RET (REARRANGED DURING TRANSFECTION) FOR THE ASSESSMENT OF STROKE

Abstract

The present invention relates to a method for aiding in the prediction of stroke and/or dementia in a subject, said method comprising a) determining the amount of the biomarker RET (Rearranged during transfection) in a sample from the subject, b) comparing the amount determined in step a) to a reference, and c) aiding in the prediction of stroke and/or dementia. The present invention further relates to a method for aiding in the assessment of the extent of white matter lesions in a subject, a method for aiding in the assessment whether a subject has experienced one or more silent strokes and to a method for aiding in the diagnosis of atrial fibrillation in a subject. Further encompassed by the present invention are the corresponding uses.

Claims

1. A method for predicting the risk of stroke and/or dementia in a subject, said method comprising a) determining the amount of the biomarker RET (Rearranged during transfection) in a sample from the subject, b) comparing the amount determined in step a) to a reference, and c) predicting the risk of stroke and/or dementia.

2. The method of claim 1, wherein the subject suffers from atrial fibrillation.

3. The method of claim 1, wherein the risk of stroke is predicted and wherein the stroke is ischemic stroke.

4. The method of claim 1, wherein the risk of dementia is predicted, and wherein dementia is vascular dementia, Alzheimer's disease, dementia with Lewy bodies and/or frontotemporal dementia.

5. The method of claim 1, wherein an amount of RET lower than the reference is indicative for a subject who is at risk of stroke and/or dementia, and/or wherein an amount of RET larger than the reference is indicative for a subject who is not at risk of stroke and/or dementia.

6. The method of claim 1, wherein the risk of the subject to suffer from stroke and/or dementia in a subject within 1 to 10 years is predicted.

7. A computer-implemented method for predicting stroke and/or dementia in a subject, said method comprising a) receiving at a processing unit a value for the amount of the biomarker RET (Rearranged during transfection) in a sample from the subject, b) processing the value received in step (a) with the processing unit, wherein said processing comprises retrieving from a memory one or more threshold values for the amount of the biomarker RET and comparing the value received in step (a) with the one or more threshold values, and c) providing a prediction of stroke and/or dementia via an output device, wherein said prediction is based on the results of step (b).

8. 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 the biomarker RET (Rearranged during transfection) in a sample from the subject, and b) combining a value for the amount of the biomarker RET with the clinical stroke risk score, whereby the prediction accuracy of said clinical stroke risk score is improved.

9. A method for assessing the extent of white matter lesions in a subject, said method comprising a) determining the amount of the biomarker RET (Rearranged during transfection) in a sample from the subject, and b) assessing the extent of white matter lesions in a subject based on the amount determined in step a).

10. A method for monitoring the extent of white matter lesions and/or the cognitive function in a subject, comprising a) determining the amount of the biomarker RET (Rearranged during transfection) in a first sample from the subject, b) determining the amount of the biomarker RET (Rearranged during transfection) in a second sample from the subject which has been obtained after the first sample, c) comparing the amount of the biomarker RET in the first sample to the amount of the biomarker RET in the second sample, and d) monitoring the extent of white matter lesions and/or the cognitive function of the subject based on the results of step c).

11. A method for assessing whether a subject has experienced one or more silent strokes, said method comprising a) determining the amount of the biomarker RET (Rearranged during transfection) in a sample from the subject, b) comparing the amount determined in step a) to a reference, and c) assessing whether a subject has experienced one or more silent strokes.

12. A method for diagnosing atrial fibrillation in a subject, said method comprising a) determining the amount of the biomarker RET (Rearranged during transfection) in a sample from the subject, b) comparing the amount determined in step a) to a reference, and c) diagnosing atrial fibrillation.

13. The method of claim 1, wherein the sample is a blood, serum or plasma sample, or wherein the sample is a heart or neural tissue sample.

14. (canceled)

15. The method of claim 1, wherein i. the biomarker RET is the RET polypeptide, ii. the subject is human, iii. the subject is 65 years or older, and/or iv. the subject has no known history of stroke and/or TIA (transient ischemic attack).

Description

[0290] In the Figures:

[0291] FIG. 1: Measurement of RET in Mapping study; Exploratory AFib panel; Patients with a history of atrial fibrillation undergoing open chest surgery and epicardial mapping of persistent AF (perAF) or Sinus Rhythm (SR), Mapping study). Atrial tissue RNA expression profiles was assessed.

[0292] FIG. 2: Measurement of RET 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 RET levels were assessed.

[0293] FIG. 3: Measurement of RET in SWISS AF study with Fazekas Score <2 (no) vs Fazekas Score≥2 (yes): Detection of WMLs/Prediction of the risk of silent stroke: Circulating RET levels were assessed.

