two or three biomarkers are shown in Table 2 in the Examples section. Further preferred marker combinations are as follows:

[0132] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is cMyBPC.

[0133] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is CHOL.

[0134] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is FGF23.

[0135] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is LDL.

[0136] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is TRIGLY.

[0137] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is APOAT.

[0138] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is HDL.

[0139] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is sFLT1.

[0140] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is GDF15.

[0141] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is CRP, for example hsCRP.

[0142] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is IL6.

[0143] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is ESM1.

[0144] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is IGFBP7.

[0145] In one embodiment, the first marker is a cardiac troponin, the second marker is a BMP10-type peptide, and the third marker is ANG2.

[0146] In one embodiment, the first marker is a cardiac troponin, the second marker is FGF23, and the third marker is cMyBPC.

[0147] In one embodiment, the first marker is a cardiac troponin, the second marker is FGF23, and the third marker is TRIGLY.

[0148] In one embodiment, the first marker is a cardiac troponin, the second marker is FGF23, and the third marker is CHOL.

[0149] In one embodiment, the first marker is a cardiac troponin, the second marker is FGF23, and the third marker is LDL.

[0150] In one embodiment, the first marker is a cardiac troponin, the second marker is FGF23, and the third marker is HDL.

[0151] In one embodiment, the first marker is a cardiac troponin, the second marker is FGF23, and the third marker is APOAT.

[0152] In one embodiment, the first marker is a cardiac troponin, the second marker is FGF23, and the third marker is ANG2.

[0153] In one embodiment, the first marker is a cardiac troponin, the second marker is FGF23, and the third marker is sFLT1.

[0154] In one embodiment, the first marker is a cardiac troponin, the second marker is FGF23, and the third marker is IGFBP7.

[0155] In one embodiment, the first marker is a cardiac troponin, the second marker is NTproBNP, and the third marker is cMyBPC.

[0156] In one embodiment, the first marker is a cardiac troponin, the second marker is NTproBNP, and the third marker is FGF23.

[0157] In one embodiment, the first marker is a cardiac troponin, the second marker is NTproBNP, and the third marker is ANG2.

[0158] In one embodiment, the first marker is a cardiac troponin, the second marker is NTproBNP, and the third marker is CHOL.

[0159] In one embodiment, the first marker is a cardiac troponin, the second marker is NTproBNP, and the third marker is LDL.

[0160] In one embodiment, the first marker is a cardiac troponin, the second marker is NTproBNP, and the third marker is TRIGLY.

[0161] In one embodiment, the first marker is a cardiac troponin, the second marker is NTproBNP, and the third marker is HDL.

[0162] In one embodiment, the first marker is a cardiac troponin, the second marker is NTproBNP, and the third marker is APOAT.

[0163] In one embodiment, the first marker is a cardiac troponin, the second marker is tNTproBNP, and the third marker is cMyBPC.

[0164] In one embodiment, the first marker is a cardiac troponin, the second marker is tNTproBNP, and the third marker is CHOL.

[0165] In one embodiment, the first marker is a cardiac troponin, the second marker is tNTproBNP, and the third marker is LDL.

[0166] In one embodiment, the first marker is a cardiac troponin, the second marker is tNTproBNP, and the third marker is TRIGLY.

[0167] In one embodiment, the first marker is a cardiac troponin, the second marker is tNTproBNP, and the third marker is HDL.

[0168] In one embodiment, the first marker is a cardiac troponin, the second marker is tNTproBNP, and the third marker is APOAT.

[0169] In one embodiment, the first marker is a cardiac troponin, the second marker is ANG2, and the third marker is cMyBPC.

[0170] In one embodiment, the first marker is a cardiac troponin, the second marker is ANG2, and the third marker is LDL.

[0171] In one embodiment, the first marker is a cardiac troponin, the second marker is ANG2, and the third marker is CHOL.

[0172] In one embodiment, the first marker is a cardiac troponin, the second marker is ANG2, and the third marker is ESM1.

[0173] The following combinations of biomarkers can be advantageously applied in diabetic patients:

[0174] In one embodiment, the first marker is a cardiac troponin and the second marker is ANG2.

[0175] In one embodiment, the first marker is a cardiac troponin, the second marker is ANG2, and the third marker is IGFBP7.

[0176] In one embodiment, the first marker is a cardiac troponin, the second marker is ANG2, and the third marker is LDL.

[0177] In one embodiment, the first marker is a cardiac troponin, the second marker is ANG2, and the third marker is APOAT.

[0178] As used herein, the term "determining" refers to the qualitative and quantitative determination of the biomarker referred to in accordance with the present invention, i.e., the term encompasses the determination of the presence or absence or the determination of the absolute or relative amount of said biomarker.

[0179] As used herein, the term "amount" refers to the absolute amount of the compound referred to herein, the relative amount or concentration of the compound, as well as any value or parameter that can be correlated to or derived therefrom. Such values ​​or parameters include intensity signal values ​​derived from any specific physical or chemical property obtained from the compound by direct measurement, such as intensity values ​​in mass or NMR spectra. In addition, all values ​​or parameters obtained by indirect measurement as specified elsewhere in this specification are included, such as the response level determined by a biological readout system in response to a compound or intensity signal obtained from a specifically bound ligand. It should be understood that values ​​correlated to the above amounts or parameters can also be obtained by all standard mathematical operations.

[0180] The determination of the amount in the method of the invention can be carried out by any technique that makes it possible to detect the presence or absence or amount of the second molecule upon its release from the first molecule. Suitable techniques depend on the nature of the molecule and the characteristics of the biomarker and are discussed in more detail elsewhere in this specification.

[0181] Typically, the amount of the biomarker referred to according to the invention can be determined by immunoassays using sandwich, competitive or other assay formats, especially when the biomarker is a protein biomarker (BNP type peptide, BMP10 type peptide, cardiac troponin, GDF-15, sFlt-1, ANG2, CRP, IL6, ESM1 or IGFBP7, APOAT, etc.). The assay generates a signal indicating the presence or absence or amount of the biomarker. Further suitable methods include measuring a physical or chemical property specific for the biomarker, such as its exact molecular mass or NMR spectrum. The methods preferably include analytical devices such as biosensors, optical devices linked to immunoassays, biochips, mass spectrometers, NMR analyzers, surface plasmon resonance measuring instruments or chromatography devices. Furthermore, the methods include microplate ELISA-based methods, fully automated or robotic immunoassays (e.g. available from Roche). Suitable measurement methods according to the present invention may also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electrogenerated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), electrochemiluminescence sandwich immunoassay (ECLIA), dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), scintillation proximity assay (SPA), nephelometry, nephelometry, latex-enhanced nephelometry or nephelometry, or solid-phase immunoassay. Further methods are known in the art, such as gel electrophoresis, 2D gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or Western blotting. More typically, the techniques specifically envisaged for determining the biomarkers referred to herein are described in the accompanying examples below.

[0182] The biomarkers that are determined according to the present invention are well known in the art.Furthermore, the method for determining the amount of biomarkers is known.For example, biomarkers can be measured as described in the Examples section (see Example 1).

[0183] Some of the biomarkers are the lipid biomarkers HDL, LDL, cholesterol and triglycerides. The amounts of these biomarkers can be determined, for example, enzymatically.

[0184] The enzymatic determination of the amount of (total) triglycerides is preferably a) contacting the sample with a lipase under conditions and for a time sufficient to permit conversion to glycerol and free fatty acids; b) contacting the sample containing glycerol with glycerokinase under conditions and for a time sufficient to permit conversion to glycerol 3-phosphate; c) contacting the sample containing glycerol-3-phosphate with glycerophosphate oxidase under conditions and for a time sufficient to permit conversion to dihydroxyacetone phosphate and H2O2; and d) enzymatically or chemically determining the amount of H2O2 produced, thereby determining the amount of triglycerides.

[0185] Thus, detection agents for triglycerides are the enzymes glycerokinase, glycerophosphate oxidase and glycerophosphate oxidase. Preferably, these enzymes are used in combination.

[0186] The amount of cholesterol can be assessed via the amount of cholesterol esters in the sample. The enzymatic determination of the amount of cholesterol esters is preferably carried out by: a) contacting the sample with cholesterol esterase under conditions and for a time sufficient to permit its conversion to cholesterol; b) contacting the sample containing cholesterol with cholesterol oxidase under conditions and for a time sufficient to allow the production of H2O2; and c) enzymatically or chemically determining the amount of H2O2 produced, thereby determining the amount of cholesterol.

[0187] Thus, detection agents for cholesterol esters are the enzymes: cholesterol esterase and cholesterol oxidase. Preferably, these enzymes are used in combination.

[0188] As mentioned above, the amount of LDL can be assessed by determining LDL cholesterol and cholesterol esters. The enzymatic determination of the amount of LDL cholesterol and cholesterol esters is preferably carried out by: a) contacting the sample in the presence of a non-ionic detergent that selectively solubilizes LDL with cholesterol esterase under conditions and for a time sufficient to permit its conversion to cholesterol; b) contacting the sample containing cholesterol with cholesterol oxidase under conditions and for a time sufficient to allow the production of H2O2; c) enzymatically or chemically determining the amount of H2O2 produced, thereby determining the amount of LDL cholesterol and cholesterol esters, and thus LDL.

