WO2018041726A1 - Biomarkers for determining the presence of an unstable atherosclerotic plaque - Google Patents

Abstract

The invention relates to a method for determining the presence of an unstable atherosclerotic plaque in an individual, comprising the steps of determining the expression level of one or more miRNA(s) selected from the group consisting of miR-223-3p, miR-125b-5p, miR-142-3p and miR-193b-3p in a biological sample from said individual; comparing said expression level(s) with a reference level, and determining presence of said unstable atherosclerotic plaque based on said information. The same miRNA species and miR-499a-5p may be used in the diagnosis of risk of suffering from or developing atherosclerosis.

Description

BIOMARKERS FOR DETERMINING THE PRESENCE OF AN UNSTABLE ATHEROSCLEROTIC PLAQUE

TECHNICAL FIELD

The present invention relates to the field of diagnostics. In particular, the invention relates to a method for determining whether a patient suffers from an unstable atherosclerotic plaque. Further, the invention relates to a method of diagnosis of atherosclerosis.

BACKGROUND

When primary prevention fails, coronary atherosclerosis either leads to stable or unstable coronary artery disease. Stable coronary artery disease (CAD) most often remains clinically silent until the atherosclerotic burden becomes so large that the patient develops cardiac complains on physical exercise. Therefore, stable CAD does not pose an immediate treat. However, on the other end of the spectrum, atherosclerosis can transform into an unstable plaque which upon rupture causes a blood clot blocking the coronary blood flow, leading to ischemia of the underlying tissue. Because especially the latter group is at high risk for mortality, it can be very important to distinguish between the mild stable CAD and the malignant unstable CAD.

In contrast to the formation of atherosclerosis, the pathophysiological mechanisms that lead to plaque instability are less well understood. Inflammation seems to play a large role in this process¹ as supported by evidence that hs-C P correlates to the number of plaque ruptures² and inflammatory cells are present at the site of plaque rupture as well as in the circulation^{3, 4}. Also changes at the plaque site such as formation of thin fibrous caps by decreased production or increased degradation of collagen are suggested to promote plaque rupture^5"7. Additionally, physical chemical stressors such as cholesterol crystallization of these thin fibrous caps have been proposed to cause plaque rupture⁸. Although many of these processes have been going before the plaque ruptures, it remains highly unpredictable in which patients plaques will become unstable and cause an acute coronary syndrome and in which patients the plaque will remain stable.

In contrast to stable atherosclerotic plaques, which can remain clinically silent for years, unstable plaques can rupture and could eventually lead to severe ischemia cardiovascular death. Since it remains highly unpredictable in which individuals the atherosclerotic plaque will evolve to become an unstable plaque and eventually will rupture, biomarkers are needed to distinguish between individuals with stable and unstable disease.

It is an objective of the invention to provide one or more biomarkers which can distinguish between patients with unstable and stable plaques. At present, no biomarkers for atherosclerosis are available. There are some imaging modules, but they are cumbersome en expensive methodes. It is another objective of the invention to provide one or more biomarkers which can be used to diagnose atherosclerosis.

SUMMARY OF THE INVENTION In the present invention, several biomarkers are identified which are associated with unstable CAD and atherosclerosis. These include miR-223-3p, miR-125b-5p, miR-142-3p and miR-193b-3p.

The invention therefore provides a method for determining the presence of an unstable

atherosclerotic plaque in an individual, comprising the steps of determining the expression level of one or more miRNA(s) selected from the group consisting of miR-223-3p, miR-125b-5p, miR-142-3p and miR-193b-3p in a biological sample from said individual; comparing said expression level(s) with a reference level, and determining presence of said unstable atherosclerotic plaque based on said information. miR-125b-5p showed a significantly higher expression level among subjects with stable CAD as compared to both patient with unstable CAD and controls. Therefore, in a preferred embodiment, downregulation of miR-125b-5p is indicative of the presence of an unstable atherosclerotic plaque. Further, the inventors found that miR-223-3p was higher expressed in patients with unstable CAD as compared to patients with stable CAD and a significantly higher expression level compared to controls. Therefore, in a preferred embodiment, upregulation of miR-223-3p is indicative of the presence of an unstable atherosclerotic plaque. In another preferred embodiment, the presence of unstable atherosclerotic plaque is based on the expression levels of at least miR-223-3p and miR-125b-5p. Besides, not only miR-223-3p and miR- 125b-5p, but also miR-142-3p was significantly associated with CAD, independently of the known classic risk factors. In a preferred embodiment, upregulation of miR-142-3p is indicative of the presence of an unstable atherosclerotic plaque. miR-193b-3p was also significantly upregulated in stable CAD. Preferably, downregulation of miR-193b-3p is indicative of the presence of an unstable atherosclerotic plaque. Moreover, when combining miR-223-3p and miR-125b-5p, the inventors observed that patients with unstable CAD had a significant higher ratio of miR-223-3p/miR-125b-5p as compared to both patients with stable CAD and controls. Therefore, not only the individual levels, but in particular the ratios of these miRNAs, could be useful biomarkers for plaque instability. Preferably, an increased miR-223- 3p/miR-125b-5p ratio is indicative of an unstable atherosclerotic plaque.

The inventors identified miR-223-3p from thrombectomy material of patients with unstable plaques, in the discovery phase. This miRNA has been described in atherosclerosis^17"20 and is proposed to be a regulator of cholesterol homeostasis²¹, but a role in plaque instability has never been described. miR- 223 acts as a feedback mechanism and inhibits cholesterol biosynthesis, efflux and HDL uptake in the liver²¹ and is therefore upregulated in individuals with atherosclerosis¹⁸.

Although less is known about miR-125b-5p in the cardiovascular field, it has been linked to atherosclerosis. Huang et al.²³ found that miR-125b was downregulated in patients with an acute myocardial infarction as compared to controls. Moreover, they found that miR-125b was downregulated in acute myocardial infarction (AMI) patients as compared to patients with stable AP. However, the inventors found that an increased expression level is associated with unstable CAD.

