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(1) Estimate risk of recurrent stroke/TIA/death in the subgroup of the Patent foramen ovale in the Cryptogenic Stroke Study (PICSS) cohort with patent foramen ovale (PFO) and antiphospholipid antibodies (aPL) and (2) Estimate risk of recurrent stroke/TIA/death in aPL positive patients who have thickened left-sided heart valves (VaT).
PFO is associated with cryptogenic ischemic stroke. Also, the presence of aPL is associated with ischemic cerebrovascular disease.
Combined data from 2 major sub studies of the Warfarin Aspirin Recurrent Stroke Trial (WARSS) were evaluated. PICSS subjects were included if they were enrolled in the Antiphospholipid Antibodies and Stroke Study (APASS) and had a baseline aPL test (lupus anticoagulant, anticardiolipin antibodies, or both) within one month of the stroke. All patients in PICSS underwent transesophageal echocardiography for PFO as well as VaT, which was performed blinded to aPL status and treatment arm (325mg/d aspirin or adjusted dose warfarin, target INR 1.4–2.8). The primary outcome event was the 2-year risk of recurrent stroke/TIA/death and was evaluated using Cox proportional hazards model. As there was no treatment effect, warfarin and aspirin groups were combined to increase power. For the combined endpoint, power to detect a HR of 2 was 47.8% for the PFO and aPL positive group, and 75.3% for the valve thickening and aPL positive group, assuming two-sided type I error of 0.05
525 subjects were tested for the combined presence of PFO and aPL and were available for evaluation. The primary outcome event rate was 23.9% (HR 1.39, 95% CI 0.75–2.59) in the PFO positive/aPL positive group, compared to 13.9% (HR 0.83, 95% CI 0.44–1.56) in the PFO positive/aPL negative group and 19.9% (HR 1.16 95% CI 0.68–1.90) in the PFO negative/aPL positive group.
545 subjects tested for combined presence of aPL and left sided cardiac VaT were available for evaluation. The primary event rate was 22.6% (HR1.65, 95% CI 0.88–3.09) in the VaT positive/aPL positive group, compared to 19.4% (HR 1.50, 95% CI 0.82–2.75) in the VaT positive/aPL negative group and 20.2% (HR 1.63, 95% CI 0.81–3.25) in the VaT negative /aPL positive group.
The combined presence of aPL with either a PFO or with left sided cardiac VaT did not significantly increase risk of subsequent cerebrovascular events in this PICCS/APASS cohort of patients.
PFO is associated with cryptogenic ischemic stroke which accounts for approximately 20–40% of all ischemic strokes1. Case control studies2–6 have consistently shown this association especially in patients less than 55 years of age, although prospective cohort7 or population based studies8, 9 have not . Similarly, the presence of antiphospholipid antibodies (aPL) is associated with ischemic cerebrovascular disease. Many case control studies10–12 and prospective cohort studies13 have shown an association between aPL and initial stroke but the relationship to recurrent or subsequent stroke is more uncertain14, 15. If paradoxical embolism is responsible for the majority of strokes in patients with a PFO, then hypercoagulable states which increase the risk of deep vein thrombosis may be overrepresented in PFO patients with a stroke. Therefore, the association of stroke with the combined presence of PFO and aPL is of interest.
Left-sided cardiac valve thickening, which is easily diagnosed by tranesophageal echocardiogram (TEE), has been suspected to be a risk factor for ischemic stroke16, 17. Moreover, Libman Sacks endocarditis is associated with aPL in some patients and may be an important mechanisms of stroke18. Little is known about stroke recurrence when these risk factors occur in combination. We hence undertook to study the risk of recurrent stroke and death associated with aPL and PFO as well as aPL and thickened left-sided heart valves.
PICSS (Patent Foramen Ovale in Cryptogenic Stroke Study)19 and APASS (Antiphospholipid Antibodies and Stroke Study)14 studies were both collaborative studies with the Warfarin Aspirin Recurrent Stroke Study (WARSS)20. Both PICSS and the APASS studies relied on the WARSS for patient recruitment as well as follow up. Patients were included in the present post-hoc analysis if they had a TEE test as part of the PICSS study, and also had tests for aPL status as part of the APASS study. Patients undergoing TEE were systematically evaluated for the presence of a PFO as well as thickened left-sided cardiac (mitral and/or aortic) valves.