[0189] Thus, detection agents for HDL are the enzymes: cholesterol esterase and cholesterol oxidase. Preferably, these enzymes are used in combination.

[0190] As mentioned above, the amount of HDL can be assessed by determining HDL cholesterol and cholesterol esters, i.e. the amount of cholesterol and cholesterol esters present in HDL. Enzymatic determination of the amount of LDL cholesterol and cholesterol esters is preferably carried out by: a) contacting the sample in the presence of a non-ionic detergent that selectively solubilizes LDL with cholesterol esterase under conditions and for a time sufficient to permit its conversion to cholesterol; b) contacting the sample containing cholesterol with cholesterol oxidase under conditions and for a time sufficient to allow the production of H2O2; c) enzymatically or chemically determining the amount of H2O2 produced, thereby determining the amount of LDL cholesterol and cholesterol esters, and thus LDL.

[0191] Thus, detection agents for LDL are the enzymes: cholesterol esterase and cholesterol oxidase. Preferably, these enzymes are used in combination.

[0192] The amount of H2O2 can be determined enzymatically, for example, by conversion in a further step (step e or d) of contacting the sample with a peroxidase (such as horseradish peroxidase) and a chromogenic substrate. The substrate is typically oxidized by the peroxidase using H2O2 as an oxidizing agent. The catalytic reaction in the presence of H2O2, peroxidase and substrate typically results in a characteristic change detectable by spectrophotometry. For example, peroxidase catalyzes the conversion of a chromogenic substrate to a colored product and produces light when acting on a chemiluminescent substrate. For example, DAOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline) and 4-aminoantipyrine in the presence of H2O2 and peroxidase results in the oxidative coupling of DAOS with 4-aminoantipyrine to form a blue chromogen. The chromogen can be detected, for example, by measuring the absorbance of light at about 590 nm. Alternatively, 10-acetyl-3,7-dihydroxyphenoxazine can be used as a substrate for peroxidase, allowing the detection of H2O2. This non-fluorescent reagent reacts with H2O2 to produce the fluorescent compound resorufin. In another option, 4-aminophenazone + 4-chlorophenol in the presence of H2O2 and peroxidase leads to the formation of 4-(p-benzoquinone-monoimino)-phenazone, a red product, which can be determined by photometric means.

[0193] It should be understood that the present invention is not limited to the above markers (first, second and third markers). Rather, the present invention may encompass the determination of additional markers.

[0194] As used herein, the term "criterion" preferably refers to an amount or score that allows a subject to be assigned to either of the groups of subjects suffering from disease or condition, such as type 2 myocardial infarction or type 1 myocardial infarction (MI). Such a criterion can be a threshold (or cut-off) amount or score that separates these groups from each other. Thus, a criterion shall be an amount or score that allows a subject to be assigned to the group of subjects suffering from type 1 or type 2 myocardial infarction. For example, a criterion shall be an amount or score that allows a subject to be assigned to the group of subjects suffering from type 2 myocardial infarction or not suffering from type 2 myocardial infarction.

[0195] In one embodiment, the criterion is a criterion amount, hi another embodiment, the criterion is a criterion score.

[0196] The term "at least one subject" refers to one subject or more than one subject, for example, at least 10, 50, 100, 200, or 1000 subjects.

[0197] Reference amounts can in principle be calculated for a cohort of subjects based on a given parameter, e.g., the mean or average value of a biomarker amount, by applying standard statistical methods. Whether a test, particularly a method aimed at diagnosing an event, is accurate or not is best described by its receiver operating characteristics (ROC) (see, in particular, Zweig 1993, Clin. Chem. 39:561-577). The ROC graph is a plot of all sensitivity / specificity pairs resulting from continuously varying the decision threshold over the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign subjects to a certain prognosis or diagnosis. The ROC plot shows the overlap between the two distributions by plotting sensitivity versus 1-specificity for the entire range of thresholds suitable for making the distinction. On the y-axis is the sensitivity, or true positive rate, defined as the ratio of the number of true positive test results to the product of the number of true positive test results and the number of false negative test results. This is also called positivity in the presence of a disease or condition. The y-axis is calculated only from the affected subgroup. On the x-axis is the false positive rate, or 1-specificity, which is defined as the ratio of the number of false positive results to the product of the number of true negative and false positive results. The x-axis is a measure of specificity, calculated only from the unaffected subgroup. The true positive rate and the false positive rate are calculated completely separately by using the test results from the two different subgroups, so the ROC plot is independent of the prevalence of the event in the cohort. Each point on the ROC plot represents a sensitivity / -specificity pair that corresponds to a particular decision threshold. A test with perfect discrimination (no overlap between the two distributions of results) would have an ROC plot that passes through the upper left corner, with a true positive rate of 1.0, or 100% (perfect sensitivity) and a false positive rate of 0 (perfect specificity). The theoretical plot for a test with no discrimination (the distribution of results for the two groups is identical) would be a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall between these two extremes. If the ROC plot falls entirely below the 45° diagonal, this is easily corrected by swapping the criteria for "positive rate" from "higher" to "lower" and vice versa.Qualitatively, the closer the plot is to the upper left corner, the higher the accuracy of the entire test. Depending on the desired confidence interval, a threshold value can be derived from the ROC curve that allows diagnosis of symptomatic disease with an appropriate balance of sensitivity and specificity, respectively. For example, the reference used in the aforementioned method of the present invention, i.e., the threshold value that allows distinguishing between type 1 and type 2 myocardial infarction, can usually be generated by establishing the ROC of the cohort in question as described above and deriving the threshold amount therefrom. Depending on the desired sensitivity and specificity of the diagnostic method, the ROC plot can lead to an appropriate threshold value.

[0198] It should be understood that the criteria referred to herein are intended to enable assessment, for example, to distinguish between type 1 and type 2 myocardial infarction or to make appropriate treatment decisions.

[0199] Step c) of the method of the present invention comprises comparing the amount of the biomarkers (i.e., the first biomarker, the second biomarker, and optionally the third biomarker) with a standard for said biomarkers and / or calculating a score for assessing myocardial infarction based on the amount of the biomarkers.

[0200] Thus, the amount of each of the first biomarker, the second biomarker and optionally the third biomarker may be compared to a standard for the first biomarker, a standard for the second biomarker and optionally a standard for the third biomarker.

[0201] Alternatively, a score can be calculated based on the amount of biomarkers, i.e., based on the amount of the first biomarker, the second biomarker, and optionally the third biomarker. The score allows the assessment of myocardial infarction, for example, to distinguish between type 1 and type 2 myocardial infarction. Optionally, the score can be compared with a suitable reference score.

[0202] As used herein, the term "comparing" encompasses comparing the determined amount of the biomarker referred to herein with a reference (or comparing the calculated score with a reference score). It should be understood that comparison as used herein refers to any kind of comparison made between an amount value and a reference. However, it should be understood that preferably, values ​​of the same type are compared with each other, e.g., if absolute amounts are determined and compared in the method of the invention, the reference is also an absolute amount, if relative amounts are determined and compared in the method of the invention, the reference is also a relative amount, etc. Alternatively, as used herein, the term "comparing" encompasses comparing the calculated score with an appropriate reference score. The comparison can be performed manually or computer-assisted. The amount and the value or score of the reference can, for example, be compared with each other, and the comparison can be performed automatically by a computer program that executes an algorithm for the comparison. The computer program that performs the evaluation provides the desired assessment in a suitable output format.

[0203] As mentioned above, it is also envisaged to combine the multiple biomarkers into a single score, i.e. a single score, based on the amount of the first and second biomarkers, or the first, second or third biomarker, and compare this score with a reference score. This results in a score. Preferably, the score is based on the amount of the first and second biomarkers in the sample from the test subject, and if the amount of the third biomarker is determined, it is based on the amount of the first, second and third biomarkers in the sample from the test subject. The score shall be based on the amount of the aforementioned biomarkers, but additional biomarkers or additional patient-specific parameters may contribute to the score.

[0204] The calculated score combines information on the amount of at least two biomarkers. Moreover, in the score, the biomarkers are preferably weighted according to their contribution to the establishment of the score. Thus, the values ​​for each marker are weighted, and the weighted values ​​are used to calculate the score. The appropriate coefficients (weights) can be determined by those skilled in the art without further difficulty. The score can also be calculated from a decision tree or a set (ensemble) of decision trees trained on at least two biomarkers. Based on the combination of biomarkers applied in the method of the present invention, the weights of each biomarker as well as the structure of the decision tree can be different.

[0205] The score can be considered as a classifier parameter for assessing a subject as described herein. In particular, it allows to provide an assessment based on a single score. The reference score is preferably a value, in particular a cut-off value that allows the assessment of myocardial infarction as described herein. Preferably, the reference is a single value. Thus, it is not necessary to interpret the entire information regarding the amount of each individual biomarker. Using the scoring system described herein, advantageously, values ​​of different dimensions or units may be used for the biomarkers, because the values ​​are mathematically converted into scores. Thus, for example, values ​​for absolute concentration may be combined with peak area ratios in the score. The applied reference score may be selected based on the desired sensitivity or the desired specificity. Methods for selecting an appropriate reference score are well known in the art.

