Application of circulating DKK3 (Dickkopf-related protein 3) in the assessment of atrial fibrillation

Abstract

The present invention relates to a method for assessing atrial fibrillation in a subject, the method comprising the steps of determining the amount of DKK3 in a sample from the subject and comparing the amount of DKK3 with a reference amount, thereby assessing atrial fibrillation. In addition, the present invention relates to a method for diagnosing heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure.

Description

Technical Field

[0001] The present invention relates to a method for assessing atrial fibrillation in a subject, the method comprising the following steps: determining the amount of DKK3 in a sample from the subject, and comparing the amount of DKK3 with a reference amount, thereby assessing atrial fibrillation. In addition, the present invention relates to a method for diagnosing heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure. Background Art

[0002] Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and one of the most prevalent conditions in the elderly population. AF is characterized by irregular heart beats and usually begins with a brief abnormal beat that increases over time and can become a permanent condition. An estimated 2.7-6.1 million Americans have AF, and approximately 33 million people worldwide have AF (Chugh SS et al., Circulation 2014; 129: 837-47). Patients with AF have a higher rate of stroke and are at a higher risk of developing congestive heart failure compared to patients in sinus rhythm.

[0003] The diagnosis of arrhythmias such as atrial fibrillation generally involves the determination of the cause of the arrhythmia and the classification of the arrhythmia. The guidelines for the classification of atrial fibrillation according to the American College of Cardiology (ACC), the American Heart Association (AHA) and the European Society of Cardiology (ESC) are based primarily on simplicity and clinical relevance. The first category is called "first detected AF". People in this category are initially diagnosed with AF and may or may not have had previously undetected episodes. If the first detected episode stops on its own in less than a week, but another episode occurs afterwards, the category becomes "paroxysmal AF". Although the episodes of patients in this category can last up to 7 days, in most cases of paroxysmal AF, the episodes will stop in less than 24 hours. If the episode lasts for more than a week, it is classified as "persistent AF". If this episode cannot be stopped, that is, it cannot be stopped by electrical cardioversion or drug cardioversion, and lasts for more than a year, the classification becomes "permanent AF". Since atrial fibrillation is an important risk factor for stroke and systemic embolism, early diagnosis of atrial fibrillation is highly desirable (Hart et al., Ann Intern Med 2007; 146(12): 857-67; Go AS et al. JAMA 2001; 285(18): 2370-5).

[0004] DKK3 is also known as Dickkopf-related protein 3, REIC, RIG, Dickkopf WNT signaling pathway inhibitor 3. DKK3 belongs to the DKK family, which encodes secretory proteins and consists of four main members in vertebrates (Dkk 1, Dkk2, Dkk 3, Dkk 4). DKK contains a signal sequence and shares two conserved cysteine-rich domains, each of which shows characteristic spacing between cysteine ​​and other conserved amino acids. In addition, DKK3 has an sgy domain, which is only found in DKKL1 (Soggy). Various studies have found that DKK family members regulate Wnt signaling.

[0005] The gene DKK3 gene has a length of 350 bp, is located on chromosome 11p15.3, consists of 9 exons, and has two promoters. Methylation of the main promoter region was detected in cell lines derived from various human tumors, in which the expression of the DKK3 gene was reduced (Kobayashi, Gene, Vol. 282, No. 1-2, January 9, 2002, pp. 151-158).

[0006] The DKK3 gene encodes multiple transcripts widely expressed in normal human tissues. The DKK3 gene is transcribed into three different isoforms; all variants have 2 to 8 exons and encode a functional protein of 350 amino acids. (Katase et al., Atlas Genet Cytogenet Oncol Haematol. 2013; 17 (10) p. 678-686). In addition, Leonard et al. (PLOS, July 24, 2017) showed the identification of DKK3b transcripts, which are isoforms of DKK3, derived from the second transcription start site located in intron 2 of the DKK3 gene.

[0007] Northern blot analysis showed that DKK3 mRNA is expressed in brain, heart, lung, liver, pancreas, spleen, kidney, small intestine, colon, skeletal muscle and placenta. Among them, DKK3 expression is particularly high in heart and brain (Katase et al., Atlas Genet Cytogenet Oncol Haematol. 2013; 17 (10) p. 678-686). DKK3 is also expressed in many tissues including heart during embryonic development. The expression of this gene is reduced in a variety of cancer cell lines, and it can be used as a tumor suppressor gene.

[0008] DKK3 levels are elevated in circulating blood in the elderly, and its expression is upregulated during cellular senescence of prostate basal epithelial cells, suggesting that DKK3 may play a role in age-related disorders (Zenzmaier et al., Experimental Gerontology, Vol. 43, No. 9, September 2008, pp. 867-870).

[0009] Loss of function and gain of function analysis performed in vivo and in vitro in mice demonstrated the regulatory role of DKK3 in protecting the heart from developing pathological myocardial hypertrophy. The results showed that the loss of DKK3 increased myocardial hypertrophy, fibrosis and dysfunction caused by pressure overload, while overexpression of DKK3 protected the heart from cardiac remodeling caused by pressure overload (Zhang et al., Cardiovascular Research, 2014, 104, p.35-45). DKK3 knockout mice further showed changes in hemoglobin and hematocrit levels and lung ventilation, especially.

[0010] Bao et al. (Basic Res Cardiol. 2015 May; 110(3)) analyzed the functional role of DKK3 in cardiac remodeling after myocardial infarction (MI). The scientists induced MI by surgical ligation of the left anterior descending coronary artery in transgenic mice expressing cardiac-specific DKK3 and DKK3 knockout (KO) mice and their non-transgenic and DKK3(+ / +) littermates. Their results showed that after MI, DKK3-deficient mice had increased mortality, larger infarct size, and exacerbated left ventricular (LV) dysfunction. In contrast, DKK3 overexpression resulted in the opposite phenotype after infarction. The authors proposed that DKK3 could represent a potential therapeutic target for the treatment of heart failure after MI.

[0011] In addition, Yu et al. (Circulation. 2017; 136: 1022-1036) found in the population-based prospective Bruneck study that people with higher levels of DKK3 were less likely to develop atherosclerosis and less likely to die from a heart attack over a five-year period. This association was independent of other atherosclerosis risk factors, such as high blood pressure and cholesterol levels. The study provides evidence for the role of DKK3 in preventing atherosclerosis involving endothelial migration and repair. The authors revealed potential therapeutic implications for atherosclerosis by using DDK3 to prevent the accumulation of fatty substances in the arteries. Therefore, DKK3 can be used as a parameter of responsive secretion to prevent heart attacks caused by atherosclerosis.

[0012] US8617877 describes a method for screening heart disease patients as candidates for stem cell therapy by measuring DDK3 protein, for example in a biological sample from the patient, and comparing the measured value to a baseline value, wherein a statistically significant amount of the protein compared to the baseline value indicates that the patient will likely benefit from stem cell therapy.

[0013] However, the involvement of DKK3 in atrial fibrillation and stroke remains unknown. Latini R. et al. (J Intern Med. 2011 Feb; 269(2): 160-71) measured various circulating biomarkers (hsTnT, NT-proBNP, MR-proANP, MR-proADM, copeptin and CT-proendothelin-1) in patients with atrial fibrillation.

[0014] Reliable methods are needed to assess atrial fibrillation, including the diagnosis of atrial fibrillation, risk stratification of patients with atrial fibrillation (such as the occurrence of stroke), assessment of the severity of atrial fibrillation, and assessment of treatment for patients with atrial fibrillation. Summary of the invention

[0015] The technical problem of the present invention can be seen as providing a method to meet the above needs. This technical problem is solved by the embodiments characterized by the claims and below.

[0016] Advantageously, it was found in the context of the present invention that the determination of the amount of DKK3 in a sample from a subject allows an improved assessment of atrial fibrillation. Due to the present invention, it is possible, for example, to diagnose whether a subject suffers from atrial fibrillation. The present invention also provides a method for stroke prediction. In addition, it is possible, for example, to distinguish between paroxysmal atrial fibrillation and persistent atrial fibrillation in a subject suffering from atrial fibrillation.

[0017] Therefore, the present invention relates to a method for assessing atrial fibrillation in a subject, the method comprising the following steps:

[0018] a) determining the amount of DKK3 in a sample from the subject, and

[0019] b) Comparing the amount of DKK3 with a reference amount to thereby assess atrial fibrillation.

[0020] In one embodiment of the method of the present invention, the method further comprises determining the amount of natriuretic peptide and / or ESM1 in the sample from the subject in step a), and comparing the amount of natriuretic peptide and / or ESM1 with a reference amount in step b).

[0021] Therefore, the present invention relates to a method for assessing atrial fibrillation in a subject, the method comprising the following steps:

[0022] a) determining the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 in a sample from the subject, and

[0023] b) comparing the amount with a reference amount, thereby assessing atrial fibrillation.

[0024] The assessment of atrial fibrillation (AF) should be based on comparison of the results of step b).

[0025] Therefore, the present invention preferably includes the following steps

[0026] a) determining the amount of DKK3, and optionally the amount of a natriuretic peptide and / or ESM1 in a sample from a subject,

[0027] b) comparing the amount of DKK3 to a reference amount, and optionally, comparing the amount of a natriuretic peptide and / or ESM1 to a reference amount, and

[0028] c) assessing atrial fibrillation based on the results of comparing step b).

[0029] The method according to the present invention comprises the method that is basically composed of the above steps or comprises other steps.In addition, the method of the present invention is preferably an in vitro method, more preferably an in vitro method.In addition, it can also include the step except the step clearly mentioned above.For example, other steps may relate to measuring other markers and / or sample pretreatment or evaluate the result obtained by the method.The method can be performed manually or assisted by automation.Preferably, step (a), (b) and / or (c) can be assisted by automation in whole or in part, for example, by suitable robot and sensory equipment to carry out the determination in step (a) or the calculation implemented by computer in step (b). BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Weighted Kaplan-Meier survival estimates for both groups were defined by baseline DKK3 measurements <= 28 NPX vs. > 28 NPX.

[0031] Figure 2 : Differential expression of DKK3 in persistent AFib vs. SR tissue samples assessed by RNAseq of the MAPPING cohort. In persistent AF, DKK3 expression was 1.496-fold higher in perAF compared to SR (FDR=0,0001).

[0032] Figure 3 : Diagnostic value of DKK3 in the PREDICTOR AFib subgroup; AUC = 0.66. Diagnosis of AF.

[0033] Figure 4: Diagnostic value of GISSI AF AFib subgroup; AUC (24 weeks) = 0.64, AUC (52 weeks) = 0,65. Diagnosis of AF. DETAILED DESCRIPTION

[0034] According to the present invention, atrial fibrillation should be assessed. As used herein, the term "assessing atrial fibrillation" preferably refers to the diagnosis of atrial fibrillation, the differentiation between paroxysmal atrial fibrillation and persistent atrial fibrillation, the prediction of the risk of adverse events associated with atrial fibrillation, the identification of subjects who should undergo an electrocardiogram (ECG) examination, or the assessment of atrial fibrillation treatment.

[0035] As will be appreciated by those skilled in the art, the assessment of the present invention is generally not intended to be correct for 100% of the subjects to be tested. The term preferably requires that a statistically significant portion of the subject can be correctly assessed (such as the diagnosis, differentiation, prediction, identification or assessment of the treatment described herein). Whether a portion is statistically significant can be determined by those skilled in the art without further effort using various well-known statistical evaluation tools (e.g., determination of confidence intervals, determination of p-values, Student's t-tests, Mann-Whitney tests, etc.). For details, see Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-value is preferably 0.4, 0.1, 0.05, 0.01, 0.005 or 0.0001.

[0036] It is known in the art that biomarkers can be increased or decreased in various diseases and disorders. This also applies to DKK3, which is known to be decreased in various cancer cell lines and may function as a suppressor gene.

[0037] However, the skilled person will take this into account. Thus, "atrial fibrillation assessment" should be understood as aiding in the assessment of atrial fibrillation, and therefore aiding in the diagnosis of atrial fibrillation, aiding in the differentiation of paroxysmal atrial fibrillation from persistent atrial fibrillation, aiding in the prediction of the risk of adverse events associated with atrial fibrillation, aiding in the identification of subjects who should undergo an electrocardiogram (ECG), or aiding in the assessment of treatment for atrial fibrillation.

[0038] In a preferred embodiment of the present invention, the assessment of atrial fibrillation is the diagnosis of atrial fibrillation. Thus, it is diagnosed whether the subject suffers from atrial fibrillation.

[0039] Accordingly, the present invention contemplates a method for diagnosing atrial fibrillation in a subject, the method comprising the steps of:

[0040] a) determining the amount of DKK3 in a sample from the subject, and

[0041] b) comparing the amount of DKK3 with a reference amount, thereby diagnosing atrial fibrillation.

[0042] In one embodiment, the aforementioned method comprises the following steps:

[0043] a) determining the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 in a sample from the subject, and

[0044] b) comparing the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 with a reference amount, thereby diagnosing atrial fibrillation.

[0045] Preferably, the subject to be tested in connection with the method for diagnosing atrial fibrillation is a subject suspected of having atrial fibrillation. However, it is also envisaged that the subject has previously been diagnosed with AF and that the previous diagnosis is confirmed by carrying out the method of the invention.

[0046] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is to distinguish between paroxysmal atrial fibrillation and persistent atrial fibrillation. Thus, it is determined whether the subject suffers from paroxysmal or persistent atrial fibrillation.

[0047] Therefore, the present invention contemplates a method for distinguishing between paroxysmal atrial fibrillation and persistent atrial fibrillation in a subject, the method comprising the steps of:

[0048] a) determining the amount of DKK3 in a sample from the subject, and

[0049] b) Comparing the amount of DKK3 with a reference amount to distinguish between paroxysmal atrial fibrillation and persistent atrial fibrillation.

[0050] In one embodiment, the aforementioned method comprises the following steps:

[0051] a) determining the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 in a sample from the subject, and

[0052] b) comparing the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 with a reference amount, thereby distinguishing between paroxysmal atrial fibrillation and persistent atrial fibrillation.

[0053] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is a prediction of the risk of an adverse event associated with atrial fibrillation, such as stroke. Thus, the risk of a subject having and / or not having said adverse event is predicted.

[0054] Thus, the present invention contemplates a method for predicting a subject's risk of an adverse event associated with atrial fibrillation, the method comprising the steps of:

[0055] a) determining the amount of DKK3 in a sample from the subject, and

[0056] b) comparing the amount of DKK3 to a reference amount to thereby predict the risk of an adverse event associated with atrial fibrillation.