WARSS was a double blind multicenter trial comparing adjusted dose warfarin (INR 1.4–2.8) versus aspirin (325mgs per day) for prevention of stroke in patients with non cardioembolic ischemic stroke. Patients were followed for two years for occurrence of stroke or death. Details of the WARSS methodology and the results have been published previously20. Briefly, patients were eligible for WARSS if they had an ischemic stroke within 30 days, were aged 30–85 years, had a moderate, mild or no deficit ( rating on Glasgow outcome scale ≤ 3), and had no contraindication to warfarin therapy. Patients were excluded if baseline INR was > 1.4, stroke was due to a procedure, attributable to a high grade carotid stenosis or cardioembolic source such as atrial fibrillation. At each WARSS center, the cryptogenic stroke patients were asked to undergo a TEE. PICSS also included patients from WARSS who underwent TEE for clinical reasons. Thus, the PICSS cohort included cryptogenic stroke patients as well as patients with other known stroke etiologies. The APASS study was a prospective study of the effect of aPL positivity on subsequent thromboembolism among patients in the WARSS study14. Forty-four of the 48 WARSS centers participated in APASS. Blood was drawn for aPL testing within one month of the ischemic event. IRB approvals were obtained at each site for the PICSS, APASS, and WARSS studies.
All patients underwent TEE according to a pre-determined protocol and the procedure is described in detail in the PICSS study19 . Biplane or multiplane probes were used and the tapes were analyzed by a single reader (Dr S.Homma) who was blinded to treatment, outcomes, and aPL status of the patients. The TEE protocol emphasized determination of cardioembolic sources including valve leaflet abnormalities including thickening. Patients were considered to have valve thickening if either the aortic or mitral valve thickness was 2mm or more. Presence of PFO was determined by agitated saline contrast study at rest and after Valsalva maneuver or cough. Presence of one or more micro bubbles in the left atrium within three cardiac cycles was considered positive for presence of PFO.
The blood for testing antiphospholipid antibodies was drawn at baseline before the patients were randomized for treatment in WARSS. This was within 30 days of stroke onset. All participating centers received video training of the procedure for drawing blood, storing, processing and shipping of aPL samples. The tests were conducted at a central laboratory. Patients were tested once at baseline for presence of the lupus anticoagulant (LA) and for the presence of anticardiolipin antibodies (aCL). Tests for LA included a sensitive activated partial thromboplastin time (aPTT), (Diagnostic Stago Inc., Parsipany, NJ), dilute Russell viper venom test (dRVVT) (American Diagnostics Inc. Pendleton, Ind.) and a hexagonal phase confirmatory test ( StaClot LA) (Diagnostic Stago Inc.). An LA test result was positive if the results of either the aPTT, or dRVVT test was positive, or if the StaClot test was positive. Testing for aCL was performed using commercial enzyme linked immunosorbent assay technique (Corgenix Inc. Denver, CO).
Cutoff values for positive results were as follows: IgM > 12 micrograms per deciliter, IgG > 21 micrograms per deciliter, and IgA > 23 micrograms per deciliter. Patients were classified as aPL positive if they tested positive at baseline blood draw for aCL alone (any isotype), LA alone, or both aCL and LA combined. Conversely, patients were aPL negative, if both LA as well as aCL tests were negative.
Two groups of patients were evaluated separately: PFO and aPL, and thickened left-sided heart valves and aPL. The primary endpoint was a composite of ischemic stroke, transient ischemic attack (TIA) or death from any cause. Patients were followed up for two years or until an endpoint occurred. The diagnosis of stroke was based on a new lesion on CT scan or MRI scan. Where imaging was negative, a clinical syndrome lasting more than 24 hours was required for the diagnosis.
The null hypotheses were:
In the WARSS study, there was no significant difference in the outcome of stroke or death at two years in the aspirin group compared to the warfarin group. Thus, for this analysis, the two groups were combined to increase power. The analysis addresses potential confounding because the aPL positive and the aPL negative groups were not randomly divided in the WARSS treatment groups. The variables tested included age, sex, hypertension, history of diabetes, history of cardiac disease, smoking status, obesity, alcohol consumption, and sedentary lifestyle. Variables were out of balance when the difference in means or proportions was significant at p < 0.1 level. The means of two groups are compared with two-sided t-test and the proportions of two groups are compared with Chi-squared test.