[0206] The score shall allow assessment of myocardial infarction, such as differentiation between type 2 and type 1 myocardial infarction. Methods for calculating the score are well known in the art. Examples are provided in the Examples section (see Example 2, Example of the use of a combination of hsTnT and BMP10). Appropriate diagnostic algorithms can also be set by those skilled in the art. For example, a calculated score greater than the reference score is indicated for subjects suffering from type 2 MI, and a calculated score less than the reference score is indicated for subjects suffering from type 1 MI.

[0207] For certain markers described herein, preferred changes ("directions") are found in Table A below. [Table A]

[0208] Thus, cardiac troponin, MyBPC3, sFlt1, LDL, TRIGL (Tryglyerides), CHOL, ANG2 and BMP10 are increased (relative to baseline) in patients with MI type 1 and decreased (relative to baseline) in patients with MI type 2. The remaining markers shown in Table A are decreased (relative to baseline) in patients with MI type 1 and increased (relative to baseline) in patients with MI type 2.

[0209] Therefore, the method of the present invention may further comprise recommending or initiating an appropriate therapeutic measure. Typically, the appropriate therapeutic measure depends on the result of the assessment described herein, i.e., whether the subject suffers from type 1 or type 2 myocardial infarction. For example, it can be determined whether the patient undergoes a treatment aimed at treating type 2 myocardial infarction or a treatment aimed at treating type 1 myocardial infarction. A patient identified as suffering from type 2 MI undergoes a treatment aimed at treating type 2 MI. A patient identified as suffering from type 1 MI undergoes a treatment aimed at treating type 1 MI.

[0210] The methods of the present invention can further guide the urgency and timing of treatment options according to various information derived from biomarkers of AMI subtypes.

[0211] In one embodiment, the therapeutic measures recommended or initiated if a patient is assessed as suffering from type 1 myocardial infarction are a reperfusion strategy to address atherothrombotic plaque rupture with drugs (antiplatelets, anticoagulation) and emergency treatment with invasive revascularization procedures (e.g., percutaneous coronary intervention [PCI], placement of a drug-eluting stent, or coronary artery bypass grafting [CABG]).

[0212] In one embodiment, the therapeutic measures recommended or initiated if the patient is assessed as suffering from type 2 myocardial infarction are procedures to improve myocardial oxygen supply demand, such as addressing symptoms of decreased myocardial oxygen supply (hypotension, anemia, bradyarrhythmia, respiratory failure) and addressing symptoms of increased myocardial oxygen demand (tachycardia, tachyarrhythmia, severe hypertension, other comorbidities) with treatments and medications such as beta-blockers, vasodilators, anticoagulants and diuretics. In one embodiment, the therapeutic measures recommended or initiated if the patient is assessed with a high biomarker score can guide the appropriate timing of intervention (immediate, early, or delayed).

[0213] The definitions given herein above apply mutatis mutandis below.

[0214] The present invention also relates to a computer implemented method for assessing myocardial infarction in a subject, the method comprising: (a) receiving a value for the amount of a first biomarker in the subject's sample, wherein the first biomarker is a cardiac troponin; (b) receiving a value for the amount of a second biomarker in the subject's sample, wherein the second biomarker is a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC, or ANG2; (c) comparing said value for said amount of said biomarker with a standard for said biomarker and / or calculating a score for assessing myocardial infarction based on said amount of said biomarker; (d) assessing myocardial infarction based on the comparison and / or calculation performed in step (c).

[0215] The term "computer-implemented" as used herein means that the method is carried out in an automated manner on a data processing unit, typically contained in a computer or similar data processing device. The data processing unit receives values ​​for the amounts of the biomarkers. Such values ​​can be amounts, relative amounts, or any other calculated values ​​that reflect the amounts described in detail elsewhere herein. It should therefore be understood that the above-mentioned method does not require the determination of amounts for the biomarkers, but rather uses values ​​for amounts that have already been predetermined.

[0216] Typically, in step (b) of the process, (i) if said value for said amount of a BMP10 type peptide is received as said second biomarker, said method may further comprise receiving as a third biomarker a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; or (ii) if the value for the amount of FGF23 is received as the second biomarker, the method may further comprise receiving a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7, as a third biomarker; or (iii) if the value for the amount of a BNP-type peptide is received as the second biomarker, the method may further comprise receiving a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2, as a third biomarker; or (iv) if the value for the amount of ANG2 is received as the second biomarker, the method further comprises receiving a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7, as a third biomarker.

[0217] The present invention also in principle contemplates a computer program, a computer program product, or a computer readable storage medium tangibly embodied with said computer program, the computer program comprising instructions which, when executed on a data processing device or computer, perform the method of the present invention as described above. a computer or computer network comprising at least one processor, the processor being configured to execute a method according to one of the embodiments described herein, - a computer-loadable data structure configured, when executed on a computer, to carry out a method according to one of the embodiments described in this specification, - a computer script, the computer program being adapted to carry out the method of one of the embodiments described herein while the program is running on a computer, a computer program comprising program means for carrying out, when the computer program is run on a computer or on a computer network, a method according to one of the embodiments described herein, a computer program comprising program means according to any preceding embodiment, the program means being stored on a computer readable storage medium; a storage medium on which a data structure is stored and adapted to carry out a method according to one of the embodiments described herein after the data structure has been loaded into a primary and / or working storage device of a computer or a computer network, a computer program product having program code means in which, or capable of being stored on a storage medium, the program code means for performing a method according to one of the embodiments described in this specification when the program code means is executed on a computer or a computer network, a typically encrypted data stream signal containing data of the parameters defined elsewhere in this specification, and - a typically encrypted data stream signal containing the assessment provided by the method of the present invention.

[0218] The present invention relates to a device for assessing myocardial infarction in a subject having a myocardial infarction, the device comprising: (a) a measuring unit for determining the amount of a first biomarker, which is a cardiac troponin, and a second biomarker in a sample from the subject, the second biomarker being a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC or ANG2, the measuring unit comprising a detection system for the first biomarker and the second biomarker; (b) an evaluation unit operably linked to the measurement unit, the evaluation unit comprising: a database having stored references for the first and second biomarkers, preferably as described above; and a data processor comprising instructions for performing a comparison of the amounts of the first and second biomarkers with a reference, preferably as described above, and / or for performing a calculation of a score for assessing myocardial infarction based on the amounts of the biomarkers or based on a calculated score, and for assessing myocardial infarction based on a comparison with a reference amount (or reference score), the evaluation unit being capable of automatically receiving values ​​for the amounts of the biomarkers from the measurement unit.

[0219] The term "device", as used herein, relates to a system comprising the above-mentioned units functionally linked to each other to allow the determination of the amount of the biomarker and its evaluation by the method of the present invention so that an assessment can be provided.

[0220] The analytical unit typically comprises at least one reaction zone having biomarker detection agents for the first and second biomarkers and preferably also for the third biomarker in immobilized form on a solid support or carrier that is brought into contact with the sample. Furthermore, in the reaction zone it is possible to apply conditions that allow specific binding of the detection agent(s) to the biomarkers contained in the sample.

[0221] The reaction zone may directly allow sample application or may be connected to a loading zone where the sample is applied. In the latter case, the sample may be actively or passively transported to the reaction zone via a connection between the loading zone and the reaction zone. Furthermore, the reaction zone is also connected to a detector. The connection is such that the detector can detect the binding of the biomarkers to their detection agent. The appropriate connection depends on the technique used to measure the presence or amount of the biomarker. For example, for optical detection, light transmission may be required between the detector and the reaction zone, and for electrochemical determination, a fluid connection may be required, for example, between the reaction zone and an electrode.

[0222] The detector is adapted to detect the determination of the amount of the biomarker. The determined amount can be subsequently transmitted to an evaluation unit comprising a data processing element, such as a computer, having an implemented algorithm for determining the amount present in the sample.

[0223] The processing unit referred to in accordance with the method of the present invention typically comprises a central processing unit (CPU) and / or one or more graphic processing units (GPUs) and / or one or more application specific integrated circuits (ASICs) and / or one or more tensor processing units (TPUs) and / or one or more field programmable gate arrays (FPGAs), etc. The data processing element may be, for example, a general-purpose computer or a portable computing device. It should also be understood that multiple computing devices may be used together, such as over a network or in other ways to transfer data, to perform one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants ("PDAs"), cellular devices, smart or mobile devices, tablet computers, and servers, etc. In general, the data processing element includes a processor capable of executing multiple instructions (e.g., a program of software).

