METHODS OF DETERMINING THE ETIOLOGY OF ACUTE ISCHEMIC STROKES

Abstract

Determining acute ischemic stroke (AIS) etiology is crucial for guidance of secondary prevention. Here, the inventors performed a correlation analysis between AIS etiology and AIS thrombus cellular composition and content, as assessed using quantitative biochemical assays. In particular, homogenates of 250 AIS patient thrombi were prepared by mechanical grinding. Platelet, red blood cell, and leukocyte content of AIS thrombi were estimated by quantification of glycoprotein (GP)VI, heme, and DNA in thrombus homogenates. AIS etiology was defined as cardioembolic, non-cardioembolic, or embolic stroke of undetermined source (ESUS), according to the TOAST classification. Cardioembolic thrombi were richer in DNA (35.8 vs 13.8 ng/mg, p<0.001) and poorer in GPVI (0.104 vs 0.117 ng/mg, p=0.045) than non- cardioembolic ones. The area under the receiver operating characteristic curve of DNA content to discriminate cardioembolic thrombi from non-cardioembolic was 0.72 (95% Cl, 0.63 to 0.81). With a threshold of 44.7 ng DNA/mg thrombus, 47% of thrombi from undetermined etiology would be classified as cardioembolic with a specificity of 90%. In conclusion, thrombus DNA content may provide an accurate biomarker for identification of cardioembolic thrombi in AIS patients with ESUS.

Claims

1. A method of determining the etiology of an acute ischemic stroke that occurred in a patient and treating the patient comprising quantifying the DNA content in a thrombus obtained from the patient and i) determining that the DNA content is higher than a predetermined reference value and treating the patient for cardioembolic stroke by administering an anticoagulant to the patient; or ii) determining that the DNA content is lower than the predetermined reference value and treating the patient for non-cardioembolic stroke by administering a diuretic, a combination of a diuretic and an ACE-inhibitor and/or a statin therapy to the patient.

2. The method of claim 1 wherein the cardioembolic stroke is of undetermined source.

3. (canceled)

4. (canceled)

5. The method of claim 1 further comprising quantifying the GPVI content in the thrombus of the patient.

6. The method of claim 5 wherein a DNA/GPVI ratio is calculated.

7. (canceled)

8. A method of determining the etiology of an acute ischemic stroke that occurred in a patient and treating the patient comprising quantifying the DNA content in a thrombus obtained from the patient, quantifying the GPVI content in the thrombus, calculating a DNA/GPVI ratio and i) determining that the DNA/GPVI ratio is higher than a predetermined reference value and treating the patient for cardioembolic stroke by administering an anticoagulant to the patient; or ii) determining that the DNA/GPVI ratio is lower than the predetermined reference value and treating the patient for non-cardioembolic stroke by administering n diuretic, a combination of a diuretic and an ACE-inhibitor and/or a statin therapy to the patient.

9. (canceled)

10. The method of claim 1, wherein the anticoagulant is selected from the group consisting of: a direct thrombin inhibitor, a direct factor Xa inhibitor, a pentasaccharide, a low molecular weight heparin, a vitamin K antagonist, and an antiplatelet drug.

11. (canceled)

12. The method of claim 10, wherein the direct thrombin inhibitor is dabigatran, hirudin, bivalirudin, lepirudin or argatroban, the direct factor Xa inhibitor is rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban or eribaxaban, the pentasaccharide is fondaparinux or idraparinux, the low molecular weight heparin is nadroparin, tinzaparin, dalteparin, enoxaparin, bemiparin, reviparin, parnaparin or certoparin or unfractionated heparin, the vitamin K antagonist is acenocoumarol, phenprocoumon, warfarin, atromentin or phenindione, and the antiplatelet drug is an irreversible cyclooxygenase inhibitor, an ADP receptor inhibitor, a phosphodiesterase inhibitor, a PAR-1 antagonist, a GPIIB/IIIa inhibitor, an adenosine reuptake inhibitor, a thromboxane inhibitor or a thromboxane receptor antagonist.

13. The method of claim 12, wherein the irreversible cyclooxygenase inhibitors is aspirin or a derivative thereof or triflusal, the ADP receptor inhibitor is clopidogrel, prasugrel, ticagrelor, ticlopedine, cangrelor or elinogrel, the phosphodiesterase inhibitor is cilostazol, the PAR-1 antagonist is voraxapar, the GPIIB/IIIa inhibitor is abciximab, eptifibatide, tirofiban, roxifiban or orbofiban, the adenosine reuptake inhibitor is dipyridamole, the thromboxane inhibitor is ifetroban or picotamide, and the thromboxane receptor antagonist is terutroban or picotamide.

