CIRCULATING SPON-1 (SPONDIN-1) IN THE ASSESSMENT OF ATRIAL FIBRILLATION

Abstract

The present invention relates to a method for assessing atrial fibrillation in a subject, said method comprising the steps of determining the amount of SPON-1 in a sample from the subject, and comparing the amount of SPON-1 to a reference amount, whereby atrial fibrillation is to be assessed. Moreover, the present invention relates to methods for the prediction of stroke based on the amount of SPON-1.

Claims

1. A method for assessing atrial fibrillation in a subject, comprising the steps of a) determining, in at least one sample from the subject, the amount of the biomarker SPON-1 (Spondin-1) and, optionally, the amount of at least one further biomarker selected from the group consisting of a natriuretic peptide, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and IGFBP7 (Insulin-like growth factor-binding protein 7), and b) comparing the amount of the biomarker SPON-1 to a reference amount for SPON-1 and, optionally, comparing the amount of the at least one further biomarker to a reference amount for said at least one further biomarker, whereby atrial fibrillation is to be assessed.

2. The method of claim 1, wherein the sample is a blood, serum or plasma sample, and/or wherein the subject is human.

3. The method according to claim 1 or 2, wherein the assessment of atrial fibrillation is the diagnosis of atrial fibrillation, in particular wherein an amount of SPON-1 and, optionally, an amount of the at least one further biomarker above the reference amount is indicative for a subject suffering from atrial fibrillation and/or wherein an amount of SPON-1 and, optionally, an amount of the at least one further biomarker below or equal to the reference amount is indicative for a subject not suffering from atrial fibrillation.

4. The method according to claim 1 or 2, wherein the subject is suffering from atrial fibrillation, and wherein the assessment of atrial fibrillation is the differentiation between paroxysmal and persistent atrial fibrillation, in particular wherein an amount of SPON-1 and, optionally, an amount of the at least one further biomarker above the reference amount is indicative for a subject suffering from persistent atrial fibrillation and/or wherein an amount of SPON-1 and, optionally, an amount of the at least one further biomarker below or equal to the reference amount is indicative for a subject suffering from paroxysmal atrial fibrillation.

5. The method according to claim 1 or 2, wherein the assessment of atrial fibrillation is the prediction of the risk of an adverse event associated with atrial fibrillation.

6. The method of claim 5, wherein the adverse event associated with atrial fibrillation is recurrence of atrial fibrillation and/or stroke.

7. The method of claims 5 and 6, wherein an amount of SPON-1 and, optionally, an amount of the at least one further biomarker above the reference amount is indicative for a subject who is at risk of suffering from an adverse event associated with atrial fibrillation and/or wherein an amount of SPON-1 and, optionally, an amount of the at least one further biomarker below or equal to the reference amount is indicative for a subject who is not at risk of suffering from an adverse event associated with atrial fibrillation.

8. The method according to claim 1 or 2, wherein the assessment of atrial fibrillation is the identification of a subject who shall be subjected to electrocardiography (ECG), or wherein the assessment of atrial fibrillation is the assessment of a therapy for atrial fibrillation such as the assessment of anticoagulation therapy.

9. A method for predicting the risk of stroke in a subject, comprising the steps of (a) determining, in at least one sample from the subject, the amount of the biomarker SPON-1 (Spondin-1) and, optionally, the amount of at least one further biomarker selected from the group consisting of a natriuretic peptide, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and IGFBP7 (Insulin-like growth factor-binding protein 7), and (b) assessing the clinical stroke risk score for said subject, and (c) predicting the risk of stroke based on the results of steps a) and b).

10. A method for improving the prediction accuracy of a clinical stroke risk score for a subject, comprising the steps of a) determining, in at least one sample from the subject, the amount of the biomarker SPON-1 (Spondin-1) and, optionally, the amount of at least one further biomarker selected from the group consisting of a natriuretic peptide, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and IGFBP7 (Insulin-like growth factor-binding protein 7), wherein the subject has a known clinical stroke risk score, and b) combining a value for the amount of SPON-1 and/or the amount of one or more biomarkers comprising of a natriuretic peptide, ESM-1, ANGT2, IGFBP7 with the clinical stroke risk score, whereby the prediction accuracy of said clinical stroke risk score is improved.