[0224] The evaluation unit typically includes a memory or has access to a memory. The memory is a computer-readable medium and may include, for example, a single storage device or multiple storage devices located locally with the computing device or accessible to the computing device over a network. The computer-readable medium may be any available medium accessible by the computing device, including both volatile and non-volatile media. Furthermore, the computer-readable medium may be one or both of removable and non-removable media. By way of example and without limitation, the computer-readable medium may include computer storage media. Exemplary computer storage media include, but are not limited to, RAM, ROM, EEPROM, flash memory or any other memory technology, CD-ROM, digital versatile disk (DVD) or other optical disk storage, magnetic cassettes, magnetic tapes, magnetic disk storage or other magnetic storage devices, or any other medium that can be accessed by a computing device and used to store a plurality of instructions that can be executed by the processor of the computing device.

[0225] According to an embodiment of the present disclosure, the software may include instructions that, when executed by a processor of a computing device, may perform one or more steps of the methods disclosed herein. Some of the instructions may be adapted to generate signals that control the operation of other machines and may thus operate via those control signals to transform materials remote from the computer itself. These descriptions and representations are the means used by those skilled in the data processing arts to most effectively convey, for example, the substance of their work to others skilled in the art.

[0226] The instructions may also include an algorithm, which is generally conceived to be a self-consistent sequence of steps leading to a desired result. These steps are those that require the physical manipulation of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic pulses or signals that can be stored, transferred, transformed, combined, compared, and otherwise manipulated. It has proven convenient at times, primarily for reasons of common usage, to refer to these signals as values, characters, representations, numbers, or the like, in reference to the physical items or representations that such signals are embodied or represented. It should be noted, however, that all of these and similar terms are associated with the appropriate physical quantities and are merely used herein as convenient labels applied to these quantities.

[0227] The evaluation unit may also include or have access to an output device. Exemplary output devices include, for example, a fax machine, a display, a printer, and a file. According to some embodiments of the present disclosure, a computing device may perform one or more steps of the methods disclosed herein and then provide output related to the results, indicators, ratios, or other factors of the method via an output device.

[0228] Typically, the measurement unit determines a third biomarker and includes a detection system for the third biomarker, and the database includes stored references for the third biomarker, the third biomarker being: (i) if a BMP10-type peptide is the second biomarker, then the second biomarker is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; (ii) when FGF23 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7; or (iii) when a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; or (iv) When ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7.

[0229] More typically, the detection system includes at least one detection agent capable of specifically detecting each of the biomarkers.

[0230] The present invention further contemplates a device for assessing myocardial infarction in a subject, the device comprising an evaluation unit comprising a database with stored references for a first biomarker, which is a cardiac troponin, and a second biomarker, the second biomarker being a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC or ANG2, and a data processor comprising instructions for comparing the amounts of the first and second biomarker with references, preferably as described above, and assessing myocardial infarction based on the comparison, the evaluation unit being capable of receiving a value for the amount of the biomarkers determined in the subject's sample.

[0231] Typically, the database includes stored references for a third biomarker, the third biomarker being (i) when a BMP10-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; or (ii) if FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, such as sFlt1, ANG2, or IGFBP7; (iii) if a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; (iv) When ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7.

[0232] The present invention also relates in principle to the use of i) a first biomarker which is a cardiac troponin and a second biomarker which is a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC or ANG2, or ii) at least one detection agent for said first biomarker and at least one detection agent for said second biomarker, for assessing myocardial infarction in a subject. Preferably, the biomarker or detection agent is used in a sample from a subject.

[0233] As used herein, the term "detection agent" refers to an agent that specifically detects a biomarker. In particular, the term typically refers to any agent that specifically binds to a biomarker, i.e., does not cross-react with other components present in the sample. Typically, the detection agent that specifically binds to a biomarker referred to herein may be an antibody, an antibody fragment or derivative, an aptamer, a ligand of the biomarker, a receptor of the biomarker, an enzyme known to bind to and / or convert the biomarker, or a small molecule known to specifically bind to the biomarker. For example, the antibodies referred to herein as detection agents include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab)2 fragments that can bind to antigens or haptens. The invention also includes single chain antibodies and humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody exhibiting the desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence usually includes at least the antigen-binding amino acid residues of the donor, but may also include other structurally and / or functionally relevant amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. For example, the aptamer detection agent can be a nucleic acid or peptide aptamer. Methods for preparing such aptamers are well known in the art. For example, random mutations can be introduced into the nucleic acid or peptide that is the basis for the aptamer. These derivatives can then be tested for binding according to screening procedures known in the art, such as phage display. Specific binding of the detection agent means that it does not substantially bind, i.e. does not cross-react, with another peptide, polypeptide or substance present in the sample being analyzed. Preferably, the specifically bound biomarker should be bound with an affinity at least 3 times higher than any other component of the sample, more preferably at least 10 times higher, and even more preferably at least 50 times higher.Non-specific binding may be tolerated if it can still be clearly distinguished and measured, for example according to its size on a Western blot or by its relatively higher abundance in the sample.

[0234] In a preferred embodiment, the detection agent is an antibody or antigen-binding fragment thereof that specifically binds to the marker.

[0235] Preferably, the biomarker or detection agent is used in a sample from a subject.

[0236] The detection agent may be permanently or reversibly fused or linked to a detectable label. Suitable labels are well known to those skilled in the art. Suitable detectable labels are any labels that can be detected by suitable detection methods. Typical labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels (including "e.g. magnetic beads", paramagnetic and superparamagnetic labels), and fluorescent labels. Enzymatically active labels include, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, and their derivatives. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate available as ready-made stock solutions from Roche Diagnostics), CDP-Star™ (Amersham Biosciences), ECF™ (Amersham Biosciences). The appropriate enzyme-substrate combination may produce a colored reaction product, fluorescence or chemiluminescence, which can be measured by methods known in the art (e.g., using a light-sensitive film or a suitable camera system). For measuring enzyme reactions, the diagnostic criteria mentioned above apply as well. Exemplary fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa568). Further fluorescent labels are available, for example, from Molecular Probes (Oregon). The use of quantum dots as fluorescent labels is also contemplated. Exemplary radioactive labels include 35S, 125I, 32P, 33P, etc. Radioactive labels can be detected by any method known and appropriate, such as a light-sensitive film or a phosphor imager.A suitable label may also be or include a tag, such as biotin, digoxygenin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose binding protein, and the like.

[0237] Determination of lipid biomarkers by enzymatic assays is described elsewhere herein. Preferred detection agents are also disclosed for these markers.

[0238] The determination of the biomarkers described herein may include mass spectrometry (MS) carried out after a separation step (e.g. by LC or HPLC). Mass spectrometry, as used herein, encompasses all techniques that allow the determination of the molecular weight (i.e. mass) or mass variable corresponding to the compound to be determined according to the invention, i.e. the biomarker. Preferably, mass spectrometry, as used herein, relates to GC-MS, LC-MS, direct injection mass spectrometry, FT-ICR-MS, CE-MS, HPLC-MS, quadrupole mass spectrometry, any sequentially coupled mass spectrometry, e.g. MS-MS or MS-MS-MS, ICP-MS, Py-MS, TOF or any combined approach using the above mentioned techniques. How to apply these techniques is well known to the skilled person. Moreover, suitable devices are commercially available. More preferably, mass spectrometry, as used herein, relates to LC-MS and / or HPLC-MS, i.e. mass spectrometry operatively coupled to a prior liquid chromatographic separation step. Preferably, mass spectrometry is tandem mass spectrometry (also known as MS / MS). Tandem mass spectrometry, also known as MS / MS, involves two or more mass spectrometry steps, with fragmentation occurring between steps. In tandem mass spectrometry, two mass spectrometers in series are connected by a collision cell. The mass spectrometer is coupled to a chromatography device. The chromatographically separated sample is sorted and weighed in the first mass spectrometer, then fragmented by an inert gas in the collision cell, and one or more pieces are sorted and weighed in the second mass spectrometer. The fragments are fractionated and weighed in the second mass spectrometer. Identification by MS / MS is more accurate.

[0239] In one embodiment, mass spectrometry as used herein encompasses quadrupole MS. Most preferably, said quadrupole MS is carried out as follows: a) selection of the mass / charge ratio (m / z) of ions produced by ionization in a first analytical quadrupole of a mass spectrometer, b) fragmentation of the ions selected in step a) by applying an accelerating voltage in an additional subsequent quadrupole filled with collision gas and acting as a collision chamber, c) selection of the mass / charge ratio of ions produced by the fragmentation process in step b) in the additional subsequent quadrupole, steps a) to c) of the method being carried out at least once, and an analysis of the mass / charge ratio of all ions present in the mixture of substances as a result of the ionization process is carried out, the quadrupole being filled with collision gas but no accelerating voltage being applied during the analysis. Details regarding said most preferred mass spectrometry used according to the present invention can be found in WO 2003 / 073464.

[0240] More preferably, the mass spectrometry is liquid chromatography (LC) MS, such as high performance liquid chromatography (HPLC) MS, in particular HPLC-MS / MS. Liquid chromatography, as used herein, refers to all techniques that allow the separation of compounds (i.e. metabolites) in a liquid or supercritical phase.