MiR-125b-5p has been suggested to be involved in vascular smooth muscle cell calcification.

Goettsch et al.²⁴ found that inhibition of miR-125b-5p promoted osteogenic transdifferentiation and found miR-125b-5p downregulated in calcified aortas of apolipoprotein E knockout mice. A role in plaque instability has not been described for miR-125b-5p. The inventors further found that the miRNAS miR-223-3p (OR: 1.66, 95% CI: 1.26-2.18, p-value: < 0.001), miR-142-3p (OR: 1.38, 95% CI: 1.07-1.78, p-value: 0.014), miR-125b-5p (OR: 1.92, 95% CI: 1.23-3.00, p-value: 0.004) and miR-499a-5p (OR: 1.84, 95% CI: 1.14-2.96, p-value: 0.012) showed an independent significant upregulation in atherosclerosis compared to controls, even after adjusting for various confounders (Figure 4, Table 5). All these miRNA markers for atherosclerosis remained significant after correction for classical risk factors age, gender, hypercholesterolemia, hypertension, smoking, diabetes and BMI (Table 6), suggesting that these markers are predictive for atherosclerosis independent of these classical risk factors and may therefore add to risk prediction. Together, these markers yielded an AUC of 0.68. Further, MiR-193b-3p showed a significant difference between groups. This study shows that individuals with an acute coronary syndrome ACS have an increased ratio of miR-223-3p/miR-125b-5p, which might be used in clinical practice to identify patients at risk for an ACS. Furthermore, miR-223-3p, miR-142-3p and miR-223-3p 125b-5p were all significantly upregulated in coronary atherosclerosis compared to controls, showing that these MIRNAS may be used to indentify subclinical atherosclerosis, without the need to do cumbersome imaging. Moreover, all these miRNAs performed independent of known classic risk factors, suggesting that they might provide an addition to established risk factors for CAD.

Therefore, the invention further provides a method for determining the risk of suffering from or developing of atherosclerosis in an individual, comprising the steps of: a. determining the expression level of one or more miRNA(s) selected from the group consisting of miR-223-3p, miR-125b-5p, miR-142-3p and miR-193b-3p in a biological sample from said individual,

b. comparing said expression level with a reference level, and

c. determining the risk of suffering from or developing atherosclerosis based on the information obtained in step b.

Preferably, the expression level of at least miR-223-3p and miR-125b-5p is determined and wherein atherosclerosis is diagnosed, based on the expression levels of miR-223-3p and miR-125b-5p.

Preferably, atherosclerosis is diagnosed, based on the miR-223-3p/miR-125b-5p ratio.

In a preferred embodiment, said biological sample is serum, plasma or blood. In a preferred embodiment, said individual is suffering from atherosclerosis. Preferably, said expression level is determined using qPCR.

The invention further provides a kit for determining the presence of an unstable atherosclerotic plaque or for diagnosing atherosclerosis in an individual comprising a first nucleic acid capable of hybridizing under stringent conditions with any of miR-125b-5p, miR-223-3p, miR-142-3p and miR- 193b-3p and a second nucleic acid capable of hybridizing under stringent conditions with any of miR- 1260, miR-1280, miR-484 and miR-718.

The invention further provides a kit according to claim comprising nucleic acids capable of hybridizing under stringent conditions with any of miR-125b-5p and miR-223-3p. Preferably, said kit further comprises one or more nucleic acid(s) capable of hybridizing under stringent conditions with any of miR-1260, miR-1280, miR-484 and miR-718.

BRIEF DESCRIPTION OF THE DRAWINGS

Table 1 provides an overview of the patients characteristics. All values are given as mean ± standard deviation for continuous variables or as absolute number (percentage). P-Values are obtained by T- test (Array cohort) or ANOVA with post hoc T-tests for numerical values and Chi2 ws performed for dichotomous and categorical variables. *,^† and Φ indicate significant differences between AP and control, Ml and control and AP and Ml groups, respectively. Hyperchol. = hypercholesterolemia, Ml = myocardial infarction, PCI = percutaneous coronary intervention, CABG = coronary artery bypass graft, AP = angina pectoris, VD = vessel disease, ACEi = Angiotensin Converting Enzyme inhibitor. ARB = Angiotensin Receptor Blocker. Oral antiDM = oral antidiabetics. Table 2(A and B) shows the normalized expression and difference in expression between groups of the top 10 candidates from the array experiment. Table 2A shows the candidates in which expression was increased in the ACS group and table 2B shows those increased in the AP group. P-values were Benjamini-Hochberg (B-H) corrected for multiple testing.

Table 3 shows the Odds Ratios (OR), 95% confidence intervals (CI) and p-values of the logistic regression analysis from the validation experiment. Odds ratios are given for the univariate analysis as well as for the age and both age and sex corrected analysis.

Table 4 shows the Odds Ratios (OR), 95% confidence intervals (CI) and p-values of the logistic regression analysis comparing the miR-223-3p/miR-125b-5p ratios between the groups of ACS patients, stable AP patients and healthy controls. Odds ratios are given for the univariate analysis as well as for the age and both age and sex corrected analysis.

Table 5. Shows the Odds Ratios (OR), 95% confidence intervals (CI) and p-values of the logistic regression analysis comparing the atherosclerosis with controls. Odds ratios are given for the raw, univariate analysis as well as for the analysis corrected for age and both age and sex.

Table 6 shows the Odds Ratios (OR), 95% confidence intervals (CI) and p-values of the multivariate logistic regression analyses on the outcome atherosclerosis corrected for the classical atherosclerotic risk factors.

Figure 1 shows the normalized expression levels of the ACS candidates both the array and the validation. Panel A and C respectively show the expression levels of miR-223-3p and miR-142-3p between atherectomy tissue of patients with stable AP (n=14) and thrombectomy material from patients with an Ml (n=25). These miRNAs were validated by qPCR in serum of patients with an ACS (n=64), stable AP (n=139) and controls (n=192). Normalized and log-transformed expression levels of miR-223-3p and miR-142-3p are shown in panel B and D, respectively. More detailed information on expression levels is found in table 3. *p < 0.05 **p< 0.01.