14. The method of claim 8, wherein the anticoagulant is selected from the group consisting of: a direct thrombin inhibitor, a direct factor Xa inhibitor, a pentasaccharide, a low molecular weight heparins, a vitamin K antagonist, and an antiplatelet drug.

15. The method of claim 14, wherein the direct thrombin inhibitor is dabigatran, hirudin, bivalirudin, lepirudin or argatroban, the direct factor Xa inhibitor is rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, letaxaban or eribaxaban, the pentasaccharide is fondaparinux or idraparinux, the low molecular weight heparin is nadroparin, tinzaparin, dalteparin, enoxaparin, bemiparin, reviparin, parnaparin or certoparin or unfractionated heparin, the vitamin K antagonist is acenocoumarol, phenprocoumon, warfarin, atromentin or phenindione, and the antiplatelet drug is an irreversible cyclooxygenase inhibitor, an ADP receptor inhibitor, a phosphodiesterase inhibitor, a PAR-1 antagonist, a GPIIB/IIIa inhibitor, an adenosine reuptake inhibitor, a thromboxane inhibitor or a thromboxane receptor antagonist.

16. The method of claim 15, wherein the irreversible cyclooxygenase inhibitors is aspirin or a derivative thereof or triflusal, the ADP receptor inhibitor is clopidogrel, prasugrel, ticagrelor, ticlopedine, cangrelor or elinogrel, the phosphodiesterase inhibitor is cilostazol, the PAR-1 antagonist is voraxapar, the GPIIB/IIIa inhibitor is abciximab, eptifibatide, tirofiban, roxifiban or orbofiban, the adenosine reuptake inhibitor is dipyridamole, the thromboxane inhibitor is ifetroban or picotamide, and the thromboxane receptor antagonist is terutroban or picotamide.

Description

FIGURES

[0028] FIG. 1. Distribution of biochemical features of AIS thrombi according to etiology. (A-D) Boxes show the 25th, 50th, and 75th, and whiskers indicate values outside the lower and upper quartile with a length equal to 1.5 interquartile range; diamond indicates the mean values. P-values for global comparison (one-way ANOVA) are reported after a log-transformation for DNA, and ratio DNA/GPVI; * indicated P-values <0.05 for post-hoc pairwise comparison between cardioembolic stroke and each other stroke subgroups (adjusted for multiple comparison using Bonferroni correction).

[0029] FIG. 2. Receiver operating characteristic (ROC) curve for differentiation of cardioembolic and non-cardioembolic strokes according to DNA and GPVI thrombus content, and to the DNA/GPVI thrombus content ratio.

EXAMPLE

Methods

Standard Protocol Approvals, Registrations, and Patient Consents

[0030] Thrombi were collected in two centers at the end of endovascular therapy (EVT). The EVT procedure was chosen at the interventionalist’s discretion, using a stent-retriever and/or a contact aspiration technique. AIS etiology was classified as described.sup.1 and determined based on cerebral magnetic resonance imaging (MRI), computed tomography or MRI angiography, transcranial and extracranial duplex sonography, coagulation tests, 1 to 3 days electrocardiography recording, and transthoracic and/or transesophageal echocardiography. Patient data were collected prospectively using a standardized questionnaire (Endovascular Treatment in Ischemic Stroke -ETIS- registry NCT03776877). All patients were provided with a written explanation of the study. The patients or their representatives were given the opportunity to refuse participation. The local Ethics Committee approved this research protocol (CPP Nord Ouest II, ID-RCB number: 2017-A01039-44).

Preparation of Thrombus Homogenates

[0031] Thrombus homogenates were prepared with stainless steel beads (5 mm, Qiagen, 69989) in cold PBS (30 .Math.L/mg thrombus) supplemented with protease inhibitor (1%, Sigma, P8340), using a tissue lyser (25 Hz, 4 minutes, TissueLyser II, Qiagen). Thrombi not completely grinded went through a second passage in the tissue lyser. The thrombus homogenates were then recovered after centifugation (14 000 g x 20 minutes, 4° C.) to eliminate non-soluble debris. Homogenates of initially cut thrombi were pooled before analysis.

Quantification of Red Blood Cell and DNA

[0032] RBC content was estimated by measurement of heme concentration in thrombus homogenates using a formic acid-based colorimetric assay, as described previously.sup.8. DNA was quantified using the Molecular Probes Quant iT Picogreen dsDNA Assay kit (Life Technologies).