[0241] For mass spectrometry, the analytes in the sample are ionized to generate charged molecules or molecular fragments. The mass-charge of the ionized analytes, particularly the ionized biomarkers, or fragments thereof, is then measured. Prior to ionization, the sample may be subjected to cleavage with a protease, for example trypsin. The protease cleaves the protein biomarkers into smaller fragments.

[0242] Thus, the mass spectrometry step preferably includes an ionization step in which the biomarkers to be determined are ionized. Naturally, other compounds present in the sample / eluate are also ionized. The ionization of the biomarkers can be carried out by any method deemed appropriate, in particular by electron impact ionization, fast atom bombardment, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI).

[0243] In a preferred embodiment, the ionization step (for mass spectrometry) is performed by electrospray ionization (ESI). Mass spectrometry is therefore preferably ESI-MS (or ESI-MS / MS, if tandem MS is performed). Electrospray is a soft ionization method that results in the formation of ions without breaking chemical bonds.

[0244] More typically, a third biomarker or at least one detection agent for said third biomarker is additionally used, said third biomarker being (i) when a BMP10-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; (ii) when FGF23 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7; or (iii) when a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; or (iv) When ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7.

[0245] The present invention also relates to a kit for assessing myocardial infarction in a subject, the kit comprising at least one detection agent for a first biomarker which is a cardiac troponin and at least one detection agent for a second biomarker, the second biomarker being a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC or ANG2.

[0246] As used herein, the term "kit" refers to a collection of the above-mentioned components, typically provided separately or in a single container. The container also typically contains instructions for carrying out the method of the invention. These instructions may be in the form of a manual or may be provided by a computer program code that, when executed on a computer or data processing device, can carry out or support the determination of the biomarkers referred to in the method of the invention. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., compact disc) or directly on the computer or data processing device, or may be provided in a download format, such as a link to an accessible server or cloud. In addition, the kit may usually include standards for reference amounts of biomarkers for calibration purposes, as described in detail elsewhere herein. The kit according to the invention may also include further components necessary for carrying out the method of the invention, such as solvents, buffers, washing solutions and / or reagents required for detection of the released second molecule. In addition, the kit may partially or entirely include the device of the invention.

[0247] More typically, the kit further comprises at least one detection agent for a third biomarker, the third biomarker being (i) when a BMP10-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; or (ii) if FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, such as sFlt1, ANG2, or IGFBP7; (iii) if a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; (iv) When ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7.

[0248] The above definitions and explanations of terms apply mutatis mutandis below.

[0249] Advantageously, some of the tested biomarkers, such as BMP10 peptide or FGF23, when measured alone, have been shown in the studies underlying the present invention to allow differentiation between type 1 and type 2 myocardial infarction (see Table 1). Furthermore, the biomarker ANG2 allows differentiation between type 1 and type 2 myocardial infarction in diabetic patients (see Table 5).

[0250] Accordingly, the present invention provides a method for assessing myocardial infarction in a subject, comprising: (a) determining the amount of a biomarker in a sample from the subject, the biomarker being BMP10 peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23) or ANG2 (angiopoietin 2); (b) comparing the amount of the biomarker to a standard for that biomarker; (c) assessing myocardial infarction based on the comparison made in step (c).

[0251] In one embodiment of the above method, the sample is obtained from the subject upon presentation to an emergency department. Preferably, the sample is a blood, serum or plasma sample.

[0252] The direction of the biomarkers (increase vs. decrease) is disclosed in connection with Table A above.

[0253] When the biomarker is ANG2, the subject is preferably a subject suffering from diabetes.

[0254] The present invention further relates to a computer-implemented method for assessing myocardial infarction in a subject, the method comprising: (a) receiving a value for the amount of a biomarker in the subject's sample, the biomarker being a BMP10-type peptide, FGF23, or ANG2; (b) comparing the value for the amount of the biomarker to a standard for that biomarker; (c) assessing myocardial infarction based on the comparison made in step (c).

[0255] The term "assessment of myocardial infarction" is described above. Accordingly, this description applies. Preferably, the assessment of myocardial infarction is a distinction between type 1 and type 2 myocardial infarction.

[0256] The present invention also provides a device for assessing myocardial infarction in a subject having myocardial infarction, the device comprising: (a) a measuring unit for determining the amount of a biomarker, which is a BMP10 type peptide, FGF23 or ANG2, in a sample from a subject, the measuring unit comprising a detection system for the biomarker; (b) an evaluation unit operably coupled to the measurement unit comprising a database having one or more stored standards for the biomarkers and a data processor including instructions for performing a comparison between the amount of the biomarker and the standards to assess myocardial infarction based on the comparison, the evaluation unit being capable of automatically receiving a value for the amount of the biomarker from the measurement unit.

[0257] In one embodiment, the detection system includes at least one detection agent for the biomarker.

[0258] The present invention further relates to a device for assessing myocardial infarction in a subject, the device comprising an evaluation unit comprising a database in which criteria for biomarkers are stored, the biomarkers being BMP10 type peptide, FGF23 or ANG2, and a data processor comprising instructions for performing a comparison of the amount of the biomarker with the criteria and assessing myocardial infarction based on said comparison, the evaluation unit being capable of receiving a value for the amount of the biomarker determined in a sample from the subject.

[0259] The present invention further relates to the use of i) a biomarker, which is a BMP10-type peptide, FGF23 or ANG2, or ii) at least one detection agent for said biomarker, for assessing myocardial infarction in a subject.

[0260] Finally, the present invention relates to a kit for assessing myocardial infarction in a subject, said kit comprising at least one detection agent for a biomarker, said biomarker being a BMP10-type peptide, FGF23 or ANG2.

[0261] It should be understood that the definitions and explanations of terms made above apply accordingly for all embodiments described in this specification and the appended claims. The following embodiments are specific embodiments contemplated in accordance with the present invention.

[0262] 1. A method for assessing myocardial infarction in a subject, comprising: (a) determining an amount of a first biomarker in a sample from the subject, wherein the first biomarker is a cardiac troponin, such as cardiac troponin T or I; (b) determining the amount of a second biomarker in the subject's sample, wherein the second biomarker is a BMP10-type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), a BNP-type peptide, such as NT-proBNP or BNP, cMyBPC (cardiac myosin-binding protein C) or ANG2 (angiopoietin 2); (c) comparing the amount of said biomarker with a standard for said biomarker and / or calculating a score for assessing myocardial infarction based on the amount of said biomarker; (d) assessing myocardial infarction based on the comparison and / or calculation performed in step (c).

[0263] 2. In step (b), (i) if the amount of a BMP10-type peptide is determined as the second biomarker, the method further comprises determining the amount of cMyBPC (cardiac myosin-binding protein C), at least one lipid biomarker, such as cholesterol (CHOL), LDL (low density lipoprotein), TRIGLY (triglyceride), APOAT (apolipoprotein A-1) or HDL (high density lipoprotein), at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6, as a third biomarker; or (ii) if the amount of FGF23 is determined as the second biomarker, the method further comprises determining the amount of at least one lipid biomarker, such as cMyBPC, CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7, as a third biomarker; or (iii) if the amount of a BNP-type peptide is determined as the second biomarker, the method further comprises determining the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2, as a third biomarker; or (iv) If the amount of ANG2 is determined as the second biomarker, the method further comprises determining the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7, as a third biomarker.

[0264] 3. The method of embodiment 1 or 2, wherein said sample is obtained from the subject upon presentation to an emergency department.

[0265] 4. The method of any one of embodiments 1-3, wherein the sample is a blood, serum or plasma sample and / or the subject is a human.

[0266] 5. The following: i. cardiac troponin and BMP10 type peptides; ii. cardiac troponin and FGF23; iii. cardiac troponin and ANG2; iv. cardiac troponin, BMP10 peptide and CRP; v. a cardiac troponin, a BMP10-type peptide, and at least one lipid biomarker, such as cholesterol (CHOL), LDL (low density lipoprotein), TRIGLY (triglyceride), APOAT (apolipoprotein A-1) or HDL (high density lipoprotein); vi. cardiac troponin and FGF23, vii. cardiac troponin and cMyBPC, viii. cardiac troponin, FGF23, and at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT, or HDL; ix. Cardiac troponin, ANG2 and cMyBPC; x. For example, in diabetic patients, cardiac troponin, ANG2 and LDL, or xi. Cardiac troponin, ANG2 and CHOL; the amount of the marker is determined; the cardiac troponin is troponin T or I, e.g., troponin T; The method according to any one of the preceding embodiments, wherein the BMP10-type peptide is BMP10, proBMP10 or NT-proBMP10, such as NT-proBMP10.

[0267] 6. A computer-implemented method for assessing myocardial infarction in a subject, comprising: (a) receiving a value for the amount of a first biomarker in the subject's sample, wherein the first biomarker is a cardiac troponin; (b) receiving a value for the amount of a second biomarker in the subject's sample, wherein the second biomarker is a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC, or ANG2; (c) comparing said value for said amount of said biomarker with a standard for said biomarker and / or calculating a score for assessing myocardial infarction based on said amount of said biomarker; (d) assessing myocardial infarction based on the comparison and / or calculation performed in step (c).