Figure 2 shows the normalized expression levels of the stable AP candidates both the array and the validation. Panel A and C respectively show the expression levels of miR-125b-5p and miR-193b-3p between atherectomy tissue of patients with stable AP (n=14) and thrombectomy material from patients with an Ml (n=25). These miRNAs were validated by qPCR in serum of patients with an ACS (n=64), stable AP (n=139) and controls (n=192). Normalized and log-transformed expression levels of mi -125b-5p and miR-193b-3p are shown in panel B and D, respectively. More detailed information on expression levels is found in table 3. *p < 0.05 **p< 0.01.

Figure 3 shows the normalized and log-transformed ratios of miR-223-3p/miR-125b-5p in patients with and Ml (n=64), stable AP (n=139) and in healthy controls (n=192). More detailed information on expression levels is found in table 4. *p < 0.05 **p< 0.01.

Figure 4 shows the normalized and log-transformed expression levels of both CAD groups (ACS + stable AP) (n= 203) compared to control (n= 193) of miR-223-3p (A), miR-142-3p (B), miR-125b-5p (C), 193b-3p (D) and miR-499a-5p (E). *p < 0.05 **p< 0.01, ***p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In accordance with the invention and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise. As used herein, "atherosclerosis" refers to a disease of the arterial blood vessels that results in hardening or furring of the arteries caused by the formation of multiple atheromatous plaques within the arteries. Pathologically, the atheromatous plaque includes nodular accumulation of a soft, flaky, yellowish material at the center of large plaques, composed of macrophages nearest the lumen of the artery, sometimes with underlying areas of cholesterol crystals, and possibly also calcification at the outer base of older/more advanced lesions. The atheromatous plaques, though compensated for by artery enlargement, eventually lead to plaque ruptures and stenosis (i.e., narrowing) of the artery and, therefore, an insufficient blood supply to the organ it feeds. Alternatively, if the compensating artery enlargement process is excessive, then a net aneurysm results. The complications associated with atherosclerosis are chronic, slowly progressing and cumulative. Most commonly, the rupture of a soft plaque causes the formation of a blood clot (e.g., thrombus) that will rapidly slow or stop blood flow, e.g. 5 minutes, leading to death of the tissues fed by the artery. A common recognized scenario is coronary thrombosis of a coronary artery causing myocardial infarction (i.e., a heart attack).

Another common scenario in advanced disease is claudication from insufficient blood supply to the legs, typically due to a combination of both stenosis and aneurysmal segments narrowed with clots. Kidney, intestinal and other arteries are also typically involved. In the context of the invention, an "atherosclerotic plaque" or " atheromatous plaque" refers to a lesion of vessel walls. Preferably, an "atherosclerotic plaque" as used herein comprises a lipid core and a fibrous cap, said cap being constituted by smooth muscle cells, collagens and an extracellular matrix, and isolating the lipid core from the arterial lumen. As known from the person skilled in the art, an atherosclerotic plaque is generally due to the accumulation and swelling in artery walls where the swelling is caused by for example macrophages, cell debris, lipids such as cholesterol and fatty acids, calcium and fibrous connective tissue. Thus, the process of atheroma development within an individual is called atherogenesis and the overall result of the disease process is termed

atherosclerosis. Atherosclerotic plaques may be found in particular in the aorta, in the carotid or in the coronary artery, and also in peripheral vessels. Atherosclerotic plaques may be further divided into "stable" atherosclerotic plaques and "unstable" or "vulnerable" atherosclerotic plaques.

The term "stable coronary heart disease" as used herein encompass coronary heart diseases including stenosis, atherosclerosis of the coronary vessels within the coronary vessel system. The coronary heart diseases shall be stable in that no symptoms of the disease shall appear at rest and, preferably, no progression or worsening of the symptoms accompanied with the diseases will be observed for the subject suffering therefrom. More preferably, a subject exhibiting a stable coronary heart disease will exhibit a reversible time period of ischemia of about 5 to 15 minutes after exercise. A subject suffering from an unstable coronary heart disease will, however, show irreversible ischemia after exercise or even at rest. In a preferred embodiment, said stable coronary heart disease is stable coronary artery disease.

As used herein, the terms "unstable atherosclerotic plaque", "vulnerable atherosclerotic plaque", "complicated atherosclerotic plaque", "complicated plaque" and "CP" are used indifferently and refer to an atherosclerotic plaque which is prone to rupture. Unstable atherosclerotic plaques may in particular be characterized by a thin fibrous cap (<100 μιτι thick) infiltrated by

monocyte/macrophage and sometimes T-cell and a lipid core accounting for 40% of the plaque's total volume. In the context of the invention, unstable atherosclerotic plaques (or vulnerable plaques) encompass thin cap fibroatheroma (TCFA), pathologic intimal thickening, thick cap fibroatheroma and calcified plaque with luminal calcified nodules. In the context of the present invention, when referring to unstable atherosclerotic plaques, the terms "vulnerable", "unstable", "dangerous" and "high-risk" are synonymous and interchangeable (Naghavi et al. (2003) Circulation 108:1664-1672; Naghavi et al. (2003) Circulation 108:1772-1778).

As used herein, the terms "stable atherosclerotic plaque", "non complicated atherosclerotic plaque", "non complicated plaque" and "NCP" are used indifferently and refer to an atherosclerotic plaque which is not prone to rupture and/or which does not show important inflammation and important lipid accumulation. In particular, an atherosclerotic plaque may contain an area which corresponds to an unstable atherosclerotic plaque and an area which corresponds to a stable atherosclerotic plaque.

In the context of the present invention, an "individual" denotes a human or non- human mammal, such as a rodent (rat, mouse, rabbit), a primate (chimpanzee), a feline (cat), a canine (dog).

Preferably, the individual is human. Preferably, said individual is suffering from atherosclerosis.