[0057] In one embodiment, the aforementioned method comprises the following steps:

[0058] a) determining the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 in a sample from the subject, and

[0059] b) comparing the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 with a reference amount, thereby predicting the risk of an adverse event associated with atrial fibrillation.

[0060] It is envisioned that various adverse events can be predicted. A preferred adverse event to be predicted is stroke.

[0061] Therefore, the present invention particularly contemplates a method for predicting the risk of stroke in a subject, the method comprising the following steps:

[0062] a) determining the amount of DKK3 in a sample from the subject, and

[0063] b) Compare the amount of DKK3 with the reference amount to predict the risk of stroke.

[0064] The aforementioned method may further include a step c) of predicting stroke based on the comparison result of step b). Therefore, steps a), b), and c) are preferably as follows:

[0065] a) determining the amount of DKK3 in a sample from the subject, and

[0066] b) comparing the amount of DKK3 to a reference amount, and

[0067] c) predicting stroke based on the comparison result of step b)

[0068] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is an assessment of treatment of atrial fibrillation.

[0069] Accordingly, the present invention contemplates a method for assessing treatment of atrial fibrillation in a subject, the method comprising the steps of:

[0070] a) determining the amount of DKK3 in a sample from the subject, and

[0071] b) Comparing the amount of DKK3 with a reference amount to assess the treatment of atrial fibrillation.

[0072] In one embodiment, the aforementioned method comprises the following steps:

[0073] a) determining the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 in a sample from the subject, and

[0074] b) comparing the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 with a reference amount to assess the treatment of atrial fibrillation.

[0075] Preferably, the subject relevant to the aforementioned differentiation, the aforementioned prediction and the assessment of atrial fibrillation treatment is a subject suffering from atrial fibrillation, in particular a subject known to suffer from atrial fibrillation (and therefore having a known history of atrial fibrillation). However, for the above prediction method, it is also envisaged that the subject has no known history of atrial fibrillation.

[0076] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is to identify subjects who should undergo an electrocardiogram (ECG). Thus, the subject who should undergo an ECG is identified.

[0077] Therefore, the present invention contemplates a method for identifying subjects who should undergo an electrocardiogram, the method comprising the steps of:

[0078] a) determining the amount of DKK3 in a sample from the subject, and

[0079] b) comparing the amount of DKK3 to a reference amount, thereby identifying subjects who should undergo an electrocardiogram.

[0080] In one embodiment, the aforementioned method comprises the following steps:

[0081] a) determining the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 in a sample from the subject, and

[0082] b) comparing the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 with a reference amount, thereby identifying subjects who should undergo an electrocardiogram.

[0083] Preferably, the subject relevant to the above method of identifying a subject who should undergo an electrocardiogram is a subject with no known history of atrial fibrillation.The expression "without known history of atrial fibrillation" is defined elsewhere herein.

[0084] The term "atrial fibrillation" (abbreviated as "AF or AFib") is well known in the art. As used herein, the term preferably refers to a supraventricular tachyarrhythmia characterized by uncoordinated activation of the atria and the resulting deterioration of the mechanical function of the atria. In particular, the term refers to an abnormal heart rhythm characterized by rapid and irregular beats. It involves the two upper chambers of the heart. In a normal heart rhythm, impulses generated by the sinoatrial node propagate through the heart and cause the heart muscle to contract and the blood to pump. In atrial fibrillation, the regular electrical impulses of the sinoatrial node are replaced by disorganized, rapid electrical impulses that cause irregular heartbeats. Symptoms of atrial fibrillation are palpitations, syncope, shortness of breath or chest pain. However, most episodes are asymptomatic. On an electrocardiogram, atrial fibrillation is characterized by the replacement of consistent P waves with rapid oscillations or tremor waves that vary in amplitude, shape and timing, which are associated with irregular, frequent rapid ventricular responses when atrioventricular conduction is intact.

[0085] The American College of Cardiology (ACC), the American Heart Association (AHA) and the European Society of Cardiology (ESC) have proposed the following classification system (see Fuster V. et al., Circulation 2006; 114(7): e257-354, the entire contents of which are incorporated herein by reference, see e.g. Figure 3 ): first detected AF, paroxysmal AF, persistent AF, and permanent AF.

[0086] All people with AF initially fall into a category known as first-detected AF. However, the subject may or may not have had previous undetected episodes. If AF persists for more than one year, and in particular, restoration of sinus rhythm does not occur (or only occurs with medical intervention), the subject has permanent AF. If AF persists for more than 7 days, the subject has persistent AF. The subject may require medication or electrical intervention to terminate atrial fibrillation. Preferably, persistent AF occurs during an episode, but the arrhythmia does not spontaneously (i.e., without medical intervention) return to sinus rhythm. Paroxysmal atrial fibrillation preferably refers to intermittent episodes of atrial fibrillation that last up to 7 days. In most cases of paroxysmal AF, the episode lasts less than 24 hours. Episodes of atrial fibrillation terminate spontaneously, i.e., terminate without medical intervention. Therefore, while episodes of paroxysmal atrial fibrillation preferably terminate spontaneously, persistent atrial fibrillation preferably does not terminate spontaneously. Preferably, persistent atrial fibrillation needs to be terminated by electrical or pharmacological cardioversion, or by other procedures, such as ablation procedures (Fuster V. et al., Circulation 2006; 114(7): e257-354). Both persistent AF and paroxysmal AF may recur, therefore, paroxysmal AF and persistent AF are distinguished by ECG recordings: AF is considered recurrent when the patient has had 2 or more episodes. AF, especially recurrent AF, is designated as paroxysmal if the arrhythmia terminates spontaneously. AF is designated as persistent if it persists for more than 7 days.

[0087] In a preferred embodiment of the invention, the term "paroxysmal atrial fibrillation" is defined as a spontaneously terminating episode of AF, wherein the episode lasts for less than 24 hours. In an alternative embodiment, the spontaneously terminating episode lasts for up to 7 days.

[0088] The "subject" referred to herein is preferably a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a human subject.

[0089] Preferably, the subject to be tested is of any age, more preferably, the subject to be tested is 50 years old or older, more preferably 60 years old or older, and most preferably 65 years old or older. Further, it is envisioned that the subject to be tested is 70 years old or older.

[0090] Furthermore, it is envisaged that the subject to be tested is 75 years of age or older. Likewise, the subject may be between 50 and 90 years of age.

[0091] In a preferred embodiment of the method for assessing atrial fibrillation, the subject to be tested shall suffer from atrial fibrillation. Thus, the subject shall have a known history of atrial fibrillation. Thus, before obtaining the test sample, the subject shall have experienced an episode of atrial fibrillation, and at least one of the previous episodes of atrial fibrillation shall have been diagnosed, for example, by ECG. For example, it is envisaged that if the assessment of atrial fibrillation is to distinguish between paroxysmal atrial fibrillation and persistent atrial fibrillation, or if the assessment of atrial fibrillation is to predict the risk of adverse events associated with atrial fibrillation, or if the assessment of atrial fibrillation is to assess the treatment of atrial fibrillation, then the subject suffers from atrial fibrillation.

[0092] In another preferred embodiment of the method of assessing atrial fibrillation, the subject to be tested is suspected of having atrial fibrillation, for example if the assessment of atrial fibrillation is diagnosing atrial fibrillation or identifying a subject who should undergo an electrocardiogram (ECG).

[0093] Preferably, the subject suspected of having atrial fibrillation is a subject who has shown at least one symptom of atrial fibrillation before the method for assessing atrial fibrillation is implemented. The symptoms are usually transient, may appear within a few seconds, and may disappear quickly. Symptoms of atrial fibrillation include dizziness, fainting, shortness of breath, and especially palpitations. Preferably, the subject has shown at least one symptom of atrial fibrillation within six months before the sample is obtained.

[0094] Alternatively or additionally, the subject suspected of having atrial fibrillation should be a subject 70 years of age or older.

[0095] Preferably, a subject suspected of having atrial fibrillation should have no known history of atrial fibrillation.

[0096] According to the present invention, a subject with no known history of atrial fibrillation is preferably a subject who has not been previously diagnosed with atrial fibrillation, i.e. before carrying out the method of the present invention (particularly before obtaining a sample from the subject). However, the subject may or may not have had an episode of atrial fibrillation that was not previously diagnosed.

[0097] Preferably, the term "atrial fibrillation" refers to all types of atrial fibrillation. Thus, the term preferably covers paroxysmal atrial fibrillation, persistent atrial fibrillation or permanent atrial fibrillation. However, in one embodiment of the present invention, the subject to be tested does not suffer from permanent atrial fibrillation. Thus, preferably, the term "atrial fibrillation" refers only to paroxysmal atrial fibrillation and persistent atrial fibrillation.

[0098] As described above, the biomarker DKK3 may be increased in various diseases and conditions other than atrial fibrillation. In one embodiment of the present invention, it is envisioned that the subject does not suffer from such diseases and conditions. For example, it is envisioned that the subject will not suffer from chronic kidney disease, diabetes, cancer, coronary artery disease, hypertension, and / or renal failure requiring dialysis. In one embodiment, it is envisioned that the subject has no history of stroke.

[0099] In one embodiment of assessing atrial fibrillation, the subject to be tested according to the method for assessing atrial fibrillation does not suffer from heart failure. The term "heart failure" is defined in conjunction with the method for diagnosing heart failure. The definitions apply accordingly.

[0100] In an alternative embodiment of atrial fibrillation assessment, the subject may have or be suffering from heart failure.

[0101] When the sample is obtained, the subject to be tested may or may not have experienced an episode of atrial fibrillation. Therefore, in a preferred embodiment of assessing atrial fibrillation (such as in the diagnosis of atrial fibrillation), when the sample is obtained, the subject is not experiencing an episode of atrial fibrillation. In this embodiment, when the sample is obtained, the subject should have normal sinus rhythm (and should be in sinus rhythm accordingly). Therefore, atrial fibrillation can be diagnosed even in the (temporary) absence of atrial fibrillation. According to the method of the present invention, the increase in DKK3 should be retained after the onset of atrial fibrillation, thereby providing a diagnosis of a subject suffering from atrial fibrillation. Preferably, AF is diagnosed within about three days, about one month, about three months or about six months after the implementation of the method of the present invention (or more precisely, after the sample is obtained). In a preferred embodiment, it is feasible to diagnose atrial fibrillation within about six months after the onset. In a preferred embodiment, it is feasible to diagnose atrial fibrillation within about six months after the onset. Therefore, the assessment of atrial fibrillation as described herein, in particular the diagnosis, risk prediction or differentiation related to the assessment of atrial fibrillation as described herein, is preferably performed after about three days, more preferably after about one month, even more preferably after about three months, and most preferably after about six months after the last episode of atrial fibrillation. Therefore, it is envisaged that the sample to be tested is preferably obtained after about three days, more preferably after about one month, even more preferably after about three months, and most preferably after about six months after the last episode of atrial fibrillation. Therefore, the diagnosis of atrial fibrillation preferably also includes the diagnosis of episodes of atrial fibrillation, which preferably occurred within about three days, more preferably within about three months, and most preferably within about six months before the sample is obtained.

[0102] However, it is also envisaged that the subject is experiencing an episode of atrial fibrillation when the sample is obtained (eg, in connection with the prediction of a stroke).

[0103] The method of the present invention can also be used to screen a larger population of subjects. Thus, it is envisaged that at least 100 subjects, in particular at least 1000 subjects, are assessed for atrial fibrillation. Thus, the amount of biomarkers is determined in samples from at least 100, in particular from at least 1000 subjects. Furthermore, it is envisaged that at least 10,000 subjects are assessed.

[0104] The term "sample" refers to a body fluid sample, a separated cell sample or a sample from a tissue or organ. Body fluid samples can be obtained by well-known techniques and include blood, plasma, serum, urine, lymph, sputum, ascites, or any other body secretions or samples of their derivatives. Tissue or organ samples can be obtained from any tissue or organ by, for example, biopsy. Separated cells can be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. For example, cells, tissues or organ samples can be obtained from those cells, tissues or organs that express or produce biomarkers. Samples can be frozen, fresh, fixed (e.g., formalin fixed), centrifuged and / or embedded (e.g., paraffin embedded), etc. Before assessing the amount of one or more biomarkers in the sample, cell samples can certainly be subjected to various well-known post-collection preparation and storage techniques (e.g., nucleic acid and / or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.).

[0105] In a preferred embodiment of the present invention, the sample is a blood (i.e., whole blood), serum or plasma sample. Serum is the liquid fraction of whole blood obtained after blood coagulation. To obtain serum, blood clots are removed by centrifugation and the supernatant is collected. Plasma is the cell-free fluid portion of blood. To obtain a plasma sample, whole blood is collected in a test tube treated with anticoagulation (e.g., a citrate-treated test tube or an EDTA-treated test tube). Cells are removed from the sample by centrifugation and a supernatant (i.e., a plasma sample) is obtained.

[0106] As described above, the subject may be in sinus rhythm or may be suffering from an AF rhythm episode when the sample is obtained.

[0107] The biomarker Dickkopf-related protein 3 (abbreviated as DKK3) is well known in the art. The biomarker is also commonly referred to as REIC, RIG, Dickkopf WNT signaling pathway inhibitor 3.

[0108] DKK3 is expressed in the heart, brain, retina, blood vessels, lungs, liver, pancreas, spleen, kidneys, skeletal muscle and placenta. Among them, DKK3 expression is particularly high in the myocardium, cerebral cortex, brain and spinal cord.

[0109] In a preferred embodiment of the present invention, the amount of human DKK3 polypeptide is determined in a sample from a subject. The sequence of human DKK3 polypeptide is well known in the art and can be assessed, for example, by the Uniprot database, see entry Q9UBP4 (DKK3_HUMAN). DKK3 is a secreted protein of 38,390 Da polypeptide, and alternative splicing results in multiple transcript variants encoding the same protein. The protein contains an sgy domain and two cysteine-rich regions that mediate interaction with LRP5 and LRP6.

[0110] The DKK3 gene is 2650bp long, located on chromosome 11p15.3, consists of 12 exons, and has at least two promoters. The DKK3 gene encodes multiple transcripts that are widely expressed in normal human tissues. The DKK3 gene is transcribed into three different isoforms (NM_015881, 2650bp, NM_013253, 2635bp, and NM_001018057, 2587bp). Two of them are caused by the alternating use of the first exon (i.e., exon 1a and exon 1b, although they are both non-coding). There is also a variant that lacks exon 1. All variants share exons 2 to 8 and encode a functional protein of 350 amino acids. (Katase et al.; Atlas Genet Cytogenet Oncol Haematol. 2013; 17 (10) p. 678-686). In addition, Leonard et al. (PLOS, July 24, 2017) showed the identification of DKK3b transcript, which is an isoform of DKK3 that originates from a second transcription start site located in intron 2 of the DKK3 gene.