Quantification of Platelet Content

[0033] Soluble GPVI levels were measured by immunoassay according to the following protocol. Ninety-six wells standard binding plate from MesoScale Discovery (MSD, Rockville, MD) were coated overnight at 4° C. with 2 .Math.g/mL sheep anti human GPVI polyclonal antibody (Bio Techne, France, AF3627). After 1 hour of incubation at room temperature with 5% MSD Blocker A (R93AA-1) and 3 washes with 150 .Math.L PBS / 0.05% Tween (PBST), 25 .Math.L of thrombus homogenate or standard were added and the plate was incubated for 1 hour at room temperature, 500 rpm. Standard curve was obtained with Recombinant Human GPVI protein (Bio techne, France, 3627-GP, 0.097-25 ng/ml). After 3 PBST washes, 25 .Math.L of biotinylated sheep anti-human GPVI antibody (Bio Techne, France, BAF3627, 0.5 .Math.g/mL in 1% MSD Blocker A) was added to each well and the plate was incubated 1 hour at room temperature. Finally, 25 uL of streptavidin Sulfo-TAG/well was added after 3 PBST washes and the plate was incubated 1 hour at room temperature. A MesoScale Quickplex Plate Scanner was used of quantification.

Statistical Analysis

[0034] Categorical variables were expressed as frequencies and percentages. Quantitative variables were expressed as mean (standard deviation, SD), or median (interquartile range, IQR) for non-normal distribution. Normality of distributions was assessed graphically and by using the Shapiro-Wilk test. We compared the different proportions of components of thrombi (heme, DNA, platelet, and DNA/platelet ratio) between the 3 AIS etiology subgroups (cardioembolic, non cardioembolic and ESUS) using one-way analysis of variance (ANOVA); post-hoc pairwise comparisons were done using linear contrast after Bonferroni correction. Primary comparison covered the overall study sample and was further performed according to use of IV alteplase prior to EVT. For thrombus content which were significant between the two group of interest (cardioembolic vs. non cardioembolic), we assessed the performance of thrombus content to determine cardioembolic from noncardioembolic etiology by calculating the area under the ROC curves (AUCs) and their 95% confidence intervals (CIs). From the ROC curves, we determined the optimal threshold value by maximizing the Youden index as well as the threshold values to reach a sensitivity and specificity of 0.90, respectively. We applied these threshold value in the cryptogenic patients. Statistical testing was conducted at the two-tailed α-level of 0.05. Data were analyzed using the SAS software version 9.4 (SAS Institute, Cary, NC).

Results

[0035] From June 2016 to November 2018, a total of 1209 consecutive AIS patients with LVO were treated by EVT in our institutions. Thrombi from 250 of these patients selected randomly were homogenized and analyzed for RBC, platelet, and leukocyte content, as estimated by quantification of heme, GPVI, and DNA, respectively. Patient and treatment characteristics of the study sample are reported in Table 1. Stroke etiology was cardioembolic in 142 (56.8%) patients, non-cardioembolic in 33 patients (13.2%), and undetermined in 75 patients (30.0%).

Thrombus Cellular Content and AIS Etiology

[0036] There was no significant difference in the heme content between thrombi from cardioembolic and non-cardioembolic origin (FIG. 1A).

[0037] Non-cardioembolic thrombi had reduced DNA content, and increased GPVI content as compared to cardioembolic thrombi (FIGS. 1B and C). As a consequence, the DNA/GPVI ratio (FIG. 1D) was higher in cardioembolic thrombi than in non-cardioembolic ones (median IQR : 322 (151 to 1132) vs 266 (151 to 1132), p<0.001). Together, these results indicate that cardioembolic thrombi contain significantly more leukocytes and less platelets than non-cardioembolic ones.

[0038] Thrombi from undetermined etiology had increased heme content compared to cardioembolic thrombi (FIG. 1A), but showed no significant differences in DNA or platelet content as compared to either of the other groups of thrombi (FIGS. 1B-D).

