CES-2 (CARBOXYLESTERASE-2) FOR THE ASSESSMENT OF AFIB RELATED STROKE

Abstract

The present invention relates to a method for assessing the risk of stroke in a subject, said method comprising the steps of determining the amount of CES-2 in a sample from the subject, and comparing the amount of CES-2 to a reference amount, whereby the risk of stroke is to be assessed. Moreover, the present invention relates to a method for assessing the efficacy of an anticoagulation therapy and a method for identifying a subject being eligible to the administration of at least one anticoagulation medicament or being eligible for increasing the dosage of at least one anticoagulation medicament.

Claims

1. A method for predicting the risk of stroke in a subject, comprising the steps of a) determining the amount of CES-2 and optionally one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 in a sample from the subject, and b) comparing the amount of CES-2 and optionally one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7, to a reference amount (or to reference amounts), whereby the risk of stroke is to be predicted.

2. The method of claim 1, wherein the amount of CES-2 in the sample from the subject is decreased as compared to the reference amount (or to reference amounts).

3. The method according to claim 1, wherein further comprising the step of recommending anticoagulation therapy or of recommending an intensification of anticoagulation therapy if the subject has been identified to be at risk to suffer from stroke.

4. The method according to claim 1, wherein the subject is human.

5. The method of according to claim 1, wherein the amounts of CES-2 and one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 are determined in step a), and wherein the method comprises the further steps of c) calculating a ratio of the amount of one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 as determined in step a) to the amount of CES-2 as determined in step a), and comparing said calculated ratio to a reference ratio.

6. A method for predicting the risk of stroke in a subject, comprising the steps of a) determining the amount of CES-2 and/or the amount of one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 in a sample from the subject having a known clinical stroke risk score, 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).

7. A method for improving the prediction accuracy of a clinical stroke risk score for a subject, comprising the steps of a) determining the amount of CES-2 and/or the amount of one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7, and b) combining a value for the amount of CES-2 and/or the amount of one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 with the clinical stroke risk score, whereby the prediction accuracy of said clinical stroke risk score is improved.

8. A method for assessing the efficacy of an anticoagulation therapy of a subject, comprising the steps of a) determining the amount of CES-2 and optionally one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 in a sample from the subject, and b) comparing the amount of CES-2 and optionally one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 to a reference amount (or to reference amounts), whereby the risk of stroke is to be assessed.

9. A method according to claim 8, wherein a decreased amount of CES-2 is significant that anticoagulation therapy is not efficient, and wherein a normal or an increased amount of CES-2 is significant that anticoagulation therapy is effective.

10. A method for identifying a subject being eligible to the administration of at least one Fifanticoagulation medicament or being eligible for increasing the dosage of at least one anticoagulation medicament, comprising a) determining the amount CES-2 and optionally one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 and b) comparing the amount as determined in step a) with a reference amount, whereby a subject being eligible to the administration of said at least one medicament or to an increased dosage of said at least one medicament is identified.

11. A method for monitoring a subject receiving an anticoagulation therapy, comprising the steps of a) determining the amount of CES-2 and optionally one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 in a sample from the subject, and b) comparing the amount of CES-2 and optionally of one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7 to a reference amount (or to reference amounts), whereby the risk of stroke is to be assessed.

12. (canceled)

13. (canceled)

14. (canceled)

15. A kit comprising an agent which specifically binds to CES-2 and an agent which specifically binds to one or more biomarkers selected from the group consisting of a natriuretic peptide, ESM-1, ANG-2, and IGFBP7.

16. The method of according to claim 1, wherein the sample is selected from the group consisting of blood, serum and plasma.

Description

[0255] The figures show:

[0256] FIG. 1: Measurement of CES-2 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). Atrial tissue RNA expression profiles were assessed.

[0257] FIG. 2: Prediction the risk of stroke CES-2 vs parameters of clinical risk scores (Beat AF study: The Figure shows, that reduced titers of CES-2 associate to increased risk of stroke. CES-2 improved the C-Index of several clinical risk scores.

[0258] FIG. 3: Correlation to NTproBNP and ESM-1 in Beat AF: FIG. 3 shows that CES-2 has almost no correlation with established markers (NTproBNP and ChadsVasc) as well as with ESM1.