[0111] In a preferred embodiment, the amount of isoform 1 of the DKK3 transcript, ie, isoform 1 having the sequence as shown in RefSeq Accession No. NM_015881, is determined.

[0112] In another preferred embodiment, the amount of isoform 2 of the DKK3 transcript, ie, isoform 2 having the sequence as shown in RefSeq Accession No. NM_013253, is determined.

[0113] In another preferred embodiment, the amount of isoform 3 of the DKK3 transcript, ie, isoform 3 having the sequence shown in RefSeq Accession No. NM_001018057, is determined.

[0114] In another preferred embodiment, the amount of isoform 4 of the DKK3b transcript, i.e., isoform 4 having the sequence as shown in Leonard et al. (PLOS, July 24, 2017), is determined.

[0115] In another preferred embodiment, the amount of isoforms 1, 2, 3 and isoform 4 of the DKK3 transcript, ie, total DKK3, is determined.

[0116] For example, the amount of DKK3 can be determined using a monoclonal antibody (such as a mouse antibody) directed against amino acids 15 to 350 of the DKK3 polypeptide and / or using a goat polyclonal antibody.

[0117] In another preferred embodiment, DKK3 is assayed in combination with natriuretic peptides and / or ESM1.

[0118] The term "natriuretic peptide" includes atrial natriuretic peptide (ANP)-type and brain natriuretic peptide (BNP)-type peptides. Thus, the natriuretic peptides according to the invention comprise ANP-type and BNP-type peptides and variants thereof (see, e.g., Bonow RO. et al., Circulation 1996; 93: 1946-1950).

[0119] ANP-type peptides include pre-proANP, proANP, NT-proANP and ANP.

[0120] BNP-type peptides include pre-proBNP, proBNP, NT-proBNP and BNP.

[0121] The precursor propeptide (134 amino acids in the case of pre-proBNP) contains a short signal peptide, which is enzymatically cleaved to release the leader peptide (108 amino acids in the case of proBNP). The leader peptide is further cleaved into the N-terminal leader peptide (NT-pro peptide, 76 amino acids in the case of NT-proBNP) and the active hormone (32 amino acids in the case of BNP and 28 amino acids in the case of ANP).

[0122] Preferred natriuretic peptides according to the invention are NT-proANP, ANP, NT-proBNP, BNP. ANP and BNP are active hormones and have a shorter half-life than their respective inactive counterparts NT-proANP and NT-proBNP. BNP is metabolized in the blood, whereas NT-proBNP circulates in the blood as an intact molecule and is therefore cleared by the kidneys.

[0123] Pre-analysis using NT-proBNP is more robust and allows samples to be easily shipped to a central laboratory (Mueller T, Gegenhuber A, Dieplinger B, Poelz W, Haltmayer M. Long-term stability of endogenous B-type natriuretic peptide (BNP) and amino terminal proBNP (NT-proBNP) in frozen plasma samples. Clin Chem Lab Med 2004; 42: 942-4.). Blood samples can be stored at room temperature for several days or can be mailed or shipped without loss of recovery. In contrast, storage of BNP for 48 hours at room temperature or 4°C results in a loss of at least 20% of the concentration (Mueller T, Gegenhuber A, et al., Clin Chem Lab Med 2004; 42: 942-4; Wu AH, Packer M, Smith A, Bijou R, Fink D, Mair J, Wallentin L, Johnston N, Feldcamp CS, Haverstick DM, Ahnadi CE, Grant A, Despres N, Bluestein B, Ghani F. Analytical and clinical evaluation of the Bayer ADVIA Centaurautomated B-type natriuretic peptide assay in patients with heart failure: amultisite study. Clin Chem 2004; 50: 867-73.). Therefore, depending on the time course or properties of interest, measurement of the active or inactive form of the natriuretic peptide may be advantageous.

[0124] The most preferred natriuretic peptides according to the present invention are NT-proBNP and BNP, in particular NT-proBNP. As briefly discussed above, human NT-proBNP according to the present invention is a polypeptide preferably comprising 76 amino acids in length corresponding to the N-terminal part of the human NT-proBNP molecule. The structure of human BNP and NT-proBNP has been described in detail in the prior art, for example, in WO 02 / 089657, WO 02 / 083913 and Bonow RO. et al., New Insights into the cardiac natriuretic peptides. Circulation 1996; 93: 1946-1950. Preferably, the human NT-proBNP used herein is human NT-proBNP as disclosed in EP 0648228 B1.

[0125] The term "ESM1" is also referred to as endothelial cell-specific molecule, which includes a proteoglycan consisting of a 20 kDa mature polypeptide and a 30 kDa O-linked glycan chain and its variants (Bechard D et al., J Biol Chem 2001; 276(51): 48341-48349)

[0126] 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, for example, Lassale P. et al., J. Biol. Chem. 1996; 271: 20458-20464 and can be assessed, for example, by the Uniprot database, see entry Q9NQ30 (ESM1_HUMAN). Two isoforms of ESM-1 are produced by alternative splicing, namely isoform 1 (with the Uniprot identifier Q9NQ30-1) and isoform 2 (with the Uniprot identifier Q9NQ30-2). Isoform 1 has a length of 184 amino acids. In isoform 2, amino acids 101 to 150 of isoform 1 are missing. Amino acids 1 to 19 form a signal peptide (which may be cleaved).

[0127] In a preferred embodiment, the amount of isoform 1 of the ESM-1 polypeptide, ie, isoform 1 having the sequence as shown in UniProt Accession No. Q9NQ30-1, is determined.

[0128] In another preferred embodiment, the amount of isoform 2 of the ESM-1 polypeptide, ie, isoform 2 having the sequence as shown in UniProt Accession No. Q9NQ30-2, is determined.

[0129] In another preferred embodiment, the amount of isoform 1 and isoform 2 of the ESM-1 polypeptide, ie, total ESM-1, is determined.

[0130] For example, the amount of ESM-1 can be determined using a monoclonal antibody (such as a mouse antibody) directed against amino acids 20 to 184 of the ESM-1 polypeptide and / or using a goat polyclonal antibody.

[0131] For example, the amount of ESM-1 can be determined using two monoclonal antibodies (such as rabbit or mouse antibodies) directed against amino acids 20 to 184 of an ESM-1 polypeptide, which reflects the two isoforms 1 or 2 (lacking residues 101-150).

[0132] The term "determining" the amount of a biomarker as described herein (such as DKK3 or a natriuretic peptide) refers to quantification of the biomarker, for example, measuring the level of the biomarker in a sample using an appropriate detection method described elsewhere herein. The terms "measuring" and "determining" are used interchangeably herein.

[0133] In one embodiment, the amount of a biomarker is determined by contacting a sample with an agent that specifically binds to the biomarker, thereby forming a complex between the agent and the biomarker, detecting the amount of the complex formed, and thereby measuring the amount of the biomarker.

[0134] The biomarkers mentioned herein (such as DKK3) can be detected using methods generally known in the art. The detection method generally includes a method for quantifying the amount of the biomarker in the sample (quantitative method). Those skilled in the art generally know which of the following methods are suitable for qualitative and / or quantitative detection of biomarkers. Commercially available Western and immunoassays, such as ELISA, RIA, fluorescence and luminescence-based immunoassays and proximity extension assays can be used to conveniently measure, for example, proteins in a sample. Other suitable methods for detecting biomarkers include measuring physical or chemical properties specific to a peptide or polypeptide, such as its precise molecular mass or NMR spectrum. The method includes, for example, a biosensor, an optical device coupled to an immunoassay, a biochip, an analytical device (such as a mass spectrometer, an NMR analyzer or a chromatographic device). Further, the method includes a microplate ELISA-based method, a fully automatic or robotic immunoassay (e.g., in an Elecsys TM Analyzers), CBA (e.g., at Roche-Hitachi TM Enzyme cobalt binding assays available on analyzers) and latex agglutination assays (e.g., on Roche-Hitachi TM available on the analyzer).

[0135] For the detection of the biomarker proteins described herein, various immunoassay techniques of this assay format can be used, see, for example, U.S. Patent Nos. 4,016,043, 4,424,279, and 4,018,653. These techniques include single-site and two-site or "sandwich" assays of the non-competitive type, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to target biomarkers.

[0136] Methods employing electrochemiluminescent labels are well known. Such methods exploit the ability of specific metal complexes to achieve an excited state by oxidation, from which they decay to a ground state, thereby emitting electrochemiluminescence. For a review, see Richter, MM, Chem. Rev. 2004; 104: 3003-3036.

[0137] In one embodiment, the detection antibody (or its antigen binding fragment) used to measure the amount of the biomarker is ruthenated or iridiumized. Therefore, the antibody (or its antigen binding fragment) should contain a ruthenium tag. In one embodiment, the ruthenium tag is a ruthenium (II) bipyridine complex. Or the antibody (or its antigen binding fragment) should contain an iridium tag. In one embodiment, the iridium tag is a complex as disclosed in WO 2012 / 107419.

[0138] In one embodiment of a sandwich assay for detecting DKK3, the assay includes a biotinylated first monoclonal antibody (as a capture antibody) that specifically binds to DKK3; and a ruthenium-containing F(ab')2 fragment of a second monoclonal antibody (as a detection antibody) that specifically binds to DKK3. The two antibodies form a sandwich immunoassay complex with DKK3 in the sample.

[0139] In one embodiment of a sandwich assay for detecting natriuretic peptides, the assay includes a biotinylated first monoclonal antibody (as a capture antibody) that specifically binds to natriuretic peptides; and a ruthenium-containing F(ab')2 fragment of a second monoclonal antibody (as a detection antibody) that specifically binds to natriuretic peptides. The two antibodies form a sandwich immunoassay complex with the natriuretic peptides in the sample.

[0140] Measuring the amount of a polypeptide (such as DKK3 or a natriuretic peptide) may preferably include the following steps: (a) contacting the polypeptide with a reagent that specifically binds to the polypeptide, (b) (optionally) removing unbound reagent, and (c) measuring the amount of bound binding reagent, i.e., the amount of the complex of the reagent formed in step (a). According to a preferred embodiment, the contacting, removing and measuring steps may be performed by an analyzer unit. According to some embodiments, the steps may be performed by a single analyzer unit of the system or by more than one analyzer unit that can communicate with each other. For example, according to a specific embodiment, the system disclosed herein may include a first analyzer unit for performing the contacting and removing steps; and a second analyzer unit, the second analyzer unit being operably connected to the first analyzer unit via a transmission unit (e.g., a robotic arm), and the second analyzer unit performing the measuring step.

[0141] The reagent that specifically binds to the biomarker (also referred to herein as a "binding reagent") can be covalently or non-covalently coupled to a label, thereby allowing detection and measurement of the bound reagent. Labeling can be performed by direct or indirect methods. Direct labeling involves coupling the label directly (covalently or non-covalently) to the binding reagent. Indirect labeling involves the binding (covalently or non-covalently) of a secondary binding reagent to the first binding reagent. The secondary binding reagent should specifically bind to the first binding reagent. The secondary binding reagent can be coupled to an appropriate label and / or be the target (receptor) of the tertiary binding reagent to which the secondary binding reagent binds. Suitable secondary and higher-order binding reagents may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The binding reagent or substrate may also be "labeled" with one or more labels known in the art. Such labels may be targets of higher-order binding reagents. Suitable labels include biotin, digoxigenin, His tag, glutathione-S-transferase, FLAG, GFP, myc tag, influenza A virus hemagglutinin (HA), maltose binding protein, etc. In the case of a peptide or polypeptide, the tag is preferably located at the N-terminus and / or C-terminus. Suitable tags are any tags that can be detected by a suitable detection method. Typical tags include gold particles, latex beads, acridan esters, luminol, ruthenium complexes, iridium complexes, enzyme activity tags, radioactive tags, magnetic tags ("such as magnetic beads", including paramagnetic tags and superparamagnetic tags) and fluorescent tags. Enzyme activity tags include, for example, horseradish peroxidase, alkaline phosphatase, β-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 a ready-made stock solution from Roche Diagnostics), CDP-Star TM (Amersham Bio-sciences), ECF TM(Amersham Biosciences). Suitable enzyme-substrate combinations can produce colored reaction products, fluorescence or chemiluminescence, which can be measured according to methods known in the art (e.g., using photographic film or a suitable camera system). For the measurement of enzyme reactions, the above-given criteria are similarly applicable. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa568). Further fluorescent labels can be purchased from Molecular Probes (Oregon). Similarly, it is also contemplated to use quantum dots as fluorescent labels. Radioactive labels can be detected by any known and appropriate method, such as photographic film or phosphorimager.

[0142] The amount of the polypeptide may also be preferably determined by (a) contacting a solid support comprising a binding reagent for the polypeptide as described elsewhere herein with a sample comprising the peptide or polypeptide, and (b) measuring the amount of the peptide or polypeptide bound to the support. Materials for making supports are well known in the art and include, among others, commercial column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon wafers and surfaces, nitrocellulose tapes, membranes, sheets, duracytes, wells and walls of reaction plates, plastic tubes, and the like.

[0143] In another aspect, the sample is removed from the complex formed between the binding reagent and at least one marker before measuring the amount of the complex formed. Thus, in one aspect, the binding reagent may be immobilized on a solid support. In another aspect, the sample may be removed from the complex formed on the solid support by applying a washing solution.

[0144] "Sandwich assays" are one of the most useful and commonly used assays, including many variations of sandwich assay technology. Briefly, in a typical assay, an unlabeled (capture) binding reagent is fixed or can be fixed to a solid substrate, and the sample to be tested is contacted with the capture binding reagent. After an appropriate incubation period, a second (detection) binding reagent labeled with a reporter molecule capable of producing a detectable signal is then added for a period of time sufficient to allow the formation of a binding reagent-biomarker complex, and incubation is allowed for a time sufficient to form another complex of a binding reagent-biomarker-labeled binding reagent. Any unreacted material can be washed away, and the presence of the biomarker is determined by observing the signal generated by the reporter molecule bound to the detection binding reagent. The results can be qualitatively characterized by simply observing the visible signal, or can be quantified by comparison with a control sample containing a known amount of the biomarker.

[0145] The incubation steps of a typical sandwich assay can be varied as needed and as appropriate. Such variations include, for example, simultaneous incubations, in which two or more binding reagents and biomarkers are co-incubated. For example, the sample to be analyzed and the labeled binding reagent are added to the immobilized capture binding reagent simultaneously. It is also possible to first incubate the sample to be analyzed and the labeled binding reagent, and then add the antibody that is bound to the solid phase or capable of binding to the solid phase.