11. A method of aiding in the assessment of atrial fibrillation, said method comprising the steps of: a) providing at least one sample from a subject, b) determining, in the at least one sample provided in step a), the amount of the biomarker SPON-1 (Spondin-1) and, optionally, the amount of at least one further biomarker selected from the group consisting of a natriuretic peptide, ESM-1 (Endocan), Ang2 and IGFBP7 (Insulin-like growth factor-binding protein 7), and c) providing information on the determined amount of the biomarker SPON-1 and optionally on the determined amount of the at least one further biomarker to a physician, thereby aiding in the assessment of atrial fibrillation.

12. A method for aiding in the assessment of atrial fibrillation, comprising: a) providing an assay for the biomarker SPON-1 and, optionally, at least one further assay for a further biomarker selected from the group consisting of a natriuretic peptide, ESM-1 (Endocan), Ang2 and IGFBP7 (Insulin-like growth factor-binding protein 7), and b) providing instructions for use of assay results obtained or obtainable by said assay(s) in the assessment of atrial fibrillation.

13. A computer-implemented method for assessing atrial fibrillation, comprising a) receiving, at a processing unit, a value for the amount of SPON-1, and, optionally at least one further value for the amount of at least one further biomarker selected from the group consisting of a natriuretic peptide, ESM-1 (Endocan), Ang2 and IGFBP7 (Insulin-like growth factor-binding protein 7), wherein said amount of SPON-1 and, optionally, the amount of the at least one further biomarker has been determined in a sample from a subject, b) comparing, by said processing unit, the value or values received in step (a) to a reference or to references, and c) assessing atrial fibrillation based in the comparison step b).

14. A kit comprising an agent which specifically binds to SPON-1 and at least one further agent selected from the group consisting of an agent which specifically binds to a natriuretic peptide, an agent which specifically binds to ESM-1, an agent which specifically binds Ang2 and an agent which specifically binds to IGFBP7.

15. In vitro use of i) the biomarker SPON-1 and optionally of at least one further biomarker selected from the group consisting of a natriuretic peptide, ESM-1 (Endocan), Ang2 and IGFBP7 (Insulin-like growth factor-binding protein 7), and/or ii) at least one agent that specifically binds to SPON-1, and, optionally, at least one further agent selected from the group consisting of an agent which specifically binds to a natriuretic peptide, an agent which specifically binds to ESM-1, an agent which specifically binds to Ang2 and an agent which specifically binds to IGFBP7, for a) assessing atrial fibrillation, b) predicting the risk of stroke in a subject, and for c) improving the prediction accuracy of a clinical stroke risk score

Description

[0304] The figures show:

[0305] FIGS. 1A&1B: Measurement of SPON-1 in Mapping study: Exploratory AFib panel: Patients with a history of atrial fibrillation undergoing open chest surgery and epicardial mapping of paroxysmal AF, persistent AF or SR (Mapping study). Circulating SPON-1 levels were assessed (Top (FIG. 1A): Boxplot, Bottom (FIG. 1B): ROC-curve).

[0306] FIG. 2: Measurement of SPON-1 in Beat AF study: AFib panel with stroke outcome: Patients with different types of atrial fibrillation paroxysmal AF, persistent AF and permanent AF. Circulating SPON-1 levels were assessed. The boxplot shows the distribution of SPON1 by AF type in the BEAT-AF study.

[0307] FIG. 3: Prediction the risk of stroke SPON-1 (Beat AF study): Kaplan-Meier curve showing the stroke-free survival in the two groups defined by having a SPON-1 value<=1.4 vs 1.4 NPX. Elevated titers of SPON-1 associate to increased risk of stroke. SPON-1 improved the C-Index of several clinical risk scores.

[0308] FIG. 4: SPON-1 values observed in the BEAT-AF study separated by intake of oral anticoagulation: Patients which use Rivaroxaban show lower concentrations of SPON-1 compared to the remaining patients.

EXAMPLES

[0309] The invention will be merely illustrated by the following Examples. The said Examples shall, whatsoever, not be construed in a manner limiting the scope of the invention.

Example 1: Assessment of AF with Circulating SPON-1

[0310] The MAPPING study related to patients undergoing open chest surgery. Samples were obtained before anesthesia and surgery. Patients were electrophysiologically characterized using high-density epicardial mapping with multi-electrode arrays (high density mapping).

[0311] Circulating SPON-1 levels have been determined in 16 patients with persistent atrial fibrillation and 30 controls, matched to best possible (on age, gender, comorbidities). SPON-1 was determined in samples of the MAPPING study.

[0312] Measurements were performed in 30 patients with sinus rhythm (SR) and in 16 persistent artrial fibrillation (persAF).