Thrombus DNA Content to Discriminate Cardioembolic Versus Non-Cardioembolic AIS

[0039] The area under the receiver operating characteristic curve (AUC) for thrombus DNA content used for differentiating thrombi of cardioembolic and non-cardioembolic origins was of 0.72 (95% CI, 0.63 to 0.81). A similar AUC value was obtained for the DNA/GPVI ratio (FIG. 2 and Table 2). These data suggest that both thrombus DNA content and DNA/GPVI ratio hold potential usefulness for identification of cardioembolic thrombi. In contrast, the AUC for the GPVI thrombus content was of 0.65 (95% CI, 0.54 to 0.77) (FIG. 2 and Table 2), indicating a poor diagnostic potential. The specificity and sensitivity of thrombus DNA content for discriminating cardioembolic thrombi from non-cardioembolic thrombi was calculated for various thresholds of DNA thrombus content (Table 2). For a threshold of 44.7 ng DNA/mg thrombus, nearly 50% of ESUS thrombi would be classified as cardioembolic with a specificity of 90%.

Discussion

[0040] In the present study conducted on 250 AIS thrombi responsible for LVO, we have explored possible relationships between AIS etiology and thrombus cell composition. In order to avoid the inherent limitations of semi-quantitative immunohistological methods.sup.7, we have analyzed cell composition using quantitative assays for markers of RBCs, platelets, and leukocytes. Our results show that cardioembolic thrombi are richer in DNA and poorer in platelets compared to non-cardioembolic thrombi. From a pathophysiological perspective, the increased DNA content of thrombi from cardioembolic origin suggests a more prominent role of leukocytes in the formation of those thrombi. Leukocytes, especially neutrophils, are indeed the primary source of DNA in blood and are now widely recognized as active players of thrombosis.sup.9,10. Interestingly, previous studies have shown that elevated neutrophil-lymphocyte ratios in patients with nonvalvular atrial fibrillation were independently associated with the presence of left atrial thrombus.sup.11, as well as with an increased risk of thromboembolic stroke.sup.12. Also consistent with our results, patients with cardioembolic stroke were reported to have increased plasma cell-free DNA levels compared to stroke patients of other etiologies.sup.13.

[0041] The increased DNA content of cardioembolic thrombi might also account for their previously reported higher leukocyte and neutrophil extracellular traps (NETs) content compared to thrombi of other origins.sup.14. Additionally, the high proportion of DNA content found in cardioembolic thrombi and the pivotal role of neutrophils and NETs in thrombosis give additional arguments for a potential benefit of DNAse 1 in AIS treatment.sup.14,15.

[0042] Importantly, our results indicate that both the thrombus DNA content and the thrombus DNA/GPVI ratio could provide biomarkers for identification of cardioembolic thrombi among thrombi of undetermined origin. In fact, specificity/selectivity calculations revealed that, by adjusting the DNA thrombus content threshold, one could classify nearly 50% of ESUS thrombi as cardioembolic with a specificity of 90%. Considering that ESUS represents 20-25% of all AIS, there is a clear interest in developing new diagnostic tools to better identify ESUS patient subgroups. A recent major secondary prevention trial found no superiority of rivaroxaban over aspirin for prevention of recurrent stroke in the overall ESUS patient population.sup.16. Identifying the subgroup of ESUS patients requiring more active cardiac screening and which could benefit from anticoagulant therapy could help to both improve patient management and refine secondary prevention studies.

[0043] In addition to be inexpensive, thrombus homogenization as performed in our study requires only moderate skills and is fairly easily feasible with common laboratory and hospital equipment, and so is the subsequent measurement of DNA in thrombus homogenates. The main limitation of this method based on mechanical grinding of AIS thrombi is that non-soluble components such as fibrin could not be directly quantified.

[0044] To date, and to our knowledge, it is the largest study on thrombus composition based on biochemical quantitative analysis of their cellular content. Our results provide a potential basis for the development of new tools and strategies for identification of ESUS patient subgroups and improved secondary prevention.