[0146] The complex formed between the specific binding agent and the biomarker should be proportional to the amount of the biomarker present in the sample. It should be understood that the specificity and / or sensitivity of the binding agent to be applied defines the degree of proportion of at least one marker contained in the sample that can be specifically bound. Further details on how the measurement can be performed can also be found elsewhere herein. The amount of the complex formed should be converted into the amount of the biomarker, thereby reflecting the amount actually present in the sample.

[0147] The terms "binding reagent", "specific binding reagent", "analyte-specific binding reagent", "detecting agent" and "agent that specifically binds to a biomarker" are used interchangeably herein. Preferably, it relates to a reagent that comprises a binding portion that specifically binds to the corresponding biomarker. Examples of "binding reagent", "detecting agent", "reagent" are nucleic acid probes, nucleic acid primers, DNA molecules, RNA molecules, aptamers, antibodies, antibody fragments, peptides, peptide nucleic acids (PNA) or compounds. Preferred reagents are antibodies that specifically bind to the biomarker to be determined. The term "antibody" herein is used in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they exhibit the desired antigen binding activity (i.e., antigen binding fragments thereof). Preferably, the antibody is a polyclonal antibody (or an antigen binding fragment thereof). More preferably, the antibody is a monoclonal antibody (or an antigen binding fragment thereof). Therefore, as described elsewhere herein, it is envisaged to use two monoclonal antibodies that bind at different positions of DKK3 (in a sandwich immunoassay). Therefore, at least one antibody is used to determine the amount of DKK3.

[0148] In one embodiment, at least one antibody is a mouse monoclonal antibody. In another embodiment, at least one antibody is a rabbit monoclonal antibody. In yet another embodiment, the antibody is a goat polyclonal antibody. In yet another embodiment, the antibody is a sheep polyclonal antibody.

[0149] The term "specific binding" or "specifically binds" refers to a binding reaction in which a binding pair of molecules exhibits binding to each other under conditions in which they do not significantly bind to other molecules. When referring to proteins or peptides as biomarkers, the term "specific binding" or "specifically binds" preferably refers to binding agents that bind to each other with a specific binding affinity of at least 10 7 M -1 The affinity (“binding constant” K a ) binding reaction with the corresponding biomarker. The term "specific binding" or "specifically binds" preferably refers to a binding reaction with at least 10 8 M -1 or even more preferably at least 10 9 M -1 The term "specific" or "specifically" is used to indicate that other molecules present in the sample do not significantly bind to the binding agent specific for the target molecule.

[0150] In one embodiment, the method of the present invention is based on detecting a protein complex comprising human DKK3 and a non-human or chimeric DKK3 specific binding agent. In such embodiments, the present invention continues to describe a method for assessing atrial fibrillation in a subject, the method comprising the following steps: (a) incubating a sample from the subject with a non-human DKK3 specific binding agent, (b) measuring the complex between the DKK3 specific binding agent and DKK3 formed in (a), and (c) comparing the measured amount of the complex with a reference amount. An amount of the complex equal to or above the reference amount indicates a diagnosis of atrial fibrillation (and therefore the presence of atrial fibrillation), the presence of persistent atrial fibrillation, a subject who should undergo an ECG examination, or a subject at risk of an adverse event. An amount of the complex below the reference amount indicates the absence of atrial fibrillation; the presence of paroxysmal atrial fibrillation, a subject who should not undergo an ECG examination, or a subject who is not at risk of an adverse event.

[0151] The term "amount" as used herein includes the absolute amount of the biomarker (such as DKK3 or natriuretic peptide) mentioned herein, the relative amount or concentration of the biomarker, and any value or parameter related thereto or derivable therefrom. Such values ​​or parameters include intensity signal values ​​from all specific physical or chemical properties obtained from the peptide by direct measurement, such as intensity values ​​in a mass spectrum or NMR spectrum. In addition, included are all values ​​or parameters obtained by indirect measurements specified elsewhere in this specification, for example, the amount of reaction measured from a biological readout system in response to an intensity signal obtained from a peptide or from a specifically bound ligand. It should be understood that values ​​related to the above amounts or parameters can also be obtained by all standard mathematical operations.

[0152] As used herein, the term "comparison" refers to comparing the amount of a biomarker (such as DKK3 and a natriuretic peptide, such as NT-proBNP or BNP and / or ESM1) in a sample from a subject with a reference amount of a biomarker specified elsewhere in this specification. It should be understood that comparison as used herein generally refers to the comparison of corresponding parameters or values, for example, comparing an absolute amount with an absolute reference amount, and comparing a concentration with a reference concentration, or comparing an intensity signal obtained from a biomarker in a sample with an intensity signal of the same type obtained from a reference sample. The comparison can be performed manually or computer-assisted. Thus, the comparison can be performed by a computing device. For example, the value of the measured or detected amount of a biomarker in a sample from a subject can be compared with a reference amount, and the comparison can be performed automatically by a computer program that executes a comparison algorithm. The computer program that performs the evaluation will provide the desired assessment in an appropriate output format. For computer-assisted comparisons, the value of the measured amount can be compared with a value corresponding to an appropriate reference stored in a database by a computer program. The computer program can further evaluate the results of the comparison, i.e., automatically provide the desired assessment in an appropriate output format. For computer-assisted comparison, the value of the determined quantity can be compared with the value corresponding to an appropriate reference stored in a database by a computer program. The computer program can further evaluate the result of the comparison, i.e. automatically provide the required assessment in an appropriate output format.

[0153] According to the present invention, the amount of the biomarker DKK3 and optionally the amount of the natriuretic peptide and / or ESM1 should be compared to a reference. The reference is preferably a reference amount. The term "reference amount" is well understood by the skilled person. It should be understood that the reference amount should allow the atrial fibrillation assessment described herein. For example, with respect to a method for diagnosing atrial fibrillation, the reference amount preferably refers to an amount that allows the subject to be assigned to (i) a group of subjects with atrial fibrillation, or (ii) a group of subjects without atrial fibrillation. An appropriate reference amount can be determined from a reference sample to be analyzed together with the test sample (i.e., simultaneously or subsequently).

[0154] It will be appreciated that the amount of DKK3 is compared to a reference amount of DKK3, whereas the amount of natriuretic peptide is compared to a reference amount of natriuretic peptide. If the amounts of both markers are determined, it is also conceivable to calculate a comprehensive score based on the amounts of DKK3 and natriuretic peptide. In a subsequent step, the score is compared to the reference score.

[0155] Furthermore, it will be appreciated that the amount of DKK3 is compared to a reference amount of ESM1, whereas the amount of ESM1 is compared to a reference amount of ESM1. If the amounts of both markers are determined, it is also conceivable to calculate a combined score based on the amounts of DKK3 and ESM1. In a subsequent step, the score is compared to the reference score.

[0156] In principle, the reference amount of the subject cohort as specified above can be calculated by applying standard statistical methods based on the average or mean of a given biomarker. In particular, the accuracy of a test (such as a method intended to diagnose an event or an event that has not occurred) is best described by its receiver operating characteristic (ROC) (see in particular Zweig MH. et al., Clin. Chem. 1993; 39: 561-577). The ROC curve is a curve of all sensitivity versus specificity pairs produced by continuously changing the decision threshold over the entire data range observed. 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 curve shows the overlap between the two distributions by plotting the sensitivity versus 1-specificity of the entire threshold range applicable to the distinction into a curve. On the y-axis is sensitivity, i.e., the true positive score, which is 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. It is calculated only from the affected subgroup. On the x-axis is the false positive fraction, 1-specificity, which is defined as the ratio of the number of false positive results to the product of the number of true negative results and the number of false positive results. This is a specificity index and is calculated entirely from the unaffected subgroup. Since the true positive fraction and the false positive fraction are calculated completely separately, the ROC curve is independent of the prevalence of events in the same group by using the test results from two different subgroups. Each point on the ROC curve represents a sensitivity / 1-specificity pair corresponding to a specific decision threshold. A test with complete distinction (no overlap between the two result distributions) has an ROC curve that passes through the upper left corner, where the true positive fraction is 1.0 or 100% (complete sensitivity) and the false positive fraction is 0 (complete specificity). The theoretical curve for a test without distinction (the result distributions of the two groups are the same) is a 45° diagonal from the lower left corner to the upper right corner. Most curves fall between these two extremes. If the ROC curve falls completely below the 45° diagonal, it can be easily corrected by reversing the criterion for "positive" from "greater than" to "less than" or vice versa. Qualitatively, the closer the curve is to the upper left corner, the higher the overall accuracy of the test. Depending on the desired confidence interval, a threshold value can be derived from the ROC curve, allowing a given event to be diagnosed at an appropriate balance of sensitivity and specificity, respectively. Therefore, preferably, by establishing the ROC of the same group as described above and deriving the threshold amount therefrom, a reference for the method of the present invention can be generated, i.e. a threshold value allowing the assessment of atrial fibrillation. Depending on the sensitivity and specificity required for the diagnostic method, the ROC curve allows a suitable threshold value to be derived. It should be understood that the optimal sensitivity is required, for example, to exclude subjects with atrial fibrillation (i.e., exclusion), while the optimal specificity is envisaged for subjects assessed to have atrial fibrillation (i.e., determination).In one embodiment, the methods of the invention allow prediction of a subject's risk for an adverse event associated with atrial fibrillation, such as the occurrence or recurrence of atrial fibrillation and / or stroke.

[0157] In a preferred embodiment, the term "reference amount" herein refers to a predetermined value. The predetermined value should allow for the assessment of atrial fibrillation, thereby diagnosing atrial fibrillation, distinguishing between paroxysmal atrial fibrillation and persistent atrial fibrillation, predicting the risk of adverse events associated with atrial fibrillation, identifying subjects who should undergo an electrocardiogram (ECG) examination, or assessing the treatment of atrial fibrillation. It should be understood that the reference amount may differ based on the type of assessment. For example, the reference amount of DKK3 used to distinguish AF will generally be higher than the reference amount used to diagnose AF. However, the skilled person will take this into account.

[0158] As mentioned above, the term "assessing atrial fibrillation" preferably refers to the diagnosis of atrial fibrillation, the differentiation between paroxysmal atrial fibrillation and persistent atrial fibrillation, the prediction of the risk of adverse events associated with atrial fibrillation, the identification of subjects who should undergo an electrocardiogram (ECG) examination, or the assessment of atrial fibrillation treatment. In the following, these embodiments of the method of the present invention will be described in more detail. The above definitions apply accordingly.

[0159] Methods used to diagnose atrial fibrillation

[0160] The term "diagnosis" as used herein refers to assessing whether a subject according to the methods of the present invention suffers from atrial fibrillation (AF). In one embodiment, the subject is diagnosed as suffering from AF. In a preferred embodiment, the subject is diagnosed as suffering from paroxysmal AF. In an alternative embodiment, the subject is diagnosed as not suffering from AF.

[0161] According to the invention, all types of AF can be diagnosed. Thus, atrial fibrillation can be paroxysmal AF, persistent AF or permanent AF. Preferably, paroxysmal or atrial fibrillation is diagnosed, especially in subjects not suffering from permanent AF.

[0162] The actual diagnosis of whether a subject suffers from AF may include other steps, such as confirming the diagnosis (e.g., by ECG confirmation such as Holter-ECG). Thus, the present invention allows for an assessment of the likelihood that a patient suffers from atrial fibrillation. A subject whose amount of DKK3 is above a reference amount is likely to suffer from atrial fibrillation, while a subject whose amount of DKK3 is below a reference amount is unlikely to suffer from atrial fibrillation. Thus, in the context of the present invention, the term "diagnosis" also encompasses helping a physician assess whether a subject suffers from atrial fibrillation.

[0163] Preferably, an increase in the amount of DKK3 (and optionally the amount of natriuretic peptides) in the sample from the test subject compared to a reference amount (or multiple reference amounts) indicates that the subject suffers from atrial fibrillation, and / or a decrease in the amount of DKK3 (and optionally the amount of natriuretic peptides) in the sample from the test subject compared to a reference amount (or multiple reference amounts) indicates that the subject does not suffer from atrial fibrillation.

[0164] In a preferred embodiment, the reference amount (ie the reference amount of DKK3, and the reference amount of the natriuretic peptide, if a natriuretic peptide is determined) should allow to distinguish between subjects with atrial fibrillation and subjects without atrial fibrillation. Preferably, the reference amount is a predetermined value.

[0165] In another preferred embodiment, the reference amount (ie the reference amount DKK3, and the reference amount of ESM1, if ESM1 is determined) should allow distinguishing subjects suffering from atrial fibrillation from subjects not suffering from atrial fibrillation. Preferably, the reference amount is a predetermined value.

[0166] In one embodiment, the method of the invention allows for diagnosis of a subject suffering from atrial fibrillation. Preferably, if the amount of DKK3 (and optionally the amount of natriuretic peptide) is above a reference amount, the subject suffers from AF. In one embodiment, if the amount of DKK3 is above a certain percentile (e.g., the 99th percentile) upper reference limit (URL) of the reference amount, the subject suffers from AF.

[0167] In another preferred embodiment, the method of the invention allows diagnosis that a subject does not suffer from atrial fibrillation. Preferably, if the amount of DKK3 (and optionally the amount of natriuretic peptide) is below a reference amount (such as a specific percentile URL), the subject does not suffer from AF. Thus, in one embodiment, the term "diagnosing atrial fibrillation" means "excluding atrial fibrillation".

[0168] Ruling out atrial fibrillation is particularly important, since further diagnostic tests for atrial fibrillation diagnosis, such as an ECG test, can be avoided. Thus, unnecessary healthcare costs can be avoided thanks to the present invention.

[0169] Therefore, the present invention also relates to a method for excluding atrial fibrillation, the method comprising the following steps:

[0170] a) determining the amount of DKK3 in a sample from the subject, and

[0171] b) Comparing the amount of DKK3 with a reference amount to exclude atrial fibrillation.

[0172] Preferably, a decrease in the amount of the biomarker DKK3 in the sample of the subject compared to a reference amount (such as a reference excluding atrial fibrillation) indicates a subject not suffering from atrial fibrillation, thus excluding atrial fibrillation in the subject. For example, the reference amount of DKK3 can be determined in a sample from a subject not suffering from AF or in a group of samples thereof.

[0173] When the biomarkers DKK3 and natriuretic peptides and / or ESM1 are determined in combination, an even more reliable exclusion can be achieved. Therefore, steps a) and b) are preferably as follows:

[0174] a) determining the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 in a sample from the subject, and

[0175] b) comparing the amount of DKK3 and the amount of natriuretic peptide and / or ESM1 with a reference amount to exclude atrial fibrillation.