[0313] FIG. 1 shows that SPON-1 is significantly elevated in patients with persAF in comparison to patients in SR (AUC 0.87). The boxplot in FIG. 1 shows the SPON-1 distribution in patients with SR vs patient with persAF. The ROC curve shows the diagnostic ability of SPON-1 to discriminate between patients with SR vs patient with persAF. Accordingly, SPON-1 could be used for aid in diagnosis of persAF. Elevated SPON-1 values would indicate a higher probability of persAF.

[0314] In addition, in the BEAT-AF study (described in example 2) SPON-1 concentrations correlated with severity of artrial fibrillation. FIG. 2 shows the distribution of SPON-1 by AF type in the BEAT-AF study.

Example 2: Prediction of Stroke

Analysis Approach

[0315] The ability of circulating SPON-1 to predict the risk for the occurrence of stroke was assessed in a prospective, multicentric registry of patients with documented atrial fibrillation (Conen D., Forum Med Suisse 2012; 12:860-862). SPON-1 was measured using a stratified case cohort design as described in Borgan (2000).

[0316] For each of the 70 patients which experienced a stroke during follow up (events), 1 matched control was selected. Controls were matched based on the demographic and clinical information of age, sex, history of hypertension, atrial fibrillation type and history of heart failure (CHF history).

[0317] SPON-1 results were available for 67 patients with an event and 66 patients without an event.

[0318] SPON-1 was measured using the Olink platform therefor no absolute concentration values are available and can be reported. Results will be reported on an arbitrary signal scale (NPX).

[0319] In order to quantify the univariate prognostic value of SPON-1 proportional hazard models were used with the outcome stroke.

[0320] The univariate prognostic performance of SPON-1 was assessed by two different incorporations of the prognostic information given by SPON-1.

[0321] The first proportional hazard model included SPON-1 binarized at the median (1.4 NPX) and therefore comparing the risk of patients with SPON-1 below or equal to the median versus patient with SPON-1 above the median.

[0322] The second proportional hazard model included the original SPON-1 levels but transformed to a log 2 scale. The log 2 transformation was performed in order to enable a better model calibration.

[0323] Because the estimates from a nave proportional hazard model on the case control cohort would be biased (due to the altered proportion of cases to controls) a weighted proportional hazard model was used. Weights are based on the inverse probability for each patient to be selected for the case control cohort as described in Mark (2006).

[0324] In order to get estimates for the absolute survival rates in the two groups based on the dichotomized baseline SPON-1 measurement (<=1.4 NPX vs >1.4 NPX) a weighted version of the Kaplan-Meier plot was created as described in Mark (2006).

[0325] In order to assess if the prognostic value of SPON-1 is independent from known clinical and demographic risk factors a weighted proportional cox model including in addition the variables age, sex, CHF history, history of hypertension, Stroke/TIA/Thromboembolism history, vascular disease history and diabetes history was calculated.

[0326] In order to assess the ability of SPON-1 to improve existing risk scores for the prognosis of stroke the CHADS.sub.2 the CHA.sub.2DS.sub.2-VASc and the ABC score were extended by SPON-1 (log 2 transformed). Extension was done by creating a portioned hazard model including SPON-1 and the respective risk score as independent variables.

[0327] The c-indices of the CHADS.sub.2, the CHA.sub.2DS.sub.2-VASc and ABC score were compared to the c-indices of these extended models. For the calculation of the c-index in the case-cohort setting a weighted version of the c-index was used as proposed in Ganna (2011).

Results

[0328] Table 1 shows the results of the two univariate weighted proportional hazard models including the binarized or the log 2 transformed SPON-1.

[0329] The association between the risk for experiencing a stroke with the baseline value of SPON1 is highly significant in both models.

[0330] The hazard ratio for the binarized SPON-1 implies a 1.51-fold higher risk for a stroke in the patient group with baseline SPON-1>=1.4 NPX versus the patient group with baseline SPON-1<1.4 NPX. This is also visible in FIG. 3 showing the Kaplan-Meier curve which depicts the probability over time to survive until the occurrence of a stroke event.

[0331] However, the p-value is above 0.05 which might indicate that binarization is sub-optimal in this case.

[0332] The results of the proportional hazard model including SPON-1 as log 2 transformed linear risk predictor suggest the log 2 transformed values SPON-1 are proportional to the risk for experiencing a stroke. The hazard ratio of 5.22 can be interpreted in a way that a 2-fold increase of SPON-1 is associated with 5.22 increase of risk for a stroke.