[0268] 7. In step (b), (i) if the value for the amount of a BMP10-type peptide is received as the second biomarker, the method further comprises receiving a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6, as a third biomarker; or (ii) if the value for the amount of FGF23 is received as the second biomarker, the method further comprises receiving a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7, as a third biomarker; or (iii) if the value for the amount of a BNP-type peptide is received as the second biomarker, the method further comprises receiving a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2, as a third biomarker; or (iv) The method of embodiment 6, wherein if the value for the amount of ANG2 is received as the second biomarker, the method further comprises receiving a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7, as a third biomarker.

[0269] 8. The method of any one of embodiments 1 to 7, wherein said assessment of myocardial infarction is a distinction between type 1 and type 2 myocardial infarction.

[0270] 9. The method of any one of embodiments 1-7, wherein said assessment of myocardial infarction is a diagnosis of type 2 myocardial infarction.

[0271] 10. The method of any one of embodiments 1-7, wherein said assessment of myocardial infarction is guidance of myocardial infarction treatment.

[0272] 11. A device for assessing myocardial infarction in a subject having myocardial infarction, comprising: (a) a measuring unit for determining the amount of a first biomarker, which is a cardiac troponin, and a second biomarker, which is a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC or ANG2, in a sample from the subject, the measuring unit comprising a detection system for the first biomarker and the second biomarker; (b) an evaluation unit operably coupled to the measurement unit, preferably comprising a database having stored references for the first and second biomarkers as described in any one of embodiments 1-10, and a data processor comprising instructions for performing a comparison of the amounts of the biomarkers and the second biomarker with the reference and / or for performing a calculation of a score for assessing myocardial infarction based on the amounts of the biomarkers or based on the calculated score, and for assessing myocardial infarction based on the comparison, preferably as described in any one of embodiments 1-10, wherein the evaluation unit is capable of automatically receiving values ​​for the amounts of the biomarkers from the measurement unit.

[0273] 12. The measurement unit determines a third biomarker and includes a detection system for the third biomarker, the database includes stored references for the third biomarker, and the third biomarker is: (i) if a BMP10-type peptide is the second biomarker, then the second biomarker is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; (ii) if FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, such as sFlt1, ANG2, or IGFBP7; (iii) when a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; or (iv) The device of embodiment 11, wherein when ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7.

[0274] 13. The device of embodiment 11 or 12, wherein the detection system comprises at least one detection agent for the first biomarker, at least one detection agent for the second biomarker, and optionally at least one detection agent for the third biomarker.

[0275] 14. A device for assessing myocardial infarction in a subject, said device comprising an evaluation unit comprising: a database with stored references for a first biomarker, said first biomarker being a cardiac troponin, and a second biomarker, said second biomarker being a BMP10 type peptide, FGF23, a BNP type peptide, cMyBPC or ANG2; and a data processor comprising instructions for performing a comparison of the amounts of said first and second biomarker with a reference, preferably as described in any one of embodiments 1 to 10, and assessing myocardial infarction based on said comparison, said evaluation unit being capable of receiving a value for the determined amount of said biomarkers in a sample of said subject.

[0276] 15. The database includes stored references for a third biomarker, the third biomarker being: (i) if a BMP10-type peptide is the second biomarker, then the second biomarker is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; (ii) when FGF23 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7; or (iii) when a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; or (iv) The device of embodiment 14, wherein when ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7.

[0277] 16. Use of i) a first biomarker which is a cardiac troponin and a second biomarker which is a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC or ANG2, or ii) at least one detection agent for the first biomarker and at least one detection agent for the second biomarker, for assessing myocardial infarction in a subject.

[0278] 17. A third biomarker or a detection agent for a third biomarker is further used, the third biomarker being: (i) when a BMP10-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; (ii) if FGF23 is the second biomarker, then cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT, or HDL, or at least one vascular biomarker, such as sFlt1, ANG2, or IGFBP7; (iii) when a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; or (iv) The device of embodiment 16, wherein when ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7.

[0279] 18. A kit for assessing myocardial infarction in a subject, comprising: i) at least one detection agent for a first biomarker which is a cardiac troponin; and ii) at least one detection agent for a second biomarker, wherein the second biomarker is a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC or ANG2.

[0280] 19. The kit further comprises a detection agent for a third biomarker, the third biomarker being: (i) when a BMP10-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; or (ii) when FGF23 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7; or (iii) if a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; (iv) The kit of embodiment 18, wherein when ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7.

[0281] 20. A method for assessing myocardial infarction in a subject, comprising: (a) determining the amount of a biomarker in a sample from the subject, wherein the biomarker is a BMP10 peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23) or ANG2 (angiopoietin 2); (b) comparing the amount of the biomarker to a reference for the biomarker; (c) assessing myocardial infarction based on the comparison made in step (c).

[0282] 21. A computer-implemented method for assessing myocardial infarction in a subject, comprising: (a) receiving a value for the amount of a biomarker in the subject's sample, wherein the biomarker is a BMP10-type peptide, FGF23, or ANG2; (b) comparing the value for the amount of said biomarker to a standard for said biomarker; (c) assessing the myocardial infarction based on the comparison made in step (c).

[0283] 22. The use of i) a biomarker, which is a BMP10-type peptide, FGF23 or ANG2, or ii) at least one detection agent for said biomarker, for assessing myocardial infarction in a subject.

[0284] 23. The method, device, use or kit according to any one of the preceding embodiments, wherein the BMP10-type peptide is BMP10, proBMP10 or NT-proBMP10.

[0285] 24. The method, device, use or kit of any one of the preceding embodiments, wherein said BNP-type peptide is NT-proBNP, proBNP or BNP.

[0286] 25. The method, device, use or kit of any one of the preceding embodiments, wherein said (second) biomarker is ANG2 and said subject is a subject suffering from diabetes.

[0287] 26. The method, device, use or kit of any one of the preceding embodiments, wherein said assessment of myocardial infarction is a distinction between type 1 and type 2 myocardial infarction.

[0288] All references referenced throughout this specification are incorporated herein in their entirety for all disclosure content not specifically mentioned above. EXAMPLES

[0289] The examples are merely illustrative of the invention and should not be construed as limiting its scope.

[0290] 1. Biomarker determination 1.1. Biomarker Determination by Sandwich Assay on the cobas Elecsys® ECLIA Platform (ECLIA Assay from Roche Diagnostics, Germany) The Elecsys® electrochemiluminescence (ECL) technology and assay method for the determination of GDF-15 are briefly described below. The concentration of GDF-15 was determined by a cobas e 801 analyzer. The detection of GDF-15 using the cobas e 801 analyzer is based on the Elecsys® electrochemiluminescence (ECL) technology. Briefly, biotin-labeled and ruthenium-labeled antibodies are combined with the respective amounts of undiluted sample and incubated in the analyzer. Subsequently, streptavidin-coated magnetic microparticles are added and incubated in the instrument to promote the binding of the biotin-labeled immunocomplexes. After this incubation step, the reaction mixture is transferred to the measurement cell, where the beads are magnetically captured on the surface of the electrode. Then, in order to separate the bound immunoassay complexes from the remaining free particles, ProCell M buffer containing tripropylamine (TPA) for the subsequent ECL reaction is introduced into the measurement cell. The induction of a voltage between the working and counter electrodes then initiates a reaction that results in the emission of photons by the ruthenium complex and TPA. The resulting electrochemiluminescent signals are recorded by photomultiplier tubes and converted to a numerical value indicative of the concentration level of each analyte.

[0291] cTNTh (high-sensitivity c-troponin T), NTpBNP (N-terminal prohormone of brain natriuretic peptide), sFLT-1 (soluble fms-like tyrosine kinase-1), GDF15 (growth / differentiation factor 15) and IL6 (interleukin 6), cMyBPC (cardiac myosin-binding protein C), BMP10 (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), IGFBP7 (insulin-like growth factor binding protein 7), ANG2 (angiopoietin 2) and ESM-1 (endothelial cell-specific molecule 1) were measured by sandwich immunoassay.

[0292] cTNThs (high-sensitivity c-troponin T), NTpBNP (N-terminal prohormone of brain natriuretic peptide), sFLT-1 (soluble fms-like tyrosine kinase-1), GDF15 (growth / differentiation factor 15) and IL6 (interleukin 6) were measured with commercially available ECLIA assays developed for the cobas Elecsys® ECLIA platform (ECLIA assays from Roche Diagnostics, Germany) in EDTA plasma samples according to the manufacturer's instructions for the cobas Elecsys® ECLIA immunoassay platform (Roche Diagnostics, Germany).

[0293] cTNThs: (Troponin T hs Elecsys G5), electrochemiluminescence immunoassay. NTproBNP: (proBNP-II Elecsys), electrochemiluminescence immunoassay. GDF15: (GDF-15 Elecsys), electrochemiluminescence immunoassay. sFLT-1: (FLT1 Elecsys), electrochemiluminescence immunoassay. IL-6: (IL6 Elecsys), electrochemiluminescence immunoassay.