[0176] Preferably, the amounts of the two biomarkers, namely the amount of the biomarker DKK3 and the amount of the natriuretic peptide,

[0177] or the amounts of two biomarkers, namely the amount of the biomarker DKK3 and the amount of ESM1,

[0178] or the amounts of three biomarkers, namely the amount of biomarker DKK3, the amount of natriuretic peptide and the amount of ESM1,

[0179] A decrease in a subject's sample compared to a corresponding reference amount (such as a reference amount excluding atrial fibrillation) indicates a subject not suffering from atrial fibrillation, thus excluding atrial fibrillation in the subject. For example, a reference amount of natriuretic peptides and / or ESM1 can be determined in a sample of a subject not suffering from AF or in samples of a group of subjects thereof.

[0180] In one embodiment of the method of diagnosing atrial fibrillation, the method further comprises the step of recommending and / or initiating atrial fibrillation treatment based on the diagnosis. Preferably, treatment is recommended or initiated if the subject is diagnosed with AF. Preferred treatments for atrial fibrillation are disclosed elsewhere herein.

[0181] Methods for distinguishing paroxysmal from persistent atrial fibrillation

[0182] The term "differentiation" as used herein refers to differentiating between paroxysmal atrial fibrillation and persistent atrial fibrillation in a subject. The term as used herein preferably includes differentially diagnosing paroxysmal atrial fibrillation and persistent atrial fibrillation in a subject. Therefore, the method of the present invention allows for the assessment of whether a subject suffering from atrial fibrillation suffers from paroxysmal atrial fibrillation or persistent atrial fibrillation. The actual differentiation may include other steps, such as confirmation of the differentiation. Therefore, in the context of the present invention, the term "differentiation" also encompasses helping physicians distinguish between paroxysmal AF and persistent AF.

[0183] Preferably, an increase in the amount of DKK3 (and optionally the amount of natriuretic peptides) in a sample from a subject compared to a reference amount (or multiple reference amounts) indicates that the subject suffers from persistent atrial fibrillation, and / or a decrease in the amount of DKK3 (and optionally the amount of natriuretic peptides) in a sample from a subject compared to a reference amount (or multiple reference amounts) indicates that the subject suffers from paroxysmal atrial fibrillation. In both types of AF (paroxysmal and persistent), the amount of DKK3 is increased compared to the reference amount in non-AF subjects.

[0184] In a preferred embodiment, the reference amount should allow distinguishing between subjects with paroxysmal atrial fibrillation and subjects with persistent atrial fibrillation.Preferably, the reference amount is a predetermined value.

[0185] In one embodiment of the above method of distinguishing between paroxysmal atrial fibrillation and persistent atrial fibrillation, the subject does not suffer from permanent atrial fibrillation.

[0186] Methods for predicting the risk of adverse events associated with atrial fibrillation

[0187] The methods of the present invention also contemplate a method for predicting the risk of an adverse event.

[0188] In one embodiment, the adverse event risk described herein can be a prediction of any adverse event associated with atrial fibrillation. Preferably, the adverse event is selected from the recurrence of atrial fibrillation (such as the recurrence of atrial fibrillation after cardioversion) and stroke. Therefore, the risk of a subject (a patient with atrial fibrillation) suffering from an adverse event (such as a stroke or atrial fibrillation recurrence) in the future should be predicted.

[0189] Furthermore, it is envisaged that the adverse event associated with atrial fibrillation is the occurrence of atrial fibrillation in a subject with no known history of atrial fibrillation.

[0190] In a particularly preferred embodiment, the risk of stroke is predicted.

[0191] Therefore, the present invention relates to a method for predicting the risk of stroke in a subject, the method comprising the following steps:

[0192] a) determining the amount of DKK3 in a sample from the subject, and

[0193] b) Compare the amount of DKK3 with the reference amount to predict the risk of stroke.

[0194] Preferably, the term "predicted risk" as used herein refers to the probability of assessing that a subject will suffer from an adverse event referred to herein (e.g., an adverse event of stroke). Typically, the subject is predicted to be at risk of suffering from the adverse event (thus increasing risk) or not having the risk of the adverse event (thus reducing risk). Therefore, the method of the present invention allows for the distinction of subjects with a risk of suffering from the adverse event and subjects without a risk of suffering from the adverse event. Further, it is envisioned that the method of the present invention allows for the distinction of subjects with reduced, average or elevated risk.

[0195] As mentioned above, the risk (and probability) of suffering from the adverse event within a certain time window should be predicted. In a preferred embodiment of the present invention, the prediction window is a period of about three months, about six months or about one year. In another preferred embodiment, the prediction window is a period of about five years (e.g., for predicting stroke). In addition, the prediction window can be a period of about six years (e.g., for predicting stroke). Alternatively, the prediction window can be about 10 years. In addition, it is envisioned that the prediction window is a period of 1 to 10 years.

[0196] Preferably, the prediction window is calculated from the completion of the method of the present invention. More preferably, the prediction window is calculated from the time point of obtaining the sample to be tested. As will be appreciated by those skilled in the art, the prediction of risk is generally not intended to be correct for 100% of the subjects to be tested. However, the term requires that the statistically significant part of the subject can be predicted in an appropriate and correct manner. Various well-known statistical evaluation tools (e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann-Whitney test, etc.) can be used by those skilled in the art to determine whether a certain part is statistically significant without further effort. For details, see Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005 or 0.0001.

[0197] In a preferred embodiment, the expression "predicting the risk of suffering from the adverse event" means that the subject to be analyzed by the method according to the present invention is assigned to a subject group at risk of suffering from the adverse event, or to a subject group at risk of not suffering from the adverse event (such as stroke). Therefore, it is possible to predict whether a subject is at risk of suffering from the adverse event. As used herein, a "subject at risk of suffering from the adverse event" preferably has an increased risk of suffering from the adverse event (preferably within the prediction window). Preferably, the risk is increased compared to the average risk in the same group of subjects. As used herein, a "subject without the risk of suffering from the adverse event" preferably has a reduced risk of suffering from the adverse event (preferably within the prediction window). Preferably, the risk is reduced compared to the average risk in the same group of subjects. Preferably, within a prediction window of about one year, a subject at risk of suffering from the adverse event preferably has a risk of at least 20% or more preferably at least 30% of suffering from the adverse event (such as recurrence or occurrence of atrial fibrillation). Preferably, within a one year prediction window, a subject not at risk of suffering said adverse event preferably has a risk of suffering said adverse event of less than 12%, more preferably less than 10%.

[0198] Regarding the prediction of stroke, preferably within a prediction window of about five years, or particularly about six years, a subject at risk of suffering from the adverse event preferably has a risk of at least 10% or more preferably at least 13% of suffering from the adverse event. Preferably within a prediction window of about five years, or particularly about six years, a subject not at risk of suffering from the adverse event preferably has a risk of less than 10%, more preferably less than 8%, or most preferably less than 5% of suffering from the adverse event. If the subject is not receiving anticoagulant therapy, the risk may be higher. The skilled person will take this into account.

[0199] Preferably, an increase in the amount of DKK3 (and optionally the amount of natriuretic peptides) in a sample from a subject compared to a reference amount (or multiple reference amounts) indicates a subject at risk for an adverse event associated with atrial fibrillation, and / or a decrease in the amount of DKK3 (and optionally the amount of natriuretic peptides) in a sample from a subject compared to a reference amount (or multiple reference amounts) indicates a subject not at risk for an adverse event associated with atrial fibrillation.

[0200] In a preferred embodiment, the reference amount should allow distinguishing between subjects at risk of an adverse event as referred to herein and subjects not at risk of said adverse event.Preferably, the reference amount is a predetermined value.

[0201] In a preferred embodiment of the aforementioned method, the risk of stroke is predicted. Preferably, the stroke should be associated with atrial fibrillation. More preferably, the stroke should be caused by atrial fibrillation.

[0202] Preferably, a stroke is associated with atrial fibrillation if there is a temporal relationship between the stroke and the onset of atrial fibrillation. More preferably, a stroke is associated with atrial fibrillation if the stroke is caused by atrial fibrillation. Most preferably, a stroke is associated with atrial fibrillation if the stroke can be caused by atrial fibrillation. For example, atrial fibrillation can cause a cardiogenic stroke (also commonly referred to as an embolic or thromboembolic stroke). Preferably, a stroke associated with AF can be prevented by oral anticoagulants.

[0203] Therefore, the stroke to be predicted is preferably a cardioembolic stroke.

[0204] Also preferably, if the subject to be tested suffers from atrial fibrillation and / or has a known history of atrial fibrillation, the stroke is considered to be associated with atrial fibrillation. Likewise, in one embodiment, if the subject is suspected of suffering from atrial fibrillation, the stroke may be considered to be associated with atrial fibrillation.

[0205] The terms "comparison", "amount", "subject" and "determination" etc. are defined elsewhere herein. These definitions apply accordingly. For example, the sample is preferably a blood, serum or plasma sample.

[0206] In a preferred embodiment of the aforementioned method for predicting adverse events (such as stroke), the subject to be tested suffers from atrial fibrillation. More preferably, the subject has a known history of atrial fibrillation. According to the method for predicting adverse events, the subject preferably suffers from permanent atrial fibrillation, more preferably suffers from persistent atrial fibrillation, and most preferably suffers from paroxysmal atrial fibrillation.

[0207] In one embodiment of the method for predicting an adverse event, the subject with atrial fibrillation is experiencing an episode of atrial fibrillation when the sample is obtained. In another embodiment of the method for predicting an adverse event, the subject with atrial fibrillation is not experiencing an episode of atrial fibrillation when the sample is obtained (and therefore should have normal sinus rhythm). In addition, the subject whose risk is to be predicted may be receiving anticoagulant therapy.

[0208] In another embodiment of the method for predicting an adverse event, the subject to be tested has no known history of atrial fibrillation. In particular, it is envisaged that the subject does not suffer from atrial fibrillation.

[0209] In the basic research of the present invention, it was further demonstrated that the determination of DKK3 allows to improve the predictive accuracy of the clinical stroke risk score of the subject. Therefore, the combination of the determination of the clinical stroke risk score and the determination of DKK3 can more reliably predict stroke compared with the determination of DKK3 or the determination of the clinical stroke risk score alone.

[0210] Therefore, the method for predicting stroke risk may further include combining the amount of DKK3 with a clinical stroke risk score. Based on the combination of the amount of DKK3 and the clinical risk score, the stroke risk of the test subject is predicted.

[0211] In one embodiment of the above method, the method further comprises comparing the amount of DKK3 with a reference amount. In this case, the comparison result is combined with a clinical stroke risk score.

[0212] Therefore, the present invention particularly relates to a method for predicting the risk of stroke in a subject, the method comprising the following steps:

[0213] a) determining the amount of DKK3 in a sample from the subject, and

[0214] b) combining the amount of DKK3 with a clinical stroke risk score to predict the stroke risk of the subject.

[0215] According to this method, it is assumed that the subject is a subject with a known clinical stroke risk score. Therefore, the value of the clinical stroke risk score is known to the subject.

[0216] Alternatively, the method may include obtaining or providing a value for a clinical stroke risk score. Preferably, the value is a number. In one embodiment, the clinical stroke risk score is generated by one of the clinical-based tools available to the physician. Preferably, the value is provided by determining the value of the clinical stroke risk score for the subject. More preferably, the value is obtained from a patient record database and medical history of the subject. Therefore, the value of the score may also be determined using historical data or published data for the subject.

[0217] According to the present invention, the amount of DKK3 is combined with a clinical stroke risk score. This means that preferably, the value of the amount of DKK3 is combined with a clinical stroke risk score. Therefore, these values ​​are effectively combined to predict the risk of a subject suffering from a stroke. By combining the values, a single value can be calculated, which can itself be used for prediction.

[0218] Clinical stroke risk scores are well known in the art. For example, the score is described in Kirchhof P. et al. (European Heart Journal 2016; 37: 2893-2962), the entire disclosure of which is incorporated herein by reference. In one embodiment, the score is CHA 2 DS 2 -VASc score. In another embodiment, the score is CHADS 2Score. (Gage BF. et al., JAMA, 285(22)(2001), pp.2864-2870) and ABC score (Hijazi Z. et al., Lancet 2016; 387(10035): 2302-2311)

[0219] Methods for improving the predictive accuracy of clinical stroke risk scores

[0220] The present invention further relates to a method for improving the predictive accuracy of a clinical stroke risk score of a subject, the method comprising the following steps:

[0221] a) determining the amount of DKK3 in the sample, and

[0222] b) combining the amount of DKK3 with a clinical stroke risk score, thereby improving the predictive accuracy of the clinical stroke risk score.

[0223] The method may comprise the further step of: c) improving the predictive accuracy of the clinical stroke risk score based on the result of step b).

[0224] The definitions and explanations given herein in connection with the method for assessing atrial fibrillation, in particular the definitions and explanations for predicting the risk of an adverse event (such as stroke) are preferably equally applicable to the above method. For example, it is envisaged that the subject is a subject with a known clinical stroke risk score. Alternatively, the method may comprise obtaining or providing the value of a clinical stroke risk score.

[0225] According to the present invention, the amount of DKK3 is combined with a clinical stroke risk score. This means that preferably, the value of the amount of DKK3 is combined with a clinical stroke risk score. Therefore, these values ​​are effectively combined to improve the predictive accuracy of the clinical stroke risk score.

[0226] Methods for identifying subjects who should undergo an electrocardiogram (ECG)

[0227] According to this embodiment of the method of the invention, it should be assessed whether the subject to be tested using the biomarker should undergo an electrocardiogram (ECG), ie an ECG assessment. The assessment should be performed for diagnosis, ie to detect the presence or absence of AF in the subject.

[0228] As used herein, the term "identifying a subject" preferably refers to using the generated information or data related to the amount of DKK3 in a sample of a subject to identify a subject who should undergo an ECG examination. The identified subject has an increased likelihood of having AF. An ECG assessment is performed as confirmation.

[0229] Electrocardiogram (ECG for short) is the process of recording the electrical activity of the heart through a suitable ECG. ECG equipment records the electrical signals generated by the heart, which propagate throughout the body to the skin. The recording of electrical signals is achieved by contacting the skin of the test subject with the electrodes contained in the ECG equipment. The process of obtaining the records is non-invasive and risk-free. ECG is performed for diagnosing atrial fibrillation, i.e., for assessing whether atrial fibrillation exists in the test subject. In an embodiment of the method of the present invention, the ECG device is a single-lead device (such as a single-lead handheld ECG device). In another preferred embodiment, the ECG device is a 12-lead ECG device, such as a Holter monitor.