[0333] In this context it is interesting to note that SPON-1 levels correlate with the intake of certain oral anticoagulants (OAKs). FIG. 4 shows that patients which use Rivaroxaban show lower concentrations of SPON-1 compared to the remaining patients. But there are also some patients with intake of Rivaroxaban which have SPON-1 values above 1.4 NPX. This could indicate that SPON-1 could be used to monitor the effectiveness of OAK intake.

TABLE-US-00001 TABLE 1 Results result of the univariate weighted proportional hazard model including the binarized and log2 transformed SPON-1. Hazard Ratio (HR) 95%-CI HR P-Value SPON-1 log2 5.2177 2.158-12.6155 0.0002 Baseline SPON-1 >= 1.5056 0.7505-3.0204 0.2494 1.4 NPX vs SPON-1 < 1.4 NPX

[0334] Table 2 shows the results of a proportional hazard model including SPON-1 (log 2 transformed) in the combination with clinical and demographic variables. It clearly shows that the prognostic effect of SPON-1 stays stable if adjusting for the prognostic effect of relevant clinical and demographic variables.

TABLE-US-00002 TABLE 2 Multivariate proportional hazard model including SPON-1 and relevant clinical and demographic variables. Hazard Ratio (HR) 95%-CI HR P-Value History hypertension 1.2951 0.5944-2.8216 0.5152 Age 1.0159 0.9689-1.0652 0.5134 History 2.3117 0.9216-5.799 0.0741 Stroke/TIA/embolism Sex = male 0.7884 0.3859-1.6107 0.5143 History CHF 0.685 0.2915-1.6097 0.3855 History vascular 1.397 0.5627-3.4685 0.4712 disease SPON-1 (log2 5.3515 1.6403-17.4592 0.0054 transformed)

[0335] Table 3 shows the results of the weighted proportional hazard model combining the CHADS.sub.2 score with SPON-1 (log 2 transformed). Also in this model SPON-1 can add prognostic information to the CHADS.sub.2 score.

TABLE-US-00003 TABLE 3 Weighted proportional hazard model combining the CHADS.sub.2 score with SPON-1 (log2 transformed) Hazard Ratio (HR) 95%-CI HR P-Value CHADS.sub.2 score 1.3851 1.0653-1.8009 0.015 SPON-1 (log2 4.3128 1.5661-11.8766 0.0047 transformed)

[0336] Table 4 shows the results of the weighted proportional hazard model combining the CHA.sub.2DS.sub.2-VASc score with SPON-1 (log 2 transformed). Also in this model SPON-1 can add prognostic information to the CHA.sub.2DS.sub.2-VASc score.

TABLE-US-00004 TABLE 4 Weighted proportional hazard model combining the CHA.sub.2DS.sub.2-VASc score with SPON-1 (log2 transformed) Hazard Ratio (HR) 95%-CI P-Value CHA.sub.2DS.sub.2-VASc 1.3497 1.0518-1.7319 0.0184 score SPON-1 (log2 3.5021 1.1755-10.4334 0.0244 transformed)

[0337] Table 5 shows the results of the weighted proportional hazard model combining the ABC score with SPON-1 (log 2 transformed). Also in this model SPON-1 can add prognostic information to the risk score.

TABLE-US-00005 TABLE 5 Weighted proportional hazard model combining the ABC score with SPON-1 (log2 transformed) Hazard Ratio (HR) 95%-CI P-Value ABC score 1.122 0.9989-1.2603 0.0521 SPON-1 (log2 3.2123 1.0574-9.7585 0.0395 transformed)

[0338] Table 6 shows the estimated C-indexes of SPON-1 (log 2) alone, the CHA.sub.2DS.sub.2-VASc score and the combination of the CHA.sub.2DS.sub.2-VASc score with SPON-1 and C-indexes of the CHADS.sub.2 and ABC score and their combination with SPON-1.

[0339] It can be seen that the addition of SPON-1 improves the c-index of all three risk models. The improvements are 0.0158, 0.0164 and 0.0410 for the CHADS.sub.2, the CHA.sub.2DS.sub.2-VASc, the ABC score respectively.

[0340] Table 6 shows the estimated c-indexes of NTproBNP alone, of ESM-1 alone, of Ang-2 alone, of IGFBP-7 alone, of the CHA.sub.2DS.sub.2-VASc score and of the weighted proportional hazard model combining the CHA.sub.2DS.sub.2-VASc score with NTproBNP (log 2), with ESM-1 (log 2), with ANG-2 (log 2), with IGFBP-7 (log 2) on the case cohort selection. It can be seen that the addition of all biomarkers improve the c-index of the CHA.sub.2DS.sub.2-VASc score. The improvements of the the CHA.sub.2DS.sub.2-VASc score are 0.002, 0.064, 0.036 and 0.006 for NTproBNP, ESM-1, Ang-2, IGFBP-7.