EXAMPLES

Example 1: Differential Expression of RET in Cardiac Tissue of AF Patients

[0294] Differential RET expression levels have been determined in tissue samples from the right atrial appendage of n=40 patients. The right atrial appendage is associated to the heart right atrium.

[0295] RNAseq Analyses

[0296] 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 atrial fibrillation and controls were matched with regard to gender, age and comorbidities.

[0297] Atrial tissue samples were prepared for [0298] AF patients, n=11 patients [0299] control patients in SR; n=28 patients

[0300] Differential expression of RET (alias CDHF12, CDHR16, HSCR1, MEN2A, MEN2B, MTCA, PTC, RET-ELE1, RETS1) was determined in RNAseq analyses applying the algorithms RSEM and DESEQ2.

[0301] As shown in FIG. 1, RET expression was found to be downregulated in the analyzed atrial tissues of the 11 patients with persistent AF versus the 28 control patients in sinus rhythm. The fold change in expression (FC) was −1.24.

[0302] The altered expression of RET was determined in the damaged end organ, the right atrial appendage, part of the atrial tissue of the heart right atrium. RET mRNA levels were compared for patients with an ongoing atrial fibrillation event in course of surgery with control samples in sinus rhythm. The status of atrial fibrillation was the result of high density mapping of the atrial tissue Reduced RET 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 RET in atrial tissue of patients suffering from atrial fibrillation supports, that RET is released in the circulation from the myocardium, in particular from the heart atrium, in setail the right atrial appendage and reduced serum/plasma titers assist the detection of episodes of AF.

[0303] It is concluded, that RET is released from the heart into the blood and may aid the detection of AF episodes and predict the risk of developing AF related stroke.

Example 2: Assessment of AF with Circulating RET

[0304] The MAPPING study related to patients undergoing open chest surgery. EDTA plasma samples were obtained before anesthesia and surgery. Patients were electrophysiologically characterized using high-density epicardial mapping with multi-electrode arrays (high density mapping).

[0305] Circulating RET protein levels have been determined in 14 patients with paroxysmal atrial fibrillation, 16 patients with persistent atrial fibrillation and 30 controls, matched to best possible (on age, gender, comorbidities). RET was determined in samples of the MAPPING study.

[0306] Measurements were performed in 30 patients with sinus rhythm (SR), in 12 patients with paroxysmal arterial fibrillation (parAF) and in 16 persistent arterial fibrillation (persAF).

[0307] FIG. 2 shows that RET titers are reduced in patients with paroxysmal AF and in patients with persAF in comparison to patients in SR (AUC 0.59 and AUC 0.62 respectively). Therefore, RET could be used for aid in diagnosis of patients with different types of AF. Reduced RET values would indicate a higher probability of AF.

Example 3: Prediction of Clinical Stroke with Circulating RET

[0308] RET in the assessment of clinical stroke provides a method to

[0309] 1. Predicting the risk of stroke in patients with atrial fibrillation based on circulating RET levels in serum/plasma (BeatAF study, SWISSAF study, Table 1)

[0310] 2. Improving the prediction of clinical accuracy of clinical stroke risk scores based on circulating RET levels in serum/plasma (e.g. CHA.sub.2DS.sub.2-VASc, CHADS.sub.2, ABC score)

[0311] The ability of circulating RET to predict the risk for the occurrence of stroke was assessed in two prospective, multicentric registry studies of patients with documented atrial fibrillation with similar inclusion criteria, the Beat AF and the SWISS AF study (Conen D., Forum Med Suisse 2012; 12:860-862; Conen et al., Swiss Med Wkly. 2017; 147). Patients of the SWISS AF cohort have a median age of 74 years, a rate of prior clinical strokes or TIA of 20%, a rate of vascular diseases of 34% and a history of diabetes of 17%. Patients of the Beat AF cohort have a median age of 70 years, a rate of prior clinical strokes or TIA of 16%, a rate of vascular diseases of 24% and a history of diabetes of 14%.

[0312] RET was measured using a stratified case cohort design. For each of the patients, which experienced a stroke during a follow up period of 5 years (70 clinical stroke patients in the Beat AF) 3 years (66 clinical stroke patients in the SWISS AF) (“events”), 1 control per event was selected.

[0313] RET were measured using the Olink platform. Therefor no absolute concentration values are available and can be reported. Results are reported on an arbitrary signal scale (NPX).

[0314] In order to quantify the univariate prognostic value of RET proportional hazard models were used with the outcome stroke.

[0315] The univariate prognostic performance of RET was assessed by two different incorporations of the prognostic information given by RET.