[0230] Preferably, an increase in the amount of DKK3 (and optionally the amount of natriuretic peptides) in the sample from the test subject compared to the reference amount (or multiple reference amounts) indicates a subject who should undergo an ECG examination, and / or a decrease in the amount of DKK3 (and optionally the amount of natriuretic peptides) in the sample from the subject compared to the reference amount (or multiple reference amounts) indicates a subject who should not undergo an ECG examination.

[0231] In a preferred embodiment, the reference amount should allow distinguishing between subjects who should undergo an ECG and subjects who should not undergo an ECG examination.Preferably, the reference amount is a predetermined value.

[0232] Methods for evaluating treatment of atrial fibrillation

[0233] As used herein, the term "assessing atrial fibrillation therapy" preferably refers to assessing a therapy aimed at treating atrial fibrillation.

[0234] The treatment to be assessed may be any treatment intended to treat atrial fibrillation. Preferably, the treatment is selected from the group consisting of: administration of at least one anticoagulant, rhythm control, heart rate control, cardioversion and ablation. The treatments are well known in the art and are reviewed, for example, in Fuster V et al. Circulation 2011; 123: e269-e367, the entire contents of which are incorporated herein by reference.

[0235] In one embodiment, treatment is to use at least one anticoagulant. Use at least one anticoagulant should be intended to reduce or prevent blood coagulation and related cerebral apoplexy. In one embodiment, at least one anticoagulant is selected from the group consisting of the following items: heparin, coumarin derivatives (such as warfarin or dicoumarol), tissue factor pathway inhibitor (TFPI), antithrombin III, factor Ixa inhibitor, factor Xa inhibitor, inhibitor of factor Va and VIIIa and thrombin inhibitor (anti-IIa type).

[0236] In a preferred embodiment, assessing atrial fibrillation treatment is monitoring said treatment. In this embodiment, the reference amount is preferably the amount of DKK3 in an earlier obtained sample (ie a sample obtained before the test sample in step a).

[0237] Optionally, in addition to the amount of DKK3, the amount of natriuretic peptide and / or ESM1 is also determined.

[0238] Therefore, the present invention relates to a method for monitoring the treatment of atrial fibrillation in a subject, preferably a subject suffering from atrial fibrillation, wherein the method comprises the following steps

[0239] a) determining the amount of DKK3 (and optionally the amount of a natriuretic peptide) in a sample from the subject, and

[0240] b) comparing the amount of DKK3 with a reference amount, wherein the reference amount is the amount of DKK3 in a sample obtained from the subject before the sample in step a), and optionally comparing the amount of natriuretic peptides and / or ESM1 with a reference amount, wherein the reference amount is the amount of natriuretic peptides and / or ESM1 in a sample obtained from the subject before the sample in step a).

[0241] The sample in step a) is also referred to herein as a "test sample" and the sample in step b) is referred to herein as a "reference sample".

[0242] The term "monitoring" as used herein preferably relates to assessing the effect of a treatment as mentioned elsewhere herein. Thus, the efficacy of a treatment, such as an anticoagulant treatment, is monitored.

[0243] The aforementioned method may include other steps of monitoring treatment based on the results of the comparison step performed in step c). As will be appreciated by those skilled in the art, the prediction of risk is generally not intended to be correct for 100% of the subjects to be tested. However, the term requires that a statistically significant portion of the subjects can be predicted in an appropriate and correct manner. Therefore, actual monitoring may include other steps such as confirmation.

[0244] Preferably, by implementing the method of the present invention, it can be assessed whether the subject responds to the treatment. If the subject's condition improves between obtaining the first sample and the second sample, the subject responds to the treatment. Preferably, if the condition worsens between obtaining the first sample and the second sample, the subject does not respond to the treatment.

[0245] Preferably, the reference sample is obtained before starting the treatment. More preferably, the sample is obtained within one week, particularly within two weeks, before starting the treatment. However, it is also contemplated that the reference sample can be obtained after the treatment begins (but before obtaining the test sample). In this case, the ongoing treatment is monitored.

[0246] Therefore, the test sample should be obtained after the reference sample.It should be understood that the test sample should be obtained after the treatment has started.

[0247] In addition, it is particularly contemplated that the test sample is obtained after a reasonable period of time after the reference sample is obtained. It should be understood that the amount of the biomarker mentioned herein will not change immediately (e.g., within 1 minute or 1 hour). Therefore, "reasonable" in this context refers to the interval between obtaining the first sample and the test sample, which allows the biomarker to be adjusted. Therefore, preferably, the test sample is obtained at least one month after the reference sample, at least three months, or in particular at least six months after the reference sample.

[0248] Preferably, an increase in the amount of the biomarker (i.e., DKK3 and optionally natriuretic peptides and / or ESM1) in the test sample compared to the amount of the biomarker in the reference sample, and more preferably a significant increase, and most preferably a statistically significant increase indicates a subject that responded to the treatment. Thus, the treatment is effective. Also preferably, a decrease in the amount of the biomarker in the test sample compared to the amount of the biomarker in the reference sample, more preferably a significant decrease, and most preferably a statistically significant decrease indicates a subject that did not respond to the treatment. Thus, the treatment is ineffective.

[0249] If the treatment reduces the risk of recurrence of atrial fibrillation in the subject, the subject is considered to have responded to the treatment. If the treatment does not increase the risk of recurrence of atrial fibrillation in the subject, the subject is considered to have not responded to the treatment.

[0250] In one embodiment, if the subject does not respond to the treatment, the intensity of the treatment is increased. In addition, it is contemplated that if the subject responds to the treatment, the intensity of the treatment is decreased. For example, the intensity of the treatment can be increased by increasing the dose of the administered drug. For example, the intensity of the treatment can be decreased by decreasing the dose of the administered drug.

[0251] In another preferred embodiment, the assessment of atrial fibrillation treatment is guidance of atrial fibrillation treatment.The term "guidance" as used herein preferably relates to adjusting the intensity of treatment, such as increasing or decreasing the dose of oral anticoagulants, based on the determination of the biomarker, ie DKK3, during treatment.

[0252] In another preferred embodiment, the assessment of atrial fibrillation treatment is a stratification of atrial fibrillation treatment. Thus, subjects who are eligible to receive a certain atrial fibrillation treatment should be identified. The term "stratification" as used herein preferably relates to selecting an appropriate treatment based on a specific risk, an identified molecular pathway, and / or the expected efficacy of a specific drug or procedure. Based on the detected risk, patients with few or no symptoms related to the arrhythmia will be eligible for ventricular rate control, cardioversion or ablation, otherwise they will only receive antithrombotic therapy.

[0253] The terms "significant" and "statistically significant" are known to the person skilled in the art. Thus, the person skilled in the art can determine whether an increase or decrease is significant or statistically significant using various well-known statistical assessment tools without further effort. For example, a significant increase or decrease is an increase or decrease of at least 10%, in particular at least 20%.

[0254] The present invention further relates to a method for assisting in the assessment of atrial fibrillation, the method comprising the following steps:

[0255] a) obtaining a sample from a subject according to the method for assessing atrial fibrillation as described herein,

[0256] b) determining the amount of the biomarker DKK3, and optionally the amount of natriuretic peptide and / or ESM1 in said sample, and

[0257] c) providing information about the determined amount of the biomarker DKK3 and optionally about the determined amount of the natriuretic peptide and / or ESM1 to the subject's attending physician, thereby assisting in the assessment of atrial fibrillation in said subject.

[0258] The attending physician should be the physician who requested the determination of one or more biomarkers. The above method should help the attending physician to assess atrial fibrillation. Therefore, the method does not include the diagnosis, prediction, monitoring, differentiation, and identification mentioned above in conjunction with the method for assessing atrial fibrillation.

[0259] Step a) of the above-mentioned sample obtaining method does not include extracting a sample from a subject. Preferably, the sample is obtained by receiving a sample from the subject. Thus, the sample can be delivered.

[0260] In one embodiment, the method is a method for assisting stroke prediction, and the method comprises the following steps:

[0261] a) obtaining a sample from a subject as described herein according to a method for assessing atrial fibrillation, in particular according to a method for predicting atrial fibrillation,

[0262] b) determining the amount of the biomarker DKK3, and optionally the amount of natriuretic peptide and / or ESM1 in said sample, and

[0263] c) providing information about the determined amount of the biomarker DKK3 and optionally about the determined amount of the natriuretic peptide and / or ESM1 to the subject's attending physician, thereby assisting in the prediction of stroke.

[0264] The present invention further relates to a method comprising:

[0265] a) providing a test for the biomarker DKK3, and optionally a test for a natriuretic peptide and / or ESM1, and

[0266] b) providing instructions for using test results obtained or obtainable by the one or more tests in the assessment of atrial fibrillation.

[0267] The foregoing method is preferably intended to aid in the assessment of atrial fibrillation.

[0268] The instructions shall contain protocols for carrying out the method of assessing atrial fibrillation as described above.Further, the instructions shall contain at least one value for a reference amount of DKK3, and optionally at least one value for a reference amount of a natriuretic peptide.

[0269] The "test" is preferably a kit suitable for performing a method for assessing atrial fibrillation. The term "kit" is explained below. For example, the kit should include at least one detection agent for the biomarker DKK3 and optionally at least one detection agent for a natriuretic peptide. The detection agents for these two biomarkers can be provided in one kit or in two separate kits.

[0270] The test result obtained or obtainable by the test is a value for the amount of one or more biomarkers.

[0271] In one embodiment, step b) comprises providing instructions for using test results obtained or obtainable by the one or more tests in the prediction of stroke (as described elsewhere herein).

[0272] Furthermore, the present invention relates to the following substances:

[0273] i) the biomarker DKK3, and optionally natriuretic peptide and / or ESM1, and / or

[0274] ii) at least one detection agent that specifically binds to DKK3, and optionally at least one detection agent that specifically binds to a natriuretic peptide,

[0275] Use for assessing atrial fibrillation in a sample from a subject.

[0276] In one embodiment, the present invention relates to the following substances:

[0277] i) the biomarker DKK3, and optionally natriuretic peptide and / or ESM1, and / or

[0278] ii) at least one detection agent that specifically binds to DKK3, and optionally at least one detection agent that specifically binds to a natriuretic peptide,

[0279] Use for predicting stroke in a sample from a subject.

[0280] The present invention further relates to the following substances:

[0281] i) biomarker DKK3, and / or

[0282] ii) at least one detection agent that specifically binds to DKK3,

[0283] In samples from subjects,

[0284] Combined with clinical stroke risk score,

[0285] Use for predicting the risk of a subject suffering from stroke.

[0286] Furthermore, the present invention relates to the use of i) the biomarker DKK3 and / or ii) at least one detection agent that specifically binds to DKK3 in a sample from a subject for improving the predictive accuracy of a clinical stroke risk score.

[0287] Terms related to the above use, such as "sample", "subject", "detection agent", "DKK3", "specifically binds", "atrial fibrillation" and "assessing atrial fibrillation" have been defined in conjunction with the method for assessing atrial fibrillation. The definitions and explanations apply accordingly.

[0288] Preferably, the above-mentioned use is an in vitro use. In addition, the detection agent is preferably an antibody, such as a monoclonal antibody (or an antigen-binding fragment thereof).

[0289] Furthermore, it has been shown in the present study that determining the amount of DKK3 in a sample from a subject allows for the diagnosis of heart failure and structural or functional abnormalities of the heart associated with heart failure. Therefore, the present invention also contemplates a method for diagnosing heart failure and / or at least one structural or functional abnormality associated with heart failure based on the biomarker DKK3.

[0290] The definitions given herein above apply mutatis mutandis to the following (unless otherwise stated).

[0291] Therefore, the present invention further relates to a method for diagnosing heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure in a subject, the method comprising the following steps:

[0292] a) determining the amount of DKK3 in a sample from the subject, and

[0293] b) comparing the amount of DKK3 with a reference amount, thereby diagnosing heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure.

[0294] As used herein, the term "diagnosis" refers to assessing whether the subject referred to in the method according to the present invention suffers from heart failure and / or suffers from at least one abnormality of cardiac structure or function associated with heart failure. In one embodiment, the subject is diagnosed with heart failure and / or at least one abnormality of cardiac structure or function associated with heart failure. In an alternative embodiment, the subject is diagnosed with not suffering from heart failure and / or at least one abnormality of cardiac structure or function associated with heart failure (excluding heart failure and / or at least one abnormality of cardiac structure and or function).

[0295] The actual diagnosis of whether a subject suffers from heart failure and / or suffers from at least one of the abnormalities may include other steps such as confirming the diagnosis. Therefore, the diagnosis of heart failure and / or an auxiliary at least one cardiac structural or functional abnormality associated with heart failure is understood as a diagnosis of heart failure and / or at least one structural or functional abnormality. Therefore, in the context of the present invention, the term "diagnosis" also encompasses helping a physician assess whether a subject suffers from heart failure and / or at least one of the abnormalities.

[0296] The term "heart failure" (abbreviated as "HF") is well known to the skilled person. As used herein, the term preferably relates to impaired systolic and / or diastolic function of the heart known to the skilled person with obvious signs of heart failure. Preferably, the heart failure referred to herein is also chronic heart failure. Heart failure according to the present invention includes overt and / or advanced heart failure. In overt heart failure, the subject exhibits symptoms of heart failure known to the skilled person.

[0297] In one embodiment of the present invention, the term "heart failure" refers to heart failure with reduced left ventricular ejection fraction (HFrEF).

[0298] In another embodiment of the present invention, the term "heart failure" refers to heart failure with preserved left ventricular ejection fraction (HFpEF).

[0299] HF can be classified into different degrees of severity.

[0300] According to the NYHA (New York Heart Association) classification, patients with heart failure are classified as belonging to NYHA I, NYHA II, NYHA III, and NYHA IV classes. A patient with heart failure has already undergone structural and functional changes in the pericardium, myocardium, coronary circulation, or heart valves. He will not be able to fully recover and will require treatment. Patients with NYHA class I have no obvious symptoms of cardiovascular disease, but there is already objective evidence of impaired function. Patients with NYHA class II have slight limitations in physical activity. Patients with NYHA class III show significant limitations in physical activity. Patients with NYHA class IV are unable to perform any physical activity without discomfort. They show symptoms of cardiac insufficiency at rest.