[0259] FIG. 4: CES-2 values observed in the BEAT-AF study separated by intake of oral anticoagulation: Patients which use Rivaroxaban show higher concentrations of CES-2 compared to the remaining patients.

[0260] a) CES-2 vs NTproBNP correlation coefficient=−0.19 [0261] b) CES-2 vs ESM1 correlation coefficient=−0.18 [0262] c) CES-2 vs CHADsVASc correlation coefficient=−0.12

[0263] These data suggest, that CES-2 provides complementary information and combinations of CES-2 and/or NTproBNP and/or ESM1 and/or ANG-2 and/or IGFBP7 and/or CHADsVASc markers may provide improved detection of patients at high risk of stroke versus each marker alone. These data further suggest that CES-2 can be used to diagnose the disease, to classify the disease, to assess the disease severity, to guide therapy (with objectives to therapy intensification/reduction), to predict disease outcome (risk prediction, e.g. stroke), therapy monitoring (e.g., effect of anti-angionetic drugs on CES-2 levels), therapy stratification (selection of therapy options; e.g. long-term from Beat AF and selection)

EXAMPLES

[0264] 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: Differential Expression of CES-2 in Cardiac Tissue of AF Patients

[0265] Differential CES-2 expression levels have been determined in myocardial tissue samples from the right atrial appendage of n=40 patients.

[0266] RNAseq analyses

[0267] Atrial tissue was sampled during open chest surgery because of CABG or valve surgery. Evidence of AF or SR (controls) was generated during surgery with simultaneous Endo-Epicardial High Density Activation Mapping. Patients with AF and controls were matched with regard to gender, age and comorbidities.

[0268] Atrial tissue samples were prepared for [0269] AF patients; n=11 patients [0270] control patients in SR; n=39 patients.

[0271] Differential expression of CES-2 was determined in RNAseq analyses applying the algorithms RSEM and DESEQ2.

[0272] As shown in FIG. 2, CES-2 expression was found to be upregulated in the analyzed atrial tissues of the 11 patients with persistent AF versus the 29 control patients.

[0273] The fold change in expression (FC) was 1,439 The FDR (false discovery rate) was 0,00000000036.

[0274] The altered expression of CES-2 was determined in the damaged end organ, the atrial tissue. CES-2 mRNA levels were compared to results of high density mapping of the atrial tissue. Elevated CES-2 mRNA levels were detected in atrial tissue samples with conduction disturbances as characterized by electrical mapping. Conductance disturbances may be caused by fat infiltration or by interstitial fibrosis. The observed differential expression of CES-2 in atrial tissue of patients suffering from atrial fibrillation supports, that CES-2 is released in the circulation from the myocardium, in particular from the right atrial appendage and elevated serum/plasma titers assist the detection of episodes of AF.

[0275] It is concluded, that CES-2 is released from the heart into the blood and may aid the detection of AF episodes.

Example 2: Prediction of Stroke

[0276] Analysis Approach

[0277] The ability of circulating CES-2 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). CES-2 was measured using a stratified case cohort design as described in Borgan (2000).

[0278] 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).

[0279] CES-2 results were available for 69 patients with an event and 69 patients without an event.

[0280] CES-2 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).

[0281] In order to quantify the univariate prognostic value of CES-2 proportional hazard models were used with the outcome stroke.

[0282] The univariate prognostic performance of CES-2 was assessed by two different incorporations of the prognostic information given by CES-2.

[0283] The first proportional hazard model included CES-2 binarized at the median (1.4 NPX) and therefore comparing the risk of patients with CES-2 below or equal to the median versus patient with CES-2 above the median.

[0284] The second proportional hazard model included the original CES-2 levels but transformed to a log 2 scale. The log 2 transformation was performed in order to enable a better model calibration.

[0285] Because the estimates from a naïve 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).

[0286] In order to get estimates for the absolute survival rates in the two groups based on the dichotomized baseline CES-2 measurement (<=1.4 NPX vs >1.4 NPX) a weighted version of the Kaplan-Meier plot was created as described in Mark (2006).

[0287] In order to assess if the prognostic value of CES-2 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.