[0294] cMyBPC (cardiac myosin binding protein C), BMP10 (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), IGFBP7 (insulin-like growth factor binding protein 7), ANG2 (angiopoietin 2) and ESM-1 (endothelial cell specific molecule 1) were measured with a robust prototype ECLIA assay. These sandwich immunoassays were developed in-house for the cobas Elecsys® ECLIA platform (ECLIA assays from Roche Diagnostics, Germany). The assays contain biotinylated and rutheniumylated monoclonal antibodies that specifically bind the analyte from EDTA plasma samples and are measured on a cobas Elecsys® ECLIA immunoassay platform analyzer (Roche Diagnostics, Germany). For example, an assay was used for BMP10 that uses an antibody that binds to NT-proBMP10. Thus, this assay detects NT-proBMP10 and BMP10 type peptides that contain the NT-proBMP10 sequence.

[0295] 1.2. Biomarker Determination on the cobas® Clinical Chemistry Analyzer Platform (Roche Diagnostics, Germany) CRPh (high-sensitivity C-reactive protein), CysC2 (cystatin C) and lipid biomarkers CHOL (cholesterol), TRIGL (triglycerides), LDL (low-density lipoprotein), HDL (high-density lipoprotein), and APOAT (apolipoprotein A-1) were measured by using commercial assays (Roche Diagnostics, Germany) in EDTA plasma samples according to the manufacturer's instructions on the cobas® clinical chemistry analyzer platform cobas c 501 (Roche Diagnostics, Germany).

[0296] The following assays were used: CRPhs: (Cat. No. 04628918 190 Cardiac-C-Reactive Protein (Latex) hs), Particle-Enhanced Immunoturbidimetric Assay APOAT: (Cat. No. 03032566 Tina quant Apolipoprotein A-1 ver 2), Immunoturbidimetric Assay CHOL: (Cat. No. 03039773 Cholesterol Gen.2), an enzymatic colorimetric assay HDL: (Cat. No. 07528566 HDL-C Gen), a homogeneous enzymatic colorimetric assay LDL: (Cat. No. 07005717 LDL-C Gen3), homogeneous enzymatic colorimetric assay TRIGL: (Cat. No. 20767107 Triglycerides), an enzymatic colorimetric assay

[0297] 2. Patient cohort, APACE study The Favorable Predictors of Acute Coronary Syndrome Evaluation (APACE) study is described by Nestelberger et al JAMA Cardiol. 2021;6(7):771-780. doi:10.1001 / jamacardio.2021.0669. In brief, the study is an international multicenter prospective cohort study of unselected patients presenting with acute chest pain at rest within the past 12 hours enrolled in the emergency department (ED) and registered with ClinicalTrials.gov (identifier: NCT00470587). The diagnosis of acute myocardial infarction (AMI) is made in consecutively enrolled patients against clinical criteria (final diagnosis) using all clinical information including cardiac imaging and serial measurements of high-sensitivity troponin, with central adjudication by two independent cardiologists according to the universal definition of AMI (Thygesen K et al, Eur Heart J. 2019;40:226).

[0298] AMI was diagnosed when there was evidence of myocardial necrosis in association with a clinical setting consistent with myocardial ischemia. Myocardial necrosis was diagnosed by at least one hs-cTnT / I value above the 99th percentile with significant elevation and / or depression. Absolute changes in high-sensitivity troponin T and I (hs-cTnT / I) were used to determine significant changes based on the diagnostic significance of absolute changes over relative changes. All other patients were classified into the categories of unstable angina, noncardiac chest pain, cardiac but noncoronary artery disease (e.g., myocarditis, takotsubo cardiomyopathy, heart failure), and symptomatic of unknown etiology with normal hs-cTnT / I levels.

[0299] Definitions of Type 1 and Type 2 Myocardial Infarction: Type 1 AMI (T1MI) and type 2 AMI (T2MI) were defined according to the universal definition of AMI (4th edition, Thygesen K et al, Eur Heart J. 2019;40:226). In brief, both diagnoses (T1 and T2 AMI) require clinical evidence of acute myocardial ischemia, such as changes in cardiac troponin, but with an atherothrombotic origin for T1MI and a nonatherothrombotic origin for T2MI. More specifically, evidence of myocardial necrosis in a clinical setting consistent with acute myocardial ischemia, type 1 MI, was defined as spontaneous MI associated with a primary atherothrombotic coronary event, such as plaque erosion or plaque rupture, intraluminal coronary thrombus, or distal microembolism. Type 2 MI was defined as secondary due to a mismatch between oxygen supply and demand. Symptoms reflecting an imbalance between myocardial oxygen supply and demand included bradycardial or tachyarrhythmias, hypoxemia, hypotension, hypertension, severe anemia, coronary artery spasm, dissection, and coronary embolism. Underlying coronary artery disease was possible but not required for the diagnosis of T2MI. To qualify for T2MI, dynamic changes in hs-cTnT / I were required, the same as for T1MI. Documentation of a clear trigger was essential for the diagnosis of T2MI, as recommended. Coronary angiography was not mandatory for the diagnosis of T1MI, to limit the possible effects of selection bias resulting from clinical referral to coronary angiography. No other subtypes of MI were reported.

[0300] Example 1: Patients in the APACE Study From a total of 4216 patients in the APACE study, N = 874 patients were diagnosed with AMI (20.7%). Of these, 708 patients were diagnosed with type 1 MI (564 patients with non-ST-elevation AMI and 144 patients with ST-elevation myocardial infarction) and 166 patients were diagnosed with type 2 MI (164 patients with non-ST-elevation myocardial infarction and 2 patients with ST-elevation myocardial infarction). Cases (N=166): Patients with type 2 MI secondary to ischemia due to either increased oxygen demand or decreased supply. Of these, 58 patients were female and 108 patients were male. Controls (N=708): Patients with type 1 MI or spontaneous MI associated with ischemia due to a primary coronary event such as plaque erosion and / or plaque rupture, fissure formation, or dissection. Of these, 191 patients were female and 517 patients were male.

[0301] Statistical analysis: Biomarker values ​​were logarithmically transformed to base 2 and mathematically combined by logistic regression. The "area under the receiver operating characteristic curve" (AUC) was used as a general measure of marker performance.

[0302] Biomarkers are differentially elevated in type 1 or type 2 myocardial infarction [Table 1]

[0303] Example 2: Use of a combination of hsTnT and BMP10 The following examples show how a score can actually be derived from the measured biomarker concentrations from patients with type 1 and type 2 MI, respectively. 1型 and BMP10 1型 are the concentrations obtained from assays measuring hsTnT and BMP10, respectively, in patients with type 1 MI. 2型 and hsTnT2型 Let be the concentrations obtained from assays measuring hsTnT and BMP10, respectively, in type 2 MI patients.

[0304] β0 is the offset, and β hsTnT and β BMP10 Let be the weighting factor (coefficient) of the mathematical model used to combine the obtained concentrations.

[0305] Furthermore, α is taken as a predefined cutoff that optimally separates type 1 and type 2 AMI patients.

[0306] Score 1型 The score of biomarker concentrations measured for type 1 AMI patients, denoted as , is obtained by calculating: Score 1型 =β0+β hsTnT log2(hsTnT 1型 )+β BMP10 log2(BMP10 1型 )

[0307] Here, log2() represents the logarithm function to base 2.

[0308] Score 1型 The score of biomarker concentrations measured for type 2 AMI patients, denoted as , is obtained by calculating: Score 2型 =β0+β hsTnT log2(hsTnT 2型 )+β BMP10 log2(BMP10 2型 )

[0309] If the score obtained is greater than the predetermined value of the cutoff α, it is suggested that the patient suffers from type 2 MI. If the score obtained is less than the predetermined value of the cutoff α, it is suggested that the patient suffers from type 1 MI.

[0310] Table 2: Univariate performance of hsTnT (AUC) considering all patient subgroups and combinations of hsTnT with a second marker (bivariate marker combinations) and a third marker (trivariate marker combinations) have an improved AUC by at least 2.0 improvement in AUC (collision AUC) over the single marker hsTnT. [Table 2-1] [Table 2-2]

[0311] Table 3: Univariate performance of hsTnT (AUC) considering all patient subgroups and combinations of hsTnT with a second marker (bivariate marker combinations) and a third marker (trivariate marker combinations) that did not have an improved AUC of at least 2.0 (collision AUC) over the single biomarker hsTnT. [Table 3]

[0312] As shown in Table 3, some biomarker combinations do not improve the AUC value over the single marker hsTnT for assessment of T1MI vs. T2MI.

[0313] Example 3: Diabetic Patients from the APACE Study Cases (N=166): Patients with type 2 MI secondary to ischemia due to either increased oxygen demand or decreased supply. Of these, 37 patients had a history of diabetes and 129 did not. Controls (N=708): Patients with type 1 MI or spontaneous MI associated with ischemia due to a primary coronary event such as plaque erosion and / or plaque rupture, fissure formation or dissection. Of these, 196 patients had a history of diabetes and 512 did not.