[0301] This functional classification is supplemented by the latest classification of the American College of Cardiology and the American Heart Association (see J. Am. Coll. Cardiol. 2001; 38; 2101-2113, updated in 2005, see J. Am. Coll. Cardiol. 2005; 46; e1-e82). Four stages are defined, A, B, C, and D. Stages A and B are not HF, but are considered to aid in the early identification of patients before the development of "true" HF. It is best to define patients in stages A and B as those who have risk factors for developing HF. For example, patients with coronary artery disease, hypertension, or diabetes who do not yet demonstrate impaired left ventricular (LV) function, hypertrophy, or geometric chamber abnormalities would be considered stage A, while patients who are asymptomatic but demonstrate LV hypertrophy and / or impaired LV function would be designated stage B. Stage C represents patients with current or past HF symptoms related to underlying structural heart disease (most patients have HF), while stage D represents patients with true refractory HF.

[0302] As used herein, the term "heart failure" preferably includes stages A, B, C and D of the above-mentioned ACC / AHA classification. In addition, the term includes NYHA I, NYHA II, NYHA III and NYHA IV classes. Therefore, a subject may or may not show typical symptoms of heart failure.

[0303] In a preferred embodiment, the term "heart failure" refers to heart failure stage A according to the above ACC / AHA classification, or in particular heart failure stage B. Identification of these early stages, in particular stage A, is advantageous because treatment can be initiated before irreversible damage occurs.

[0304] At least one abnormality of cardiac structure or function associated with heart failure is preferably selected from functional and / or structural damage to the myocardium, epicardium, valves or coronary circulation; impaired pumping or filling capacity, usually systolic or diastolic dysfunction; altered geometry of the left ventricle; hypertension associated with geometric chamber malformations; left ventricular hypertrophy; altered left ventricular structure with or without left ventricular hypertrophy; increased septal diameter; increased posterior wall diameter, concentric myocardial enlargement, eccentric myocardial enlargement, left ventricular diastolic dysfunction.

[0305] In one embodiment of the invention, the structural or functional abnormality of the heart associated with heart failure is left ventricular hypertrophy.

[0306] As described elsewhere herein, the biomarker DKK3 may be increased in various diseases and conditions other than atrial fibrillation. In one embodiment of the aforementioned methods of the present invention, it is envisioned that the subject does not suffer from such diseases and conditions. For example, it is envisioned that the subject will not suffer from chronic kidney disease, diabetes, cancer, coronary artery disease, hypertension, and / or renal failure requiring dialysis. Further, it is envisioned that the subject has no history of stroke.

[0307] The subject being tested according to the method for diagnosing heart failure and / or at least one abnormality of cardiac structure or function associated with heart failure preferably does not suffer from atrial fibrillation. However, it is also envisaged that the subject suffers from atrial fibrillation. The term "atrial fibrillation" is defined in conjunction with the method for assessing heart failure.

[0308] Preferably, the subject to be tested is suspected of suffering from heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure.

[0309] The term "reference amount" has been defined in connection with the method for assessing atrial fibrillation. In principle, the reference amount used in the method for diagnosing heart failure and / or determining at least one cardiac structural or functional abnormality associated with heart failure can be as described above.

[0310] Preferably, an increase in the amount of DKK3 in the sample from the subject compared to the reference amount indicates that the subject suffers from heart failure and / or suffers from at least one cardiac structural or functional abnormality associated with heart failure, and / or a decrease in the amount of DKK3 in the sample from the subject compared to the reference amount indicates that the subject does not suffer from heart failure and / or does not suffer from at least one cardiac structural or functional abnormality associated with heart failure.

[0311] In one embodiment of the method for diagnosing heart failure in a subject and / or at least one cardiac structural abnormality or functional abnormality in a sample associated with heart failure, step a) further comprises determining the amount of natriuretic peptide and / or ESM1 from the subject. In step b) of the method, the amount of the marker thus determined is compared with a reference amount.

[0312] Preferably, the NT-proBNP / DKK3 ratio is indicative of differentiating subjects with atrial fibrillation from those with heart failure - as described below.

[0313] Furthermore, the present invention relates to the following substances:

[0314] i) the biomarker DKK3, and optionally natriuretic peptide and / or ESM1, and / or

[0315] ii) at least one detection agent that specifically binds to DKK3, and optionally at least one detection agent that specifically binds to a natriuretic peptide,

[0316] Use of a sample for diagnosing heart failure and / or at least one abnormality of cardiac structure or function associated with heart failure in a subject.

[0317] The present invention further relates to a method for assisting in diagnosing heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure in a subject, the method comprising the following steps:

[0318] a) obtained from a subject as described herein according to a method for diagnosing heart failure in a subject and / or at least one abnormality in cardiac structure or function associated with heart failure,

[0319] b) determining the amount of the biomarker DKK3, and optionally the amount of natriuretic peptide and / or ESM1 in said sample, and

[0320] c) providing information about the determined amount of the biomarker DKK3 and optionally about the determined amount of the natriuretic peptide and / or ESM1 to the subject's attending physician, thereby assisting in diagnosing heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure in said subject.

[0321] The attending physician is the physician who requests the determination of one or more biomarkers. The above method should help the attending physician to assess atrial fibrillation. Therefore, the method does not include the diagnosis, prediction, monitoring, differentiation, identification referred to in the above method in conjunction with diagnosing heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure.

[0322] Step a) of the above-mentioned sample obtaining method does not include extracting a sample from a subject. Preferably, the sample is obtained by receiving a sample from the subject. Therefore, the sample should have been delivered.

[0323] The present invention further relates to a method comprising:

[0324] a) providing a test for the biomarker DKK3, and optionally a test for a natriuretic peptide, and

[0325] b) providing instructions for using test results obtained or obtainable by said test in diagnosing heart failure and / or at least one abnormality of cardiac structure or function associated with heart failure.

[0326] The purpose of the aforementioned methods is preferably to aid in the diagnosis of heart failure and / or at least one abnormality of cardiac structure or function.

[0327] The instructions shall contain a protocol for performing the method of diagnosing heart failure and / or at least one cardiac structural or functional abnormality as described herein above. Further, the instructions shall contain at least one value for a reference amount of DKK3, and optionally at least one value for a reference amount of a natriuretic peptide.

[0328] The "test" is preferably a kit suitable for performing a method for diagnosing heart failure and / or at least one abnormality of cardiac structure or function. The term "kit" is explained below. For example, the kit should include at least one detection agent for the biomarker DKK3 and optionally at least one detection agent for a natriuretic peptide. The detection agents for these two biomarkers can be provided in one kit or in two separate kits.

[0329] The test result obtained or obtainable by the test is a value for the amount of one or more biomarkers.

[0330] The present invention also relates to a kit. Preferably, the kit is suitable for performing the method of the present invention, i.e., a method for assessing atrial fibrillation, or a method for diagnosing heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure. The kit should contain at least one reagent that specifically binds to DKK3. In a preferred embodiment, the kit should further contain a reagent that specifically binds to a natriuretic peptide (such as NT-proBNP) and / or ESM1. Alternatively, the kit includes instructions for implementing the method.

[0331] The term "kit" as used herein refers to a collection of the above-mentioned components, preferably, provided separately or in a single container. The container also includes instructions for implementing the method of the present invention. These instructions can be in the form of a manual, or can be provided by a computer program code, which can perform the calculations and comparisons mentioned in the method of the present invention, and establish an assessment or diagnosis accordingly when implemented on a computer or data processing device. The computer program code can be provided on a data storage medium or device such as an optical storage medium (e.g., a CD) or directly on a computer or data processing device. In addition, the kit may preferably include a standard amount of the biomarker DKK3 for calibration purposes. In a preferred embodiment, the kit also includes a standard amount of a natriuretic peptide and / or ESM1 for calibration purposes.

[0332] In one embodiment, the kit is for use in in vitro assessment of atrial fibrillation.

[0333] In another embodiment, the kit is for in vitro diagnosis of heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure.

[0334] The ratio of DKK3 to natriuretic peptides was calculated.

[0335] Atrial fibrillation and heart failure share common risk factors that predispose to the development of diseases such as coronary artery disease. It is also well established that patients with heart failure often develop atrial fibrillation and vice versa.

[0336] The examples show that DKK3 is a marker for both atrial fibrillation and heart failure (see Figure 6). Therefore, biomarkers can be used for the diagnosis of heart failure and the assessment of atrial fibrillation. For example, based on the determination of DKK3 (as described above), the presence of heart failure or atrial fibrillation in a subject can be excluded.

[0337] If the level of DKK3 is increased compared to the reference amount, the subject may in principle suffer from atrial fibrillation or heart failure, or both. In this case, it would be advantageous to distinguish between heart failure and atrial fibrillation. The distinction can be made by calculating the ratio of the amount of natriuretic peptide (e.g. NT-proBNP or BNP) and / or ESM1 in a sample from a subject to the amount of DKK3 in a sample from a subject.

[0338] Although the amount of natriuretic peptides is increased in samples from subjects with atrial fibrillation, the amount of natriuretic peptides in samples from patients with heart failure is generally much higher than in patients with atrial fibrillation. For example, in samples from subjects with atrial fibrillation but not with heart failure, NT-proBNP levels of up to 3000 pg / ml were observed in the studies underlying the present invention. In contrast, NT-proBNP levels in subjects with heart failure were as high as 15000 pg / ml.

[0339] Since there are differences in the levels of natriuretic peptides, in particular NT-proBNP, in subjects with atrial fibrillation and subjects with heart failure, it is possible to distinguish between heart failure and atrial fibrillation in subjects with elevated DKK3 levels (i.e., in subjects in which the amount of DKK3 in the sample is increased compared to a reference amount).

[0340] Preferably, the diagnosis of heart failure or atrial fibrillation is further supported or verified by performing the following further step: calculating the ratio of the amount of natriuretic peptides and / or ESM1 to the amount of DKK3 determined in step a) of the method for assessing atrial fibrillation described above or in step a) of the method for diagnosing heart failure and / or at least one abnormality of cardiac structure or function associated with heart failure. In a further step, the calculated ratio is compared with a reference ratio (amount of natriuretic peptides and / or ESM1 to amount of DKK3). The reference ratio should allow differentiation between heart failure and atrial fibrillation (in particular subjects with increased amounts of DKK3).

[0341] Thus, both the method of assessing atrial fibrillation (such as the method of diagnosing atrial fibrillation) and the method of diagnosing heart failure and / or at least one abnormality of cardiac structure or function associated with heart failure may include the following additional steps:

[0342] - calculating the ratio of the amount of natriuretic peptide and / or ESM1 determined in step a) to the amount of DKK3 determined in step a), and

[0343] - comparing said calculated ratio with a reference ratio.

[0344] The calculating step is preferably step c) and the comparing step is preferably step d).

[0345] If steps c) and d) are performed, the amount of biomarker DKK3 and natriuretic peptide and / or ESM1 is determined in step a) of the method for assessing atrial fibrillation and diagnosing heart failure. However, in this case, it is not necessary to compare the amount of natriuretic peptide and / or ESM1 with a reference amount in step b). The method of the invention can be performed with and without this comparison in step b).

[0346] Thereby, the diagnosis of atrial fibrillation or the diagnosis of heart failure and / or at least one cardiac structural or functional abnormality associated with heart failure is confirmed, confirmed or supported. In particular, the ratio allows the number of false positives to be reduced.

[0347] The above-mentioned calculating and comparing steps are preferably performed in a subject in which the amount of DKK3 in the sample determined in step a) is increased compared to the reference amount.

[0348] With regard to the method of assessing atrial fibrillation, in particular with regard to the diagnosis of atrial fibrillation, the following applies: Preferably, a ratio that is reduced compared to a reference ratio indicates, i.e. further indicates, a subject suffering from atrial fibrillation. Such a ratio thus confirms the diagnosis of atrial fibrillation based on steps a) and b). An increased ratio compared to a reference ratio indicates heart failure. Such a ratio thus indicates that the subject is likely not suffering from atrial fibrillation. Therefore, further diagnostic measures, such as an ECG for diagnosing atrial fibrillation, should be recommended or initiated.

[0349] With regard to the method for diagnosing heart failure, the following applies: Preferably, an increased ratio compared to a reference ratio indicates a subject with heart failure. Thus, an increased ratio confirms the diagnosis of heart failure based on steps a) and b). A decreased ratio compared to a reference ratio indicates that the subject does not have a diagnosis of heart failure. Thus, such a ratio indicates that the subject does not have atrial fibrillation.

[0350] The present invention thus reports a method for diagnosing both diseases and detecting said risk factors in a subject, the method comprising the steps of determining the amount of DKK3 and NT-proBNP in a sample from the subject, and comparing the amount of DKK3 and NT-proBNP as a ratio. A subject suspected of having heart failure should have a higher ratio of NT-proBNP / DKK3 than a subject with atrial fibrillation.

[0351] The entire disclosures of all references cited in this specification and the disclosures specifically mentioned in this specification are incorporated herein by reference.

[0352] Examples

[0353] The present invention is illustrated by the following examples only. The examples are not to be construed in any way as limiting the scope of the present invention.

[0354] Example 1: Differential expression of DKK3 in cardiac tissue of AF patients

[0355] Differential DKK3 expression levels have been determined in myocardial tissue samples from the right atrial appendage of n=40 samples

[0356] RNAseq analysis

[0357] Atrial tissue was sampled during open-chest surgery for CABG or valvular surgery. Evidence of AF or SR (control) was generated by simultaneous endocardial and epicardial hyperdense activation mapping during surgery. AF patients and controls were matched for sex, age, and comorbidities.

[0358] Atrial tissue samples are ready for

[0359] AF patients; n = 11 samples

[0360] Control SR patients; n = 29 samples

[0361] The algorithms RSEM and DESeq2 were applied to determine the differential expression of DKK3 in RNAseq analysis.

[0362] like Figure 2As shown, DKK3 expression was found to be upregulated in atrial tissue analyzed from 11 patients with persistent AF and 29 controls in sinus rhythm.

[0363] The fold change (FC) of expression was 1.496 and the FDR (false discovery rate) was 0.00001.

[0364] The altered expression of DKK3 was determined in the damaged end organ, atrial tissue. The DKK3 mRNA levels were compared with the results of high-density mapping of atrial tissue. As shown by electrical mapping, elevated DKK3 mRNA levels were detected in atrial tissue samples with conduction disturbances. Fatty infiltration or interstitial fibrosis may cause conduction disturbances. The differential expression of DKK3 observed in atrial tissue of patients with atrial fibrillation supports the release of FABP from the myocardium, especially from the right atrial appendage, in the circulation, and elevated serum / plasma titers assist in detecting AF attacks.

[0365] In conclusion, DKK3 is released from the heart into the blood and may aid in detecting AF episodes and predicting the risk of developing AF-related stroke.