As used herein, the term "biological sample" refers to a sample of tissue or fluid isolated from an individual. In particular a biological sample may be a liquid biological sample or a tissue sample. Examples of biological samples include, but are not limited to, pieces of organs or of tissues such as kidney, liver, heart, lung, and the like, arteries veins and the like, blood and components thereof such as plasma, platelets, serum, subpopulations of blood cells and the like, tears, urine and saliva.

Preferably, when the biological sample is a liquid biological sample, it is selected from the group consisting of blood sample, serum, plasma and urine. More preferably, when the biological sample is a liquid biological sample, it is a blood, serum or plasma sample. As used herein "determining the level of a certain miRNA in a sample" means assaying a test sample, e.g. a serum sample from a patient, in vitro to determine the concentration or amount of the miRNAs in the sample. Any convenient qualitative, semi-quantitative or, preferably, quantitative detection method for determining nucleic acids can be used to determine the concentration or amount of the miRNAs in the sample. A variety of methods for determining nucleic acids are well known to those of skill in the art, e.g. determination by nucleic acid hybridization and/or nucleic acid amplification. Exemplary methods to determine the concentration or amount of the miRNAs in the sample are provided below.

The term "miRNAs" refers to small non-coding RNAs (17-24 nucleotides) that regulate gene expression by binding to partly complementary sequences in messenger RNA transcripts (mRNAs) thereby preventing the mRNAs from being translated into protein. Due to their function as regulators of gene expression they play a critical role in fundamental biological processes, including

hematopoietic differentiation, cell cycle regulation, metabolism, cardiovascular biology, and immune function, and have been suggested to be involved in pathological processes. It has been found that the expression level (or expression pattern) of miRNAs varies over time and between tissues/cells. The terms "miR-223-3p", "125b-5p", " miR-142-3p" , "miR-499a-5p" and "miR-193b-3p" as used herein refer to the miRNAs as retrieved in miRBase version 21. An exemplary sequence of miR-223- 3p is UGUCAGUUUGUCAAAUACCCCA (SEQ ID NO:l). An exemplary sequence of miR-125b-5p is UCCCUGAGACCCUAACUUGUGA (SEQ ID NO:2). An exemplary sequence of miR-142-3p is UGUAGUGUUUCCUACUUUAUGGA (SEQ ID NO:3). An exemplary sequence of miR-193b-3p is AACUGGCCCUCAAAGUCCCGCU (SEQ ID NO:4). An exemplary sequence of miR-499a-5p is

UUAAGACUUGCAGUGAUGUUU (SEQ ID NO:5).

As used herein, the term "PCR" refers to polymerase chain reaction. PCR is a method for the detection of a template sequence in an amplified product from a reaction determined by the addition of a pair of oligonucleotide primers.

As used herein, the term "quantitative PCR" ("qPCR") refers to the quantitative polymerase chain reaction. Quantitative PCR is a means for quantifying the amount of template DNA present in the original mixture, usually achieved by the addition of a known amount of a target sequence that is amplified by the same primer set but can be differentiated at the end of the reaction.

As used herein, the term "real-time PCR" refers to the real-time polymerase chain reaction. Realtime PCR is a method for the detection and quantitation of an amplified PCR product based on a fluorescent reporter dye; the fluorescent signal increases in direct proportion to the amount of PCR product produced and is monitored at each cycle, 'in real time', such that the time point at which the first significant increase in the amount of PCR product correlates with the initial amount of target template.

Embodiments

The invention provides a method for determining the presence of an unstable atherosclerotic plaque in an individual, comprising the steps of: a) determining the expression level of one or moremiRNA(s) selected from the group consisting of miR-125b-5p, miR-223-3p, miR-142-3p and miR-193b-3p in a biological sample from said individual,

b) comparing said expression level with a reference level, and

c) determining presence of said unstable atherosclerotic plaque based on the information obtained in step b.

The invention further provides a method for diagnosis of atherosclerosis in an individual, comprising the steps of: a) determining the expression level of one or more miRNA(s) selected from the group consisting of miR-223-3p, miR-125b-5p, miR-142-3p and miR-193b-3p in a biological sample from said individual,

b) comparing said expression level with a reference level, and

c) diagnosing atherosclerosis based on the information obtained in step b. The concentration or amount of the miRNAs in the biological sample may be directly determined in the sample, that is, without an RNA extraction step. Alternatively, RNA may be extracted from the sample prior to miRNA processing for detection. RNA may be purified using a variety of standard procedures as described, for example, in RNA Methodologies, A laboratory guide for isolation and characterization, 2nd edition; 1998, Robert E. Farrell, Jr., Ed., Academic Press. In addition, there are various processes as well as products commercially available for isolation of small molecular weight RNAs, including miRNeasy™ kit (Qiagen), MagMAX™ kit (Life Technologies), Pure Link™ kit (Life Technologies), TRIzol LS reagent (Invitrogen Corp., Carlsbad, CA), and mirVANA™ miRNA Isolation Kit (Ambion). For example, small molecular weight RNAs may be isolated by organic extraction followed by purification on a glass fiber filter. Alternative methods for isolating miRNAs include hybridization to magnetic beads.

The determination of stability of the plaque is based on comparing the expression level(s) of the miRNAs in the patient's sample with those obtained using relevant controls, e.g. internal standards, biological samples from subjects diagnosed with an unstable atherosclerotic plaque, or from individuals with an acute phase of a myocardial infarction. In a highly preferred embodiments, biological samples from individuals suffering from unstable CAD are used as control.

The diagnosis of atherosclerosis is based on comparing the expression level(s) of the miRNAs in the patient's sample with those obtained using relevant controls, e.g. internal standards, biological samples from subjects diagnosed with an atherosclerosis.