[0316] The first proportional hazard model included RET binarized at the median and therefore comparing the risk of patients with RET below or equal to the median versus patient with RET above the median.

[0317] The second proportional hazard model included the original RET levels but transformed to a log 2 scale. The log 2 transformation was performed in order to enable a better model calibration.

[0318] 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. In order to get estimates for the absolute survival rates in the two groups based on the dichotomized baseline RET measurement (<=median vs >median) a weighted version of the Kaplan-Meier plot was created.

[0319] In order to assess the ability of RET 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 RET (log 2 transformed). Extension was done by creating a portioned hazard model including RET and the respective risk score as independent variables.

[0320] 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).

Example 4. Prediction of Silent Stroke with Circulating RET

[0321] Data in the SWISS-AF data shows that RET correlates with existence of white matter lesions (WML) in patients. The extent of matter lesions can be expressed by the Fazekas score (Fazekas, J B Chawluk, A Alavi, H I Hurtig, and R A Zimmerman American Journal of Roentgenology 1987 149:2, 351-356). The Fazekas score is ranging from 0 to 3.0 indicates no WML, 1 mild WML, 2 moderate WML and 3 severe WML. In order to compare the association of RET with WML patients were classified in two groups, Fazekas Score <2 (no) vs Fazekas Score ≥2 (yes). FIG. 3 shows that RET is reduced in patients with moderate or severe WMLs versus patients with mild or no WMLs.

[0322] WML extent can be caused by clinical silent strokes (Wang Y, Liu G, Hong D, Chen F, Ji X, Cao G. White matter injury in ischemic stroke Prog Neurobiol. 2016; 141:45-60. doi:10.1016/j.pneurobio.2016.04005). This further advocates the usefulness of RET to predict the risk for clinical stroke.

[0323] The ability of circulating RET to discriminate between patients with Fazekas Score <2 (no) versus Fazekas Score ≥2 (yes) is indicated by the AUC of 0.64. White matter changes in the brain of dementia patients Advanced age and changes in WML scores have been described to be associated with severity of dementia in Alzheimers disease patients (Kao et al., 2019).

[0324] Age is also an important predictor of clinical stroke. Therefor it is plausible that data of significantly reduced RET levels in the circulation indicate not only moderate or severe WML, but also indicate age related brain diseases, e.g. vascular dementia.

[0325] Results

[0326] Table 1 shows the results of univariate weighted proportional hazard model including log transformed values of RET.

[0327] The association between the risk for experiencing a stroke with the baseline value of RET is highly significant.

[0328] The hazard ratio for RET implies a 0.39 fold higher risk for a stroke in the patients of the Beat AF study and a 0.43 fold higher risk for a stroke in patients of the SWISS AF cohort.

[0329] The results of the proportional hazard model including RET as log 2 transformed linear risk predictor suggest the log 2 transformed values RET are proportional to the risk for experiencing a stroke

TABLE-US-00001 TABLE 1 Measurement of circulating RET in Beat AF and in SWISS AF patients; Case control of patients experiencing a clinical stroke event in the follow up Beat AF SWISS AF HR AUC p-Value HR AUC p-Value RET 0.39 0.69 0.0007 0.43 0.67 0.0223

[0330] As demonstrated in Table 1, Beat AF study data show the surprising finding of highly significantly reduced levels of circulating RET titers in samples of patients with atrial fibrillation, that experienced a stroke in the follow up period. This finding was replicated in an independent study cohort (SWISS AF). Patients of the SWISS AF cohort have a higher number of risk factors for stroke including e.g. higher age, more comorbidities, shorter time until the experience of stroke versus patients of the Beat AF cohort.

[0331] Obviously circulating RET levels associate with the severity of the risk. The finding of significantly reduced RET levels in patients at risk of experiencing a stroke in the next years was consistent between the two cohorts (Beat AF and SWISS AF). It is remarkable that circulating RET levels indicate the risk of stroke independent of differences in the burden of comorbidities, which is different in the investigated cohorts.

[0332] As demonstrated in Table 1 reduced RET levels in the circulation associate with the severity of the risk of experiencing a clinical stroke.

[0333] These data suggest that RET can be used to assess the risk of stroke, 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 anticoagulation drugs on RET levels), therapy stratification (selection of therapy options).

[0334] Table 2 shows the estimated c-indexes of RET alone, of the CHADS.sub.2, the CHA.sub.2DS.sub.2-VASc, the ABC score and of the weighted proportional hazard model combining the CHADS.sub.2, the CHA.sub.2DS.sub.2VASc, the ABC score with RET (log 2) on the case cohort selection.