[0288] In order to assess the ability of CES-2 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 CES-2 (log 2 transformed). Extension was done by creating a portioned hazard model including CES-2 and the respective risk score as independent variables.

[0289] 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).

[0290] Results

[0291] Table 1 shows the results of the two univariate weighted proportional hazard models including the binarized or the log 2 transformed CES-2.

[0292] The association between the risk for experiencing a stroke with the baseline value of CES-2 is significant in both models.

[0293] The hazard ration for the binarized CES-2 implies a 0.4-fold lower risk for a stroke in the patient group with baseline CES-2>1.4 NPX versus the patient group with baseline CES-2<=1.4 NPX. The results of the proportional hazard model including CES-2 as log 2 transformed linear risk predictor suggest the log 2 transformed values CES-2 are negatively correlated to the risk for experiencing a stroke. The hazard ratio of 0.14 can be interpreted in a way that a 2-fold increase of CES-2 is associated with 0.14 decrease of risk for a stroke. In this context it is interesting to note that CES-2 level correlate with the intake of certain oral anticoagulants (OAKs). FIG. 4 shows that patients which use Rivaroxaban show higher concentrations of CES-2 compared to the remaining patients. But there are also some patients with intake of Rivaroxaban which have CES values below 1.4 NPX. This could indicate that CES-2 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 CES-2. Hazard Ratio (HR) 95%-CI HR P-Value CES-2 log2 0.138 0.0235-0.8055 0.028 Baseline CES-2 > 1.4 0.4116 0.1966-0.8618 0.019 NPX vs CES-2 <= 1.4 NPX

[0294] Table 2 shows the results of a proportional hazard model including CES-2 (log 2 transformed) in the combination with clinical and demographic variables.

[0295] The effect of CES-2 remains significant and the HR is now 0.09 for the lo2 transformed CES-2.

TABLE-US-00002 TABLE 2 Multivariate proportional hazard model including CES-2 and relevant clinical and demographic variables. Hazard Ratio (HR) 95%-CI HR P-Value History hypertension 1.7327  0.655-4.5835 0.2681 Age 1.0225 0.9791-1.0679 0.3145 History 1.8311 0.7158-4.6843 0.2069 Stroke/TIA/embolism Sex = male 0.5124 0.2218-1.1837 0.1175 History CHF 0.7825 0.3404-1.7984 0.5634 History vascular 1.1212 0.4705-2.6718 0.7962 disease CES-2 (log2 0.0947 0.0144-0.6237 0.0142 transformed)

[0296] Table 3 shows the results of the weighted proportional hazard model combining the CHADS.sub.2 score with CES-2 (log 2 transformed). Also in this model CES-2 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 CES-2 (log2 transformed) Hazard Ratio (HR) 95%-CI HR P-Value CHADS.sub.2 score 1.3892 1.0733-1.7980 0.0125 CES-2 (log2 0.1271 0.0203-0.7964 0.0276 transformed)

[0297] Table 4 shows the results of the weighted proportional hazard model combining the CHA.sub.2DS.sub.2-VASc score with CES-2 (log 2 transformed). Again CES-2 adds prognostic information.

TABLE-US-00004 TABLE 4 Weighted proportional hazard model combining the CHA.sub.2DS.sub.2-VASc score with CES-2 (log2 transformed) Hazard Ratio (HR) 95%-CI HR P-Value CHA.sub.2DS.sub.2-VASc 1.3862 1.1191-1.7172 0.0028 score CES-2 (log2 0.1113 0.0180-0.6874 0.0181 transformed)

[0298] Table 5 shows the results of the weighted proportional hazard model combining the ABC score with CES-2 (log 2 transformed). The prognostic additional value of CES-2 decreases slightly but stays significant.

TABLE-US-00005 TABLE 5 Weighted proportional hazard model combining the ABC score with CES-2 (log2 transformed) Hazard Ratio (HR) 95%-CI HR P-Value ABC score 1.1289 1.0171-1.2530 0.0227 CES-2 (log2 0.1804 0.0338-0.9613 0.0448 transformed)

[0299] Table 6 shows the estimated c-indexes of CES-2 alone, of the CHADS.sub.2, the CHA.sub.2DS.sub.2-VASc and the ABC score and of the weighted proportional hazard model combining the CHADS.sub.2, the CHA.sub.2DS.sub.2-VASc and the ABC score with CES-2 (log 2).