It will be understood that it is not absolutely essential that an actual control sample be run at the same time that assays are being performed on a test sample. Once "normal," i.e., control, levels of the miRNAs (or of miRNA ratios) have been established, these levels can provide a basis for comparison without the need to rerun a new control sample with each assay. The step of determining whether a subject has an unstable atherosclerotic plaque is based on the information obtained by comparison between the expression level with a reference level. The expression level(s) of the analysed miRNA(s) when statistically analysed will have a threshold whereby expression levels of the individual miRNA(s) below or above the threshold are indicative for respectively the presence or absence of an unstable atherosclerotic plaque. The step of determining whether a subject is at risk of suffering from atherosclerosis or of developing atherosclerosis is based on the information obtained by comparison between the expression level with a reference level. The expression level(s) of the analysed miRNA(s) when statistically analysed will have a threshold whereby expression levels of the individual miRNA(s) below or above the threshold are indicative for an increased risk of suffering from or developing atherosclerosis.

Threshold miRNA levels for each of the analysed miRNAs can be determined by any suitable algorithm. Such an algorithm may involve classifying a sample between at risk and not at risk groups. For example, samples may be classified on the basis of threshold values, or based upon Mean and/or Median miRNA levels in at risk patients versus not at risk (e.g., a cohort from the general population or a patient cohort with diseases unrelated to unstable atherosclerotic plaque). Various classification schemes are known for classifying samples between two or more groups, including Decision Trees, Logistic Regression, Principal Components Analysis, Naive Bayes model, Support Vector Machine model, and Nearest Neighbour model. In addition, the predictions from multiple models can be combined to generate an overall prediction.

The miRNA expression level (miRNA signature, level, or miRNA concentration) is generated

(determined) from (in) the biological-sample using any of various methods known in the art for quantifying miRNA levels. Such methods include polymerase-based assays, such as Real-Time PCR (e.g., Taqman™), hybridization-based assays, for example using microarrays (e.g. miRNome microRNA Profilers QuantiMir Human PCR array (Biocat)), nucleic acid sequence based amplification (NASBA), flap endonuclease-based assays, as well as direct RNA capture with branched DNA

(QuantiGene™), Hybrid Capture™ (Digene), or nCounter™ miRNA detection (nanostring). The assay format, in addition to determining the miRNA levels will also allow for the control of, inter alia, intrinsic signal intensity variation. Such controls may include, for example, controls for background signal intensity and/or sample processing, and/or hybridization efficiency, as well as other desirable controls for quantifying miRNA levels across samples (e.g., collectively referred to as "controls"). Many of the assay formats for amplifying and quantitating miRNA sequences, and thus for generating miRNA levels are commercially available and/or have been described, e.g. in WO 2008/153692, WO 2010/139810, and WO 2011/163214, or references cited therein. In a preferred embodiment, expression level is determined using QPCR.

In all cases, unless otherwise explicitly specified, the miRNAs of the invention refer to human sequences, which may be indicated with the prefix "hsa" (Homo sapiens), i.e. hsa-miR-223-3p and has-miR-125b-5p, under the standard nomenclature system. Although the miRNAs tested for are indicated as RNA sequences, it will be understood that, when referring to hybridizations or other assays, corresponding DNA sequences can be used as well. For example, RNA sequences may be reverse transcribed and amplified using the polymerase chain reaction (PCR) in order to facilitate detection. In these cases, it will actually be DNA and not RNA that is directly quantitated. It will also be understood that the complement of the reverse transcribed DNA sequences can be analysed instead of the sequence itself. In this context, the term "complement" refers to an oligonucleotide that has an exactly complementary sequence, i.e. for each adenine there is a thymine, etc. Although assays may be performed for the miRNAs individually, it is generally preferable to assay several miRNAs or to compare the ratio of two or more of the miRNAs. In a preferred embodiment, the miR- 223-3p/miR-125b-5p ratio is used as a biomarker indicative of an unstable atherosclerotic plaque.

The method of the invention can comprise differentiating between patients with unstable atherosclerotic plaque and patients with a stable atherosclerotic plaque, wherein miR-223-3p and/or miR-142-3p are up-regulated (increased concentration) in the biological sample from said individual compared to a normal control. In other preferred embodiments wherein miR-125b-5p and/or miR- 193b-3p are upregulated in the biological sample, said upregulation is indicative of a stable atherosclerotic plaque.

In a preferred embodiment, said method for determining the risk of suffering from or developing of atherosclerosis in an individual comprise differentiating between patients at risk and not at risk, wherein any of miR-125b-5p, miR-223-3p, miR-142-3p, miR-499a-5p and miR-193b-3p are upregulated in the biological sample from said individual compared to a normal control. In a preferred embodiment, miR-223-3p, miR-142-3p and miR-125b-are up-regulated (increased concentration), which is indicative of an increased risk of suffering from or developing

atherosclerosis.

QPCR of circulating miRNAs in plasma or serum are sensitive to false or inaccurate signals, which is partly explained by the often low concentrations of miRNAs in plasma and serum¹⁵. Therefore, it is preferred to use a quality assessment algorithm to ensure the validity of each measurement. Such quality assessment algorithms are known in the art and described extensively elsewhere (inter alia in de Ronde et al. In a preferred embodiment, measurements of expression levels are distinguished three groups of measurements: 'valid', 'invalid', or 'nondetectable' (signal too low). In case of a nondetectable, the sample is preferably be set to a low value, which is preferably defined as the highest Cq (i.e. the lowest concentration) that produces a reliable result + 1. For example, if the highest reliable Cq is 36, the Cq of the nondetectable sample is preferably set to 37. If the measurement do not pass the quality controls of the algorithm (e.g. there is too much difference between duplicates or a bad melting curve), it may be defined as 'invalid'. When a measurement is considered invalid, it is preferably taken into the analysis as missing at random and is therefore imputed using multiple imputations. If the measurement passes all the quality checks, it can be marked as 'valid' and may be included in the analysis.

In a preferred embodiment, the method of the invention can further comprise determining the level of one or more normalization control(s) in the sample. Preferably, the sample can be spiked with the normalization control(s).