Tables

[0045] TABLE-US-00001 Patients and treatment characteristics, in overall and according to suspected acute ischemic stroke etiology Characteristics Suspected AIS etiology Overall Cardioembolic Non-cardioembolic ESUS Number of patients 250 142 33 75 Demographics Age, years, mean (SD) 70.1 (15.5) 74.4 (14.6) 62.2 (12.9) 65.3 (15.5) Men, n (%) 129/250 (51.6) 66/142 (46.5) 24/33 (72.7) 39/75 (52.0) Medical history Hypertension 144/247 (58.3) 92/141 (65.2) 14/32 (43.8) 38/74 (51.4) Diabetes 42/248 (16.9) 25/142 (17.6) 6/32 (18.8) 11/74 (14.9) Hypercholesterolemia 79/247 (32.0) 52/141 (36.9) 9/32 (28.1) 18/74 (24.3) Current smoking 50/238 (21.0) 22/134 (16.4) 7/32 (21.9) 21/72 (29.2) Coronary artery disease 32/245 (13.1) 21/139 (15.1) 3/33 (9.1) 8/73 (11.0) Previous stroke or TIA 36/246 (14.2) 23/139 (16.5) 5/33 (15.2) 7/74 (9.5) Previous antithrombotic medications 103/244 (42.2) 81/140 (57.9) 7/31 (22.6) 15/73 (20.5) Antiplatelet 47/244 (19.3) 29/140 (20.7) 5/31 (16.1) 13/73 (17.8) Anticoagulant 48/244 (19.7) 44/140 (31.4) 2/31 (6.5) 2/73 (2.7) Current stroke event NIHSS score, median (IQR).sup.a 17 (12 to 20) 18 (14 to 21) 16 (9 to 19) 16 (12 to 20) Pre-stroke mRS≥1 23/248 (9.2) 30/141 (21.3) 5/33 (15.2) 8/74 (10.8) ASPECTS, median (IQR).sup.b 7 (5 to 8) 7 (6 to 8) 6 (5 to 8) 6 (5 to 8) Site of occlusion M1-MCA 134/246 (54.5) 80/139 (57.6) 7/33 (21.2) 47/74 (63.5) M2-MCA 20/246 (8.1) 14/139 (10.1) 0 (0.0) 6/74 (8.1) Intracranial ICA or tandem 53/246 (21.5) 28/139 (20.1) 7/33 (21.2) 18/74 (24.3) Tandem 19/246 (7.7) 5/139 (3.6) 14/33 (42.4) 0 (0.0) extracranial ICA 6/246 (2.4) 4/139 (2.9) 1/33 (3.0) 1/74 (1.4) Vertebro-Basilar 12/246 (4.9) 6/139 (4.3) 4/33 (12.1) 2/74 (2.7) Others 2/246 (0.8) 2/139 (1.4) 0 (0.0) 0 (0.0) Treatment characteristics Intravenous Alteplase 131/250 (52.4) 62/142 (43.7) 20/33 (60.6) 49/75 (65.3) General anesthesia 38/242 (15.7) 22/138 (15.9) 7/30 (23.3) 9/74 (12.2) Onset to groin puncture time, min, median (IQR).sup.c 240 (186 to 286) 222 (170 to 279) 262 (217 to 308) 250 (205 to 295) Values expressed as no/total no. (%) unless otherwise indicated. .sup.a3 missing data (2 in cardioembolic group and 1 in Non-cardioembolic group) .sup.b18 missing data (12 in cardioembolic group, 1 in Non-cardioembolic group and 5 in Cryptogenic group) .sup.c7 missing data (4 in cardioembolic group, 1 in Non-cardioembolic group and 2 in Cryptogenic group). Abbreviations: ASPECTS= Alberta stroke program early computed tomography score; ICA=internal carotid artery; IQR=interquartile range; MCA=middle cerebral artery; NIHSS=National Institutes of Health Stroke Scale; rt-PA=recombinant tissue plasminogen activator; TIA=transient ischemic attack; mRS=modified Rankin scale, SD=standard deviation.

TABLE-US-00002 Accuracy of thrombus cell marker content for identification of cardioembolic thrombi AUC (95%CI) Threshold Sensitivity (95%CI) Specificity (95%CI) % of patients with ESUS DNA 0.72 (0.63 to 0.81) >22.4.sup.1 66.0 (57.5 to 73.7) 69.7 (51.3 to 84.4) 62.5 >8.9 90.0 27.3 (13.3 to 45.5) 84.7 >44.7 44.0 (35.6 to 52.3) 90.0 47.2 GPVI 0.65 (0.54 to 0.77) <11.5.sup.1 56.2 (37.7 to 73.6) 89.2 (82.6 to 94.0) 71.9 <13.4 90.0 28.1 (13.7 to 46.7) 90.6 <7.7 10.0 (5.4 to 16.5) 90.0 12.5 DNA/GPVI 0.73 (0.63 to 0.82) >161.sup.1 72.9 (64.3 to 80.3) 65.6 (46.8 to 81.4) 65.6 >81 90.0 34.4 (18.6 to 53.2) 81.2 >614 36.4 (28.1 to 45.4) 90.0 31.2 .sup.1 cut-value who maximize the Youden index. Abbreviations: AUC=area under the Receiver Operating Curve; CI=confidence interval.

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