[0366] Example 2: Detecting Atrial Fibrillation in the Predictor Cohort - Specifically screening and identifying patients with atrial fibrillation in the elderly general population

[0367] The PREDICTOR study was a population-based trial of elderly (≥65 years), apparently healthy subjects (n=2001). Participants were referred to a cardiology center for a clinical examination and comprehensive Doppler echocardiographic and electrocardiographic measurements. Patients predominantly had heart failure stage B. However, some patients had heart failure stages A or C. The atrial fibrillation subcohort included 29 subjects who had sustained episodes of atrial fibrillation during their visit and 83 matched controls.

[0368] DKK3 was measured in an atrial fibrillation subcohort selected from the PREDICTOR study. Elevated circulating DKK3 levels were observed in samples from subjects with ongoing atrial fibrillation compared to controls.

[0369] Example 3: Detection of paroxysmal atrial fibrillation

[0370] In the biomarker substudy of the GISSI-AF trial, blood samples were collected at the beginning of the study and after 6 and 12 months of follow-up. For more details on the GISSI-AF trial, see the main publication: GISSI-AF Investigators, New Engl J Med 2009;360:1606-17. For more details on the biomarker substudy, see: Latini R et al., J Intern Med 2011;269:160-71.

[0371] For 382 patients, DKK3 values ​​from plasma samples were obtained at baseline. After 24 weeks, 38 of 360 patients developed paroxysmal atrial fibrillation. After 52 weeks, 48 ​​of 357 patients developed atrial fibrillation.

[0372] DKK3 was measured in an atrial fibrillation subcohort selected from the GISSI AF study. Elevated circulating DKK3 levels were observed in samples from subjects with ongoing atrial fibrillation compared to controls.

[0373] Example 4: Prediction of Stroke

[0374] Analytical methods

[0375] The ability of circulating DKK3 to predict the risk of incident stroke was assessed in a prospective multicenter registry of patients with documented atrial fibrillation (Conen D., Forum Med Suisse 2012; 12: 860-862).DKK3 was measured using a stratified case cohort design as described in Borgan (2000).

[0376] For each of the 70 patients who experienced a stroke during the follow-up period ("event"), 1 matched control was selected. Controls were matched based on demographic and clinical information on age, sex, history of hypertension, type of atrial fibrillation, and history of heart failure (CHF history).

[0377] DKK3 results were available for 69 patients with events and 69 patients without events.

[0378] DKK3 was measured using the Olink platform, therefore no absolute concentration values ​​are available and can be reported. Results will be reported on an arbitrary signal scale (NPX).

[0379] To quantify the univariate prognostic value of DKK3, a proportional hazards model with the outcome of stroke was used.

[0380] The univariate prognostic performance of DKK3 was assessed using two different combinations of the prognostic information given by DKK3.

[0381] The first proportional hazards model included DKK3 binarized at the median (28NPX), thus comparing the risk of patients with DKK3 below or equal to the median to those with DKK3 above the median.

[0382] The second proportional hazards model included the original DKK3 levels but transformed to the log2 scale. The log2 transformation was performed to achieve better model calibration.

[0383] Because estimates from a simple proportional hazards model on the case-control cohort will be biased (due to the varying proportions of cases and controls), a weighted proportional hazards model was used. The weights were based on the inverse probability of each patient being selected for the case-control cohort, as described in Mark (2006).

[0384] To obtain estimates of absolute survival in both groups based on dichotomous baseline DKK3 measurements (<=28 NPX vs. >28 NPX), a weighted version of the Kaplan-Meier curve was created as described in Mark (2006).

[0385] To assess whether the prognostic value of DKK3 was independent of known clinical and demographic risk factors, a weighted proportional Cox model was calculated that also included the following variables: age, sex, history of CHF, history of hypertension, history of stroke / TIA / thromboembolism, history of vascular disease, and history of diabetes.

[0386] To assess the ability of DKK3 to improve existing risk scores for stroke prognosis, we extended CHADS with DKK3 (log2-transformed) 2 , CHA 2 DS 2 -VASc and ABC scores. The risk-sharing model was extended by creating a risk-sharing model including DKK3 and the corresponding risk score as independent variables.

[0387] The CHADS 2 , CHA 2 DS 2 - The c-indexes for VASc and ABC scores were compared with those for these extended models. To calculate the c-index in the case cohort, a weighted form of setting the c-index proposed by Ganna (2011) was used.

[0388] result

[0389] Table 1 shows the results of two univariate weighted proportional hazards models including either binarized or log2-transformed DKK3.

[0390] In both models, the association between the risk of incident stroke and baseline DKK3 values ​​was highly significant.

[0391] The hazard ratio for binarized DKK3 means that the risk of stroke in the group of patients with baseline DKK3>28NPX was 1.8 times higher than that in the group of patients with baseline DKK3<=28NPX. However, the confidence interval includes 1, which means that the median is not the optimal cutoff value or that binarization is not suitable for DKK3.

[0392] Results of a proportional hazards model including DKK3 as a log2-transformed linear hazard predictor showed that log2-transformed values ​​of DKK3 were positively associated with the risk of incident stroke. The hazard ratio of 2.8 could be interpreted as a 2-fold increase in DKK3 being associated with a 2.8 increase in the risk of stroke.

[0393]

[0394] Table 1: Results of univariate weighted proportional hazards models including binarized and log2-transformed DKK3.

[0395] Figure 1 Weighted Kaplan-Meier curves for the two patient groups with baseline DKK3 measurements (<=28 NPX vs. >28 NPX) are shown.

[0396] The results of the proportional hazards model including DKK3 (log2 transformed) combined with clinical and demographic variables are shown in Table 2. Although the point estimate of the hazard ratio for DKK3 is still significantly above 1, the p-value is now above 0.05.

[0397] However, considering that the hazard ratios remained high and the c-index for the model including only the clinical variables shown in Table 2 improved by 0.0055 with the addition of DKK3, it can be expected that the effect of DKK3 would be significant in a larger cohort with more observed events.

[0398]

[0399] Table 2: Multivariable proportional hazards models including DKK3 and related clinical and demographic variables.

[0400] Table 3 shows the results of the weighted proportional hazards model, which combined the CHADS 2 score and DKK3 (log2-transformed). In addition, in this model, DKK3 can add prognostic information to CHADS 2 Similar to Table 2, the hazard ratio for DKK3 is still above 1, but the p value still does not reach 0.05. The relatively small number of events should also be taken into account here.

[0401]

[0402] Table 3: Combined CHADS 2 Weighted proportional hazards model for score and DKK3 (log2 transformed)

[0403] Table 4 shows the results of the weighted proportional hazards model, which combined CHA 2 DS 2 - VASc score and DKK3 (log2 transformed). Similar to Table 2, the hazard ratio for DKK3 is still above 1, but the p value still does not reach 0.05. The relatively small number of events should also be taken into account here.

[0404]

[0405] Table 4: Binding to CHA 2 DS 2 -Weighted proportional hazards model for VASc score and DKK3 (log2 transformed)

[0406] Table 5 shows the results of a weighted proportional hazards model combining the ABC score with DKK3 (log2 transformed). Similar to Table 2, the hazard ratio for DKK3 is still above 1, but the p value still does not reach 0.05. The relatively small number of events should also be taken into account here.

[0407]

[0408] Table 5: Weighted proportional hazards model combining ABC score and DKK3 (log2 transformed)

[0409] Table 6 shows DKK3 alone, CHADS 2 , CHA 2 DS 2 -VASc and ABC scores, and CHADS 2 , CHA 2 DS 2 - Estimated c-index of the weighted proportional hazards model (log2) for the combination of VASc and ABC scores with DKK3.

[0410] To CHA 2 DS 2 - Addition of DKK3 to the VASc score improved the c-index by 0.0028, which can still be considered a clinically meaningful improvement in risk prediction.

[0411] For CHADS 2For the score, the c-index improved higher at 0.0090 and was the highest for ABC at 0.0163.

[0412] C-index DKK3 Univariate 0.6301 <![CDATA[CHADS 2 ]]> 0.6541 <h2 style=";text-align:left;direction:ltr"><![CDATA[CHADS <h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> +DKK3]]><h2 style=";text-align:left;direction:ltr"> 0.6632 <![CDATA[CHA 2 DS 2 -VASc]]> 0.6800 <![CDATA[CHA 2 DS 2 -VASc+DKK3]]> 0.6828 ABC Rating 0.6531 ABC score + DKK3 0.6694

[0413] Table 6DKK3, CHADS 2 , CHA 2 DS 2 - C-index of VASc and ABC scores and their combination with DKK3.

[0414] The results suggest that DKK3 could be used in multiple ways to predict future stroke risk in new patients, either alone or in combination to significantly improve clinical scores in predicting stroke risk, such as the CHADS 2 and CHA 2 DS 2 -VASc).

[0415] For new patients, DKK3 can be measured and compared to a predefined cutoff. If the new patient's measured value is above the predefined cutoff, the patient is considered to be at high risk for stroke and appropriate clinical measures can be initiated.

[0416] More than two risk groups can also be defined based on an increasing set of cutoff values. Patients are then assigned to one of the risk groups based on the measured value of DKK3. The risk of stroke is expected to increase in different risk groups.

[0417] Alternatively, the DKK3 result can be directly converted into a continuous risk score based on a pre-defined appropriate conversion function.

[0418] In addition, DKK3 values ​​can be correlated with risk scores based on clinical and demographic variables (e.g., CHA 2 DS 2 -VASc score) to improve the accuracy of risk prediction.

[0419] For new patients, a value for the risk score should be assessed and combined with the measured DKK3 value (potentially log2-transformed) in an appropriate manner, for example by creating a weighted sum of the risk score result and the DKK3 value with appropriate predefined weights (e.g. as shown in Table 3).

[0420] Example 3: Biomarker Measurement

[0421] DKK3 was measured in the commercially available O-link multi-marker panel of Dickkopf-related protein 3 (DKK3); proximity extension assay from Swedish O-link.

Claims

1. Use of a reagent for determining the amount of DKK3 and optionally the amount of a natriuretic peptide and / or ESM1 in a sample from a subject in the preparation of a kit for performing a method for assessing atrial fibrillation in the subject, the method comprising the following steps a) determining the amount of DKK3 and optionally the amount of natriuretic peptide and / or ESM1 in a sample from said subject, and b) comparing the amount of DKK3 and optionally the amount of the natriuretic peptide and / or ESM1 to a reference amount (or reference amounts), thereby assessing atrial fibrillation.

2. The use according to claim 1, wherein an increase in the amount of DKK3 (and optionally the amount of natriuretic peptide and / or ESM1) in the sample from the subject compared to the reference amount (or multiple reference amounts) indicates that the subject suffers from atrial fibrillation, and / or wherein a decrease in the amount of DKK3 (and optionally the amount of natriuretic peptide and / or ESM1) in the sample from the subject compared to the reference amount (or multiple reference amounts) indicates that the subject does not suffer from atrial fibrillation.

3. The use according to claim 2, wherein the amount of DKK3 and the amount of the natriuretic peptide and / or ESM1 are determined in step a), and wherein the method comprises the following further step: c) calculating the ratio of the amount of the natriuretic peptide and / or ESM1 determined in step a) to the amount of DKK3 determined in step a), and comparing the calculated ratio with a reference ratio.

4. The use according to claim 1, wherein the subject suffers from atrial fibrillation, and wherein the assessment of atrial fibrillation is to distinguish between paroxysmal atrial fibrillation and persistent atrial fibrillation, in particular, wherein an increase in the amount of DKK3 in the sample from the subject compared to the reference amount indicates that the subject suffers from persistent atrial fibrillation, and / or wherein a decrease in the amount of DKK3 in the sample from the subject compared to the reference amount indicates that the subject suffers from paroxysmal atrial fibrillation.

5. The use of claim 1, wherein the subject suffers from atrial fibrillation, and wherein the assessment of atrial fibrillation is an assessment of treatment for atrial fibrillation.

6. Use of a reagent for determining the amount of the biomarker DKK3 and optionally the amount of a natriuretic peptide and / or ESM1 in a sample in the preparation of a kit for performing a method for assisting in the assessment of atrial fibrillation, the method comprising the following steps: a) obtaining a sample from a subject, b) determining the amount of the biomarker DKK3 and optionally the amount of the natriuretic peptide and / or ESM1 in the sample, and c) providing information about the determined amount of the biomarker DKK3 and optionally about the determined amount of the natriuretic peptide and / or ESM1 to the subject's attending physician, thereby assisting in the assessment of atrial fibrillation in the subject.

7. Use of a reagent for testing the biomarker DKK3 and optionally for testing a natriuretic peptide and / or ESM1 in the preparation of a kit for performing a method for assisting in the assessment of atrial fibrillation, the method comprising: a) providing a test for said biomarker DKK3 and optionally a test for a natriuretic peptide and / or ESM1, and b) providing instructions for using test results obtained or obtainable by said one or more tests in said assessment of atrial fibrillation.

8. Use of a preparation for determining the amount of DKK3 in a sample from a subject in the preparation of a kit for performing a method for predicting the risk of stroke in the subject, the method comprising the following steps a) determining the amount of said DKK3 in a sample from said subject, and b) comparing the amount of DKK3 with a reference amount to thereby predict the risk of stroke.

9. Use of a reagent for determining the amount of DKK3 in a sample from a subject in the preparation of a kit for performing a method for improving the predictive accuracy of a clinical stroke risk score for the subject, the method comprising the following steps c) determining the amount of said DKK3 in a sample from said subject having a known clinical stroke risk score, and a) combining the amount of DKK3 with the clinical stroke risk score, thereby improving the predictive accuracy of the clinical stroke risk score.

10. Use of a reagent for determining the amount of DKK3 in a sample from a subject in the preparation of a kit for performing a method for predicting the risk of stroke in the subject, the method comprising the following steps a) determining the amount of said DKK3 in a sample from said subject having a known clinical stroke risk score, and b) combining the amount of DKK3 with the clinical stroke risk score to thereby predict the stroke risk of the subject.

11. Use of i) the biomarker DKK3 and / or ii) at least one detection agent that specifically binds to DKK3 in a sample from a subject in the preparation of a kit for improving the predictive accuracy of a clinical stroke risk score. 12.i) Biomarkers DKK3 and / or ii) at least one detection agent that specifically binds to DKK3, In samples from subjects, Combined with clinical stroke risk score, Use in preparing a kit for predicting the risk of a subject suffering from stroke.

13. A kit comprising a reagent that specifically binds to DKK3 and a reagent that specifically binds to a natriuretic peptide and / or ESM1.

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