Preferred according to the invention, the normalization control can be a non-endogenous RNA or miRNA, or a miRNA not expressed in the sample. For example, the normalization control may be one or more exogenously added RNA(s) or miRNA(s) that are not naturally present in the biological sample, e.g. an RNA or miRNA from another organism, and/or one or more human miRNAs not expressed in the sample-sample undergoing analysis. In a highly preferred embodiment, said level of the miRNA of the invention is normalized using one or more reference miRNAs which are stably expressed in serum. Preferably, said one or more reference miRNAs is selected from the group consisting of miR-1260, miR-1280, miR-484 and miR-718. The normalization may suitably be performed as described herein and as described in European patent application EP15166257. The miRNA level may be determined with the use of a custom kit or array, e.g., to allow particularly for the profiling of the miRNAs of the invention. Accordingly, the present invention further provides a kit (or test) for determining the presence of an unstable atherosclerotic plaque in an individual based upon the miRNA levels in the biological samples as described herein.

The kit for determining the presence of an unstable atherosclerotic plaque of the invention may comprise means for determining the concentration (expression level) of miR-125b-5p, miR-223-3p, miR-142-3p, miR-499a-5p and/or miR-193b-3p in a biological sample from the individual. The means for determining the concentration of said miRNAs can be nucleic acids capable of hybridizing under stringent conditions with any of miR-125b-5p, miR-223-3p, miR-142-3p, miR-499a-5p and miR-193b- 3p. Such nucleic acids may include miRNA-specific primers for reverse transcribing or amplifying each of miR-125b-5p, miR-223-3p, miR-499a-5p, miR-142-3p and/or miR-193b-3p. For example, the means for determining the concentration of miR-125b-5p, miR-223-3p, miR-142-3p, miR-499a-5p and/or miR-193b-3p may be TaqMan probes specific for each miRNA of the kit. In a preferred embodiment, said kit comprises the means for determining the expression levels of miR-125b-5p and miR-223-3p. In another preferred embodiment, said kit comprises the means for determining the expression levels of miR-223-3p, miR-142-3p and miR-125b. In a further preferred embodiment, said kit further contains the means for determining the expression levels of miR-1260, miR-1280, miR-484 and miR-718. The design of oligonucleotide probes specific for miR-125b-5p, miR-223-3p, miR-142-3p, miR-499a- 5p and miR-193b-3p; or miRNA-specific primers for reverse transcribing or amplifying each of miR- 125b-5p, miR-223-3p, miR-142-3p, miR-499a-5p and miR-193b-3p to detect their expression levels (concentrations) in accordance with suitable assay formats is well known to those of skill in the art, and appropriate probes and/or primers can be commercially purchased.

Further, the kit may comprise an enzyme for cDNA preparation (e.g. , reverse transcriptase) and/or PCR amplification (e.g., Taq polymerase), and/or a reagent for detecting and/or quantifying miRNA. Additionally, the kit may further comprise include a reagent for miRNA isolation from samples. The kit can also comprise one or more normalization control(s). The normalization control(s) can, for example, be provided as one or more separate reagent(s) for spiking samples or reactions.

Preferably, the normalization control(s) is/are selected from non-endogenous RNA or miRNA, or a miRNA not expressed in the sample. In a preferred embodiment, said kit comprises a specific primer for reverse transcribing or amplifying one or more reference miRNAs is selected from the group consisting of miR-1260, miR-1280, miR-484 and miR-718. The above disclosure generally describes the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE

Methods

Source populations All CAD subjects were obtained from consecutive patients who underwent a percutaneous coronary intervention (PCI) at the Academic Medical Center - University of Amsterdam between February 1993 and October 2010. Our institution is a high volume tertiary referral hospital with on-site cardiac surgery. PCI was performed according to the standard PCI guidelines by highly experienced operators. All patients received aspirin, a loading dose of clopidogrel, prasugrel or ticagrelor and unfractioned heparin 5000 IU at the start of the procedure.

Controls were first degree relatives (FD s) of individuals with premature CVD, obtained from the outpatient clinic for cardiovascular risk assessment of the Academic Medical Center in Amsterdam between July 2009 and July 2014. All controls underwent coronary CT-scanning.

Blood withdrawal and tissue collection

Blood was drawn in vails just before performing the PCI and before the unfractioned heparin 5000 IU was administered.

Between 1993 and 1996, atherectomy was performed using high frequency rotablation as described by Erbel et al¹². Tissue collected during the procedure and stored in a -80°C until further use.

Study populations

For the array cohort, in total 39 subjects were selected from the source population that underwent either atherectomy (n=14) during elective PCI because of stable angina complaints (stable AP) or thrombectomy (n=25) during an acute PCI because of an acute coronary syndrome (ACS group). Patients were matched for age and gender.

In the validation cohort, 203 CAD patients who underwent an acute PCI because of unstable AP (ACS group, n=64) or an elective PCI because of stable AP (stable AP group, n=139) were randomly selected from the source population between January and December of 2008. PCR-based array

Frozen Atherectomy and thrombectomy tissue was sliced, thawed and dissolved into 1ml of Trizol LS reagent (Invitrogen Corp., Carlsbad, CA) and was incubated for 10 minutes at room temperature followed by 200μΙ chloroform. The mixture was centrifuged at 12,000 g for 10 minutes, and the aqueous layer was transferred to a new tube. RNA was precipitated by isopropanol and washed with 75% ETOH subsequently. RNA pellet was collected in 30μΙ RNAse free water and checked by Nanodrop (Thermofischer scientific, Nanodrop products, Wilmington, USA).

To obtain cDNA, 8 μΙ of RNA was reverse transcribed using the miRCURY LNA™ Universal RT microRNA PCR, Polyadenylation and cDNA synthesis kit (Exiqon, Vedbaek, Denmark). cDNA was diluted 50 x and in total, 742 microRNAs (human panel I and II) and negative controls were measured PCR according to the protocol for miRCURY LNA™ Universal RT microRNA PCR (Exiqon, Vedbaek, Denmark) on the LightCycler^® 480 Real-Time PCR System (Roche, Basel, Switzerland).