[0341] In this context it is interesting, that SPON-1 has only low correlation with established markers (NTproBNP and ChadsVasc) as well as with ESM-1: a) SPON-1 vs NTproBNP correlation coefficient=0.44, b) SPON-1 vs ESM1 correlation coefficient=0.37 c) SPON-1 vs CHADsVASc. correlation coefficient=0.25. These data suggest, that SPON-1 provides complementary information and combinations of SPON-1 and/or NTproBNP and/or ESM1 and/or CHADsVASc markers may provide improved detection of patients at high risk of stroke vs each marker alone.

TABLE-US-00006 TABLE 6 C-indexes of SPON-1, the CHADS.sub.2 and CHA.sub.2DS.sub.2-VASc score and their combination with SPON-1. C-Index SPON-1 univariate 0.634 CHADS.sub.2 0.650 CHADS.sub.2 + SPON-1 0.666 CHA.sub.2DS.sub.2-VASc 0.674 CHA.sub.2DS.sub.2-VASc + SPON-1 0.690 ABC score 0.648 ABC score + SPON-1 0.689 NTproBNP univariate 0.651 CHA.sub.2DS.sub.2-VASc + NTproBNP 0.676 ESM-1 univariate 0.708 CHA.sub.2DS.sub.2-VASc + ESM-1 0.738 Ang-2 univariate 0.696 CHA.sub.2DS.sub.2-VASc + Ang-2 0.710 IGFBP-7 univariate 0.652 CHA.sub.2DS.sub.2-VASc + IGFBP-7 0.680

Case Studies

[0342] There is a growing interest in knowing and reducing the ischemic stroke risk also in patients without atrial fibrillation (Yao X et al, Am Heart J. 2018 May; 199:137-143). Identifying these patients at high stroke risk is essential to include them in drug studies (oral anticoagulation) and to establish optimum treatment strategies. For example, there is insufficient clinical guidance at what CHA2DS2-VASc level these patients at high stroke risk and without atrial fibrillation should receive oral anticoagulationand at which dose (Yao X et al, Am Heart J. 2018 May; 199:137-143). The CHA2DS2-VASc score, for example, predicts the incidence of ischemic strokes also in patients without atrial fibrillation, but with a lower absolute event rate (Mitchell L B et al, Heart. 2014; 100:1524-30), so that additional stroke risk markers, such as SPON-1, help to assess the stroke risk and provide guidance for oral anticoagulation.

[0343] A 70-year old male patient with hypertension and no history of atrial fibrillation presents in sinus rhythm. SPON-1 is determined in an EDTA plasma sample obtained from the patient. The clinical information (advanced age and hypertension) indicate a certain stroke risk, but also the SPON-1 value is measured above a reference value (being indicative of high stroke risk). As consequence the patient is admitted to an anticoagulation therapy.

[0344] A 75-year old female patient without a history of atrial fibrillation requests a checkup at the doctor's office. The patient presents in sinus rhythm, however structural heart disease is diagnosed. Because of a history of stroke and high overall CHA2DS2-VASc score, the patient receives direct oral anticoagulation (low dose). In order to determine the residual stroke risk at the second visit and to conclude on eventual change of drug dose, SPON-1 is measured in a serum sample obtained from the patient. The observed SPON-1 value is above a reference value. The elevated SPON-1 titers in combination of other risk parameters (history of stroke) are indicative of a high residual stroke risk that is higher than the bleeding risk (assessed with other clinical information). As a consequence the dosage of the anticoagulation therapy is increased.

[0345] A 68-year old obese female patient with Diabetes Mellitus and heart failure with reduced ejection fraction presents with acute symptoms of shortness of breath. In prior visits, the patient had no history of atrial fibrillation. According to a high overall CHA2DS2-VASC risk score, the physician decided to start oral anticoagulation (low dose) even in the absence of atrial fibrillation. The SPON-1 level is determined before and after onset of anticoagulation. The patient is wondering whether the anticoagulation therapy is effective and still necessary. In order to specify the current stroke risk SPON-1 is determined in an EDTA sample obtained from the patient. The observed SPON-1 value is below a reference value. The reduced SPON-1 titers are indicative of an effective anticoagulation therapy and the anticoagulation therapy is maintained.