[0314] Table 5 provides the performance of the marker combinations in patients with and without a history of diabetes. Table 5 shows the improved performance of hsTNT in combination with several biomarker panels. It is particularly interesting that all biomarker panels that improve the performance of hsTNT include ANG2. The hsTNT and ANG2 biomarker panel shows an improvement over hsTNT with a significant AUC delta change of 4.875 (AUC 66.870 -> AUC 73.745). The addition of a third biomarker, LDL, to hsTNT and ANG2 further improves compared to hsTNT with an AUC delta change of 6.805 (AUC 68.870 -> AUC 75.675) in the subgroup of diabetic patients.

[0315] It can be concluded from these results that in a subgroup of diabetic patients it is beneficial to use a biomarker combination of hsTNT and a second biomarker, ANG2.

[0316] Table 4: Type 1 MI patients with and without a history of diabetes and Type 2 MI patients with and without a history of diabetes [Table 4]

[0317] Table 5: Univariate performance of hsTnT (AUC) considering all patients with a history of diabetes, as well as combinations of hsTnT with a second marker (bivariate marker combinations) and a third marker (trivariate marker combinations). All bivariate and trivariate combinations have an improvement in AUC of at least 3.0 in patients with a history of diabetes versus those without a history of diabetes. All bivariate and trivariate combinations with an improvement in AUC of at least 3.0 over the univariate performance of hsTnT in both subgroups are reported for the markers used in the combination for reference. [Table 5]

[0318] Table 6: Univariate performance of hsTnT (AUC) considering all patients with a history of diabetes and combinations of hsTnT with a second marker (bivariate marker combinations) and a third marker (trivariate marker combinations). All bivariate and trivariate combinations did not have an improvement in AUC of at least 3.0 in patients with a history of diabetes vs. those without a history of diabetes. Univariate performance in both subgroups is reported for the markers used in combination for the criteria. [Table 6]

Claims

1. 1. A method for assessing myocardial infarction in a subject, comprising: (a) determining an amount of a first biomarker in a sample from the subject, wherein the first biomarker is a cardiac troponin, such as cardiac troponin T or I; (b) determining the amount of a second biomarker in the subject's sample, wherein the second biomarker is a BMP10 type peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23), a BNP type peptide, e.g. NT-proBNP or BNP, cMyBPC (cardiac myosin binding protein C) or ANG2 (angiopoietin 2); (c) comparing the amount of said biomarker with a standard for said biomarker and / or calculating a score for assessing myocardial infarction based on the amount of said biomarker; (d) assessing myocardial infarction based on the comparison and / or calculation performed in step (c); A method comprising:

2. In step (b), (i) if the amount of a BMP10 type peptide is determined as the second biomarker, the method further comprises determining the amount of cMyBPC (cardiac myosin binding protein C), at least one lipid biomarker, such as cholesterol (CHOL), LDL (low density lipoprotein), TRIGLY (triglyceride), APOAT (apolipoprotein A-1) or HDL (high density lipoprotein), at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6, as a third biomarker; or (ii) if the amount of FGF23 is determined as the second biomarker, the method further comprises determining the amount of at least one lipid biomarker, such as cMyBPC, CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7, as a third biomarker; or (iii) if the amount of a BNP-type peptide is determined as the second biomarker, the method further comprises determining the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2, as a third biomarker; or (iv) if the amount of ANG2 is determined as the second biomarker, the method further comprises determining the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7, as a third biomarker; The method of claim 1.

3. 3. The method of claim 1 or 2, wherein the sample is obtained from the subject upon presentation to an emergency department.

4. The method according to any one of claims 1 to 3, wherein the sample is a blood, serum or plasma sample and / or the subject is a human.

5. below: i. Cardiac troponin and BMP10 type peptides; ii. cardiac troponin and FGF23; iii. Cardiac troponin and ANG2; iv. cardiac troponin, BMP10 peptide and CRP; v. a cardiac troponin, a BMP10 peptide, and at least one lipid biomarker, such as cholesterol (CHOL), LDL (low density lipoprotein), TRIGLY (triglyceride), APOAT (apolipoprotein A-1) or HDL (high density lipoprotein); vi. Cardiac troponin and cMyBPC; vii. cardiac troponin, FGF23, and at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT, or HDL; viii. cardiac troponin, ANG2 and cMyBPC; ix. For example, in diabetic patients, cardiac troponin, ANG2 and LDL, or x. cardiac troponin, ANG2 and CHOL; the amount of the marker is determined; the cardiac troponin is troponin T or I, e.g., troponin T; The BMP10 type peptide is BMP10, proBMP10 or NT-proBMP10, for example NT-proBMP10; The method according to any one of claims 1 to 4.

6. 1. A computer-implemented method for assessing myocardial infarction in a subject, comprising: (a) receiving a value for the amount of a first biomarker in the subject's sample, wherein the first biomarker is a cardiac troponin; (b) receiving a value for the amount of a second biomarker in the subject's sample, wherein the second biomarker is a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC, or ANG2; (c) comparing said value for said amount of said biomarker with a standard for said biomarker and / or calculating a score for assessing myocardial infarction based on said amount of said biomarker; (d) assessing myocardial infarction based on said comparison and / or said calculation performed in step (c); Including, Optionally, in step (b), (i) if the value for the amount of BMP10 peptide is received as the second biomarker, the method further comprises receiving as a third biomarker a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; or (ii) if the value for the amount of FGF23 is received as the second biomarker, the method further comprises receiving as a third biomarker a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7; or (iii) if the value for the amount of a BNP-type peptide is received as the second biomarker, the method further comprises receiving a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2, as a third biomarker; or (iv) if the value for the amount of ANG2 is received as the second biomarker, the method further comprises receiving a value for the amount of cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7, as a third biomarker. Computer-implemented method.

7. A device for assessing myocardial infarction in a subject, said device comprising an evaluation unit comprising a database with stored references for a first biomarker, said first biomarker being a cardiac troponin, and a second biomarker, said second biomarker being a BMP10 type peptide, FGF23, a BNP type peptide, cMyBPC or ANG2, and a data processor comprising instructions for performing a comparison of the amounts of said first and second biomarker with a reference, preferably as claimed in any one of claims 1 to 10, and assessing myocardial infarction based on said comparison, said evaluation unit being able to receive a value for the determined amount of said biomarkers in a sample of said subject, and optionally The database includes stored references for a third biomarker, the third biomarker being: (i) when a BMP10 type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; (ii) when FGF23 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7; or (iii) when a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; or (iv) when ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7; A device for assessing myocardial infarction in a subject.

8. The use of i) a first biomarker which is a cardiac troponin, and a second biomarker which is a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC or ANG2, or ii) at least one detection agent for the first biomarker and at least one detection agent for the second biomarker, for assessing myocardial infarction in a subject.

9. A third biomarker or a detection agent for a third biomarker may also be used, said third biomarker being: (i) when a BMP10 type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; (ii) when FGF23 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7; (iii) when a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; or (iv) when ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7; 9. The use according to claim 8.

10. 1. A kit for assessing myocardial infarction in a subject, comprising: i) at least one detection agent for a first biomarker which is a cardiac troponin; and ii) at least one detection agent for a second biomarker, wherein said second biomarker is a BMP10-type peptide, FGF23, a BNP-type peptide, cMyBPC or ANG2; and optionally The kit further comprises a detection agent for a third biomarker, the third biomarker being: (i) when a BMP10 type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, at least one vascular biomarker, such as FGF23, sFlt1, GDF15, ESM1, ANG2 or IGFBP7, or at least one inflammatory biomarker, such as hsCRP or IL6; or (ii) when FGF23 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as sFlt1, ANG2 or IGFBP7; or (iii) when a BNP-type peptide is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as FGF23 or ANG2; (iv) when ANG2 is the second biomarker, it is cMyBPC, at least one lipid biomarker, such as CHOL, LDL, TRIGLY, APOAT or HDL, or at least one vascular biomarker, such as ESM1 or IGFBP7; kit.

11. 1. A method for assessing myocardial infarction in a subject, comprising: (a) determining the amount of a biomarker in the subject's sample, wherein the biomarker is BMP10 peptide (bone morphogenetic protein type 10 peptide), FGF23 (fibroblast growth factor 23) or ANG2 (angiopoietin 2); (b) comparing the amount of the biomarker to a reference for the biomarker; (c) assessing myocardial infarction based on the comparison made in step (c); A method comprising:

12. 1. The use of i) a biomarker, which is a BMP10 type peptide, FGF23 or ANG2, or ii) at least one detection agent for said biomarker, for assessing myocardial infarction in a subject.

13. The BMP10 type peptide is BMP10, proBMP10 or NT-proBMP10; the BNP-type peptide is NT-proBNP, proBNP or BNP, and / or the (second) biomarker is ANG2 and the subject is a subject suffering from diabetes; 13. A method, device, use or kit according to any one of the preceding claims.

14. The assessment of myocardial infarction is a) Differentiating between type 1 and type 2 myocardial infarction; b) a diagnosis of type 2 myocardial infarction, or c) Guidance on the treatment of myocardial infarction; 13. A method, device, use or kit according to any one of the preceding claims.

15. The kit of any one of claims 12 to 14, wherein the detection agent is an antibody or antigen-binding fragment thereof that specifically binds to the biomarker.

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