Amplification curves were analyzed using the Roche LC software, both for determination of Cp (by the 2nd derivative method) and for melting curve analysis. Amplification efficiency was calculated using algorithms similar to the LinRegPCR software. Furthermore assays must be detected with 5 Cqs less than the negative control, and with Cq <37 to be included in the data analysis. Data that did not pass these criteria were omitted from any further analysis. Using NormFinder the best normalizer was found to be the average of assays detected in all samples. All data was normalized to the average of assays detected in all samples (average - assay Cp). miRNAs were only included in the analysis when both groups contained at least 14 measurements.

qPCR measurements of miRNAs: Validation phases Validation phase: selection of miRNAs

Top candidates selected from the PCR based array were selected for validation and comprised miR- 223, miR-142-3p, miR-126-5p (ACS candidates), miR-125b-5p, miR-455-3p, miR-193b-3p (AP candidates).

RNA isolation

For qPCR, RNA was extracted from 250μΙ serum using 750μΙ TRIzol LS reagent (Invitrogen Corp., Carlsbad, CA) and was incubated for 10 minutes at room temperature followed by 200μΙ chloroform. The mixture was centrifuged at 12,000 g for 10 minutes, and the aqueous layer was transferred to a new tube. RNA was precipitated by isopropanol and washed with 75% ETOH subsequently. RNA pellet was collected in 40μΙ RNAse free water. Nucleic acid quantification could not be performed due to the low concentration of RNA in plasma. DNAse and RNAse treatment was omitted since previous experiments showed no difference of miRNA expression in plasma between with and without these treatments.

Reverse transcriptase ofmiRNAs

Complementary DNA was synthesized using the Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems, Gent, Belgium) and miRNA-specific stem-loop primers using the manufacturer's instructions in a total reaction volume of 7,5μί. Each reaction contained 2.5μί of RNA, 1.5μί reverse transcriptase primers and 3.5μί of master mix. The reaction mixture was incubated for 30 minutes at 16°C, 30 minutes at 42°C, 5 minutes at 85°C, 5 minutes at 15°C and finally held at 4°C for 10 minutes. cDNA was diluted in a 1:15 ratio using nuclease free water.

Quantification of miRNA expression by RT-qPCR

MiRNA expression levels were quantified in triplicates by RT-qPCR using Taqman microRNA assays (Applied Biosystems), according to the manufacturer's instructions. Each reaction consisted of 5μί 480 probe master, 0.5μί Taqman primers, 1.33μί diluted cDNA and 3.17μί nuclease free water, totaling a volume of ΙΟμί. On a LightCycler 480 system II (Roche, Basel, Switzerland), the reaction mixture was incubated for 10 minutes at 95°C followed by 50 cycles of 15 seconds of 95°C and 60 seconds at 60°C. PCR efficiencies were between 1.87 and 2.01 for all miRNAs. Data were analysed using LinRegPCR quantitative PCR data analysis software, version ll¹⁶.

qPCR data handling and normalization miRNA expression was normalised to the geometric mean of a previously established miRNA normalisation panel for serum samples consisting of miR-1260, -1280, -484 and -718¹⁶.

Statistical analysis

Baseline characteristics are expressed as mean ± standard deviation for continuous variables and number (%) for dichotomous variables, except when indicated otherwise. ANOVA with post-hoc Student's t-tests, Mann-Whitney U-tests and Fisher's exact test were used to calculate differences in baseline characteristics as appropriate. Variables with a skewed distribution, e.g. the miRNA expression levels, were log-transformed before they were analysed. T-tests were used to calculate differences in array expression which were expressed in fold change. P-values of PCR based array were Benjamini-Hochberg corrected for multiple testing.

Logistic regression was used to analyse the differences in miRNA expression between AP, Ml and the control group in validation 1 and 2. Results from the array cohort were matched for age and gender. Results in cohort 2 were adjusted for age and gender.

All statistical analyses were performed using SPSS for Windows Version 23. A p-value < 0.05 was considered statistically significant.

Results

Description of the cohorts:

Clinical characteristics are displayed in Tablel. In the array cohort (n=39) the ACS group used significantly more beta-blockers, calcium antagonists and nitrates compared to the AP group. In the validation cohort, the stable AP group and the ACS group were both significantly older than the control group, were more often male and had more often diabetes, more often hypertension, and did more often use all types of medication, except for insulin. Compared to controls, the stable AP group had more often hypercholesterolemia. Furthermore, the ACS group showed significantly more 3 vessel disease compared to the Ml group.

Array:

In the array experiment, the inventors aimed to find miRNA candidates that showed tissue specific differences in expression between stable and unstable plaques. Therefore, a PCR-based miRNA array was performed on RNA extracted from atherectomy tissue (n=14) of patients with stable angina pectoris (AP) and thrombectomy tissue from patients with an a myocardial infarction (n=25) (Ml), measuring 742 distinct miRNAs.

Clinical characteristics are displayed in Table 1. There were no significant differences in age and sex between both groups. Based on expression levels, a list of top candidates with high expression in the Ml groups and one of candidates with high expression in the AP group was composed. Among top candidates, mi -223-3p was found most upregulated in the Ml group compared to the AP group (mean diff: 2.55, 95%CI: 2.00-3.10, corr. p-value <0.001 ) and miR-125b-5p was most upregulated in the AP group compared to the Ml group (mean diff: 5.94, 95%CI: 5.04-6.85, corr. p-value < 0.001). Other top candidates are displayed in table 2.

Number of valid, invalid and nondetectable miRNA measurements

As described before, raw qPCR data was processed using a prespecified algorithm. QPCR measurements were classified as 'valid' (reliably measurable), 'invalid' (unreliable measurement) or 'nondetectable' (unmeasurably low) as described above. As shown in supplemental table 2, all miRNAs used for normalization had high 'valid' rates. miR-142-3p, miR-125-5p and miR-193b-3p showed a 'valid' rate of 42%, 48% and 41% respectively with low rates of 'invalids', showing that these miRNAs were reliable measurable, however a substantial number of measurements were below the detection limit.