[0300] The addition of CES-2 to CHA.sub.2DS.sub.2-VASc score improves the c-index by 0.0611 which can be considered as a clinical meaningful improvement of the risk prediction.

[0301] For the CHADS.sub.2 score the c-index improvement is comparable with 0.0646 as for the ABC score with 0.0617.

TABLE-US-00006 TABLE 6 C-indexes of CES-2, the CHADS.sub.2, CHA.sub.2DS.sub.2-VASc and ABC score and their combination with CES-2. C-Index CES-2 univariate 0.7080 CHADS.sub.2 0.6505 CHADS.sub.2 + CES-2 0.7151 CHA.sub.2DS.sub.2-VASc 0.6740 CHA.sub.2DS.sub.2-VASc + CES-2 0.7350 ABC score 0.6484 ABC score + CES-2 0.7101

Example 3: Biomarker Measurements

[0302] CES-2 was measured in a commercially available O-link multi-marker panel for (Carboxy-lesterase-2 (CES-2); Proximity Extension Assay from O-link, Sweden.

[0303] Case Studies

[0304] The CHA2DS2-VASc score predicts incidence of stroke in patients with and also without atrial fibrillation (https://www.ncbi.nlm nih.gov/pubmed/29754652); however, it is less clear, if and at what CHA2DS2-VASc score the patients without atrial fibrillation should receive oral anticoagulation (OAC) and at which dose, so that biomarkers such as CES-2 help to assess the need for therapy and effectiveness of OAC.

[0305] A 70-year-old male patient with hypertension and no history of atrial fibrillation presents in sinus rhythm. CES2 is determined in an EDTA plasma sample obtained from the patient. The CES2 value is below a reference value. The reduced CES2 titers in combination of other stroke risk parameters (advanced age and hypertension) are indicative of high risk to experience a stroke. As consequence the patient is admitted to an anticoagulation therapy.

[0306] 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. The patient already receives direct oral anticoagulation therapy (at low starting dose) because of a history of stroke and high overall CHA2DS2-VASc score. In order to determine the current risk of stroke, CES2 is measured in a serum sample obtained from the patient. The observed CES2 value is below a reference value. The reduced CES2 titers in combination of other risk parameters (history of stroke) are indicative of a high risk of stroke. As consequence the dosage of the anticoagulation therapy is increased.

[0307] 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, he patient has 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 AFib. The CES-2 level was determined before and after onset of anticoagulation. The patient now is wondering whether the anticoagulation therapy is effective and still necessary. In order to specify the acute risk of stroke CES2 is determined in a EDTA sample obtained from the patient. The observed CES2 value is above a reference value. The increased CES2 titers are indicative of an effective anticoagulation therapy. As consequence the anticoagulation therapy is maintained. [0308] Related, very recent research question “Does CHA2DS2VASc score predict incidence of stroke in patients without A-Fib/Flutter?” or “how much risk points does AFib adds to the CHA.sub.2DS.sub.2-VASc (eg, a 7-fold risk, but how many points]” and first results from 2014-2019: [0309] “The event rates were 0.67%/y for ischemic stroke or MI, 0.96%/y for AF, and 0.52%/y for major bleeding “https://www.ncbi.nlm.nih.gov/pubmed/29754652 (related also: Circulation. 2017; 136:A20985) [0310] “In patients with ACS but no AF, the CHADS2 and CHA2DS2-VASc scores predict ischaemic stroke/TIA events with similar accuracy to that observed in historical populations with non-valvular AF, but with lower absolute event rates.” https://www.ncbi.nlm nih.gov/pubmed/24860007 [0311] “The CHA2DS2-VASc tool predicts thromboembolic events and overall mortality in patients without atrial fibrillation who have implantable devices” https://www.ncbi.nlm nih.gov/pubmed/28259228 [0312] “The absolute risk of thromboembolic complications was higher among patients without AF compared with patients with concomitant AF at high CHA2DS2-VASc scores.” https://www.ncbi.nlm nih.gov/pubmed/26318604.