Validation phase: expression levels ACS vs stable AP

After the discovery of candidates, the top 3 differently expressed candidates from both the list of ACS vs Ml and vice versa as depicted in table 2 were selected and expression was measured in serum to confirm the differences between stable and unstable CAD.

Similar to the array experiment, an upregulation of miR-223-3p, was found in the ACS group versus the stable AP group (Figure 1A and B, table 3A). Compared to controls, both the stable AP group and the ACS group showed a significant difference (stable AP vs control OR: 1.61, 95% CI: 1.20-2.15, p- value: 0.002; Ml vs control: OR: 2.38, 95% CI: 1.41-4.01, p-value: 0.001). The upregulation of miR-125b-5p in the stable AP group that was found in the array experiment could be replicated in the validation phase (Figure2A and 2B, table 3B); the stable AP group showed significantly higher expression levels compared to the ACS group (OR: 1.64 , 95% CI: 1.09-2.48, p- value: 0.019). Moreover, the expression in the AP group was significantly higher than in control subjects (OR: 2.09 , 95% CI: 1.30-3.36, p-value 0.002). The ACS group showed an expression halfway between the AP and the control group. Validation phase: miR-223-3p/miR-125b-5p ratios

Next, since in clinical practice, it would be most useful to identify unstable CAD, the inventors investigated whether a combination of the mi NAs was able to identify unstable plaque. For that matter the inventors calculated the ratios of miR-223-3p/miR-125b-5p were able to distinguish the ACS group from both the stable AP group and the control group. MiR-223-3p/miR-125b-5p ratios showed a significant increase in the ACS group compared to both the AP group (OR: 1.37 , 95% CI: 1.06-1.77, p-value: 0.018) and the control group (OR: 1.78, 95% CI: 1.11-2.86, p-value: 0.016)(Figure 3, Table 4).

Markers for atherosclerosis

Since above markers not only distinguished between stable and unstable disease, but were also different in expression as compared to controls, these markers could serve as markers for atherosclerosis as well. Nowadays, atherosclerotic disease is estimated from risk models, for which it is known that they are fairly unreliable. The only other way to detect the atherosclerotic process, is by coronary CT scanning, identifying coronary calcifications. Therefore, biomarkers detecting the presence atherosclerotic process are very welcome. The inventors showed, that when combining the AP and Ml groups, miRNA-223-3p (OR: 1.66, 95% CI: 1.26-2.18, p-value: < 0.001), miR-142-3p (OR: 1.38, 95% CI: 1.07-1.78, p-value: 0.014) and miR-125b-5p (OR: 1.92, 95% CI: 1.23-3.00, p-value:

0.004. showed a significant upregulation in atherosclerosis compared to controls (Figure 4, Table 5). MiR-193b-3p also showed a significant difference between groups. All these miRNA markers for atherosclerosis remained significant after correction for classical risk factors age, gender, hypercholesterolemia, hypertension, smoking, diabetes and BMI (Table 6), suggesting that these markers are predictive for atherosclerosis independent of these classical risk factors and may therefore add to risk prediction. Together, these markers yielded an AUC of 0.68.

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Claims

A method for determining the stability of an atherosclerotic plaque in an individual, comprising the steps of: a. determining the expression level of one or more miRNA(s) selected from the group consisting of miR-223-3p, miR-125b-5p, miR-142-3p and miR-193b-3p in a biological sample from said individual,

b. comparing said expression level with a reference level, and

c. determining the stability of said atherosclerotic plaque based on the information obtained in step b.

The method according to claim 1, wherein upregulation of miR-223-3p and/or miR-142-3p is indicative of the presence of an unstable atherosclerotic plaque.

The method according to claim 1 or 2, wherein upregulation of miR-125b-5p and/or miR-

193b-3p is indicative of the presence of a stable atherosclerotic plaque.

The method according to any one of claims 1-3, wherein the presence of unstable atherosclerotic plaque is based on the expression levels of at least miR-223-3p and miR-

125b-5p.

The method according to any one of claims 1-4, wherein an increased miR-223-3p/miR-125b- 5p ratio is indicative of the presence of an unstable atherosclerotic plaque.

A method for determining the risk of suffering from or developing of atherosclerosis in an individual, comprising the steps of: a. determining the expression level of one or more miRNA(s) selected from the group consisting of miR-223-3p, miR-125b-5p, miR-142-3p, miR-499a-5p and miR-193b-3p in a biological sample from said individual,

b. comparing said expression level with a reference level, and

c. determining said risk based on the information obtained in step b.

The method according to claim 6, wherein the expression level of at least miR-223-3p and miR-125b-5p is determined, and wherein an increased risk of suffering from or developing atherosclerosis is determined, based on the expression levels of miR-223-3p and miR-125b- 5p.

The method according to claim 6 or 7, wherein an increased risk of suffering from or developing atherosclerosis is determined, based on the upregulation of miR-223-3p, miR- 142-3p and miR-125b.

9. The method according to any one of claims 1-8, wherein said biological sample is serum, plasma or blood.

10. The method according to any one of claims 1-5, wherein said individual is suffering from atherosclerosis.

11. The method according to any one of claims 1-10, wherein said expression level is determined using qPCR.

12. A kit for use in the method according to any one of claims 1-11, comprising a first nucleic acid capable of hybridizing under stringent conditions with any of miR-125b-5p, miR-223-3p, miR- 142-3p, miR-499a-5p and miR-193b-3p and a second nucleic acid capable of hybridizing under stringent conditions with any of miR-1260, miR-1280, miR-484 and miR-718.

13. Kit according to claim comprising nucleic acids capable of hybridizing under stringent

conditions with any of miR-125b-5p and miR-223-3p.

14. Kit according to claim 13, further comprising one or more nucleic acid(s) capable of

hybridizing under stringent conditions with any of miR-1260, miR-1280, miR-484 and miR- 718.