Reported event rates were actuarized estimates from the Kaplan-Meier curves that adjust for censoring. Differences among groups in the two year risk of the composite endpoint were calculated. A Cox proportional hazards model was used for calculating the hazards ratios (HR) and the 95% confidence intervals (CI) after adjusting for covariates that had p-values less than 0.2 in univariate analysis.
For the combined endpoint, power to detect a HR of 2 for the aPL positive and PFO positive group with the sample size 525 was 47.8% after adjustment of age, history of prior stroke, heart disease status, BMI, current smoking, sedentary life style and diabetes, assuming 2-sided type I error of 0.05. The power to detect a HR of 2 for the VaT positive/ aPL positive group with the sample size 545 was 75.3% after adjustment of age, gender, history of prior stroke, heart disease status, BMI, current smoking, sedentary life style and diabetes, assuming 2-sided type I error of 0.05.
The APASS study enrolled a total of 1770 patients and the PICSS study enrolled 630 patients. Subjects enrolled in both those studies formed the combined PICCS/APASS cohort which was used for evaluation in the present study.
There were 525 subjects from the APASS/PICSS cohort available for evaluation. The baseline characteristics of the patients with and without PFO are described in Table 1. PFO was present in 175 and there were 350 subjects who did not have a PFO. The subjects were divided into four subgroups based on presence or absence of both PFO and aPL. The primary outcome event rates did not statistically differ in the various groups compared to the PFO negative/aPL negative group.
Hypertension,, diabetes mellitus, and sedentary lifestyle as risk factors were more prevalent in the group without PFO and consequently were adjusted for in the multi-variate analysis. The hazard ratios (HR) for combined risk of TIA, ischemic stroke or death in the three groups were: PFO positive/aPL positive group 1.39 (95% CI 0.75–2.59), PFO negative /aPL positive group was 1.14 (95% CI 0.68–1.90) and 0.83 (95% CI 0.44–1.56) for the PFO positive/aPL negative group, all of which were not statistically significant. This did not change even after adjusting for the variables which were statistically different in the various subgroups.
The baseline characteristics of the patients with and without left-sided cardiac valve thickening are described in table 2. Of 545 subjects available for evaluation, 313 had left sided cardiac VaT, whereas 232 did not. Those with valve thickening were older, more likely to have diabetes mellitus, and be current smokers. Based on combined presence or absence of VaT and aPL, the subjects were divided into four subgroups. The primary outcome event rates did not differ significantly in the different subgroups when compared to the VaT negative/aPL negative group. The HRs for the risk of TIA, ischemic stroke or death in the various subgroups were: 1.65 (95% CI 0.88–3.09) for VaT positive /aPL positive group, 1.50 (95%CI 0.82–2.75) for the VaT positive/aPL negative group, and 1.63 (95% CI 0.81–3.25) for the VaT negative/aPL positive group.
The aPL tests included testing for the lupus anticoagulant (LA) as well as the anticardiolipin antibodies (aCL). 312 of the 525 patients tested were negative for both the LA and the aCL, while the remaining 213 were classified as aPL+ (40.6%). Of these, 34 were positive for both LA and the aCL (6.5%), and the rest were positive for either the LA or the aCL test (34%).
In this post-hoc, exploratory analysis from the PICSS and APASS studies, we did not detect a significant increase in the risk of ischemic stroke/TIA/death in ischemic stroke patients with presence of PFO and aPL either individually or in combination compared to those without a PFO nor aPLs. Similarly, the study failed to show a significant risk of recurrent stroke/TIA /death in ischemic stroke patients who had presence of VaT and aPL either separately or in combination compared to patients with neither VaT nor aPL.
PFOs are associated with cryptogenic stroke, and the mechanism is postulated to be paradoxical embolism, with clots forming and originating in the venous circulation and traveling to the arterial side via a right to left shunt. Systemic hypercoagulable states can potentially facilitate this process by increased formation of clots21, 22. In the PELVIS study23, cryptogenic stroke patients were found to have increased evidence of pelvic vein thrombosis. The study did not look for hypercoagulable states, but did add to the notion that paradoxical embolism may be important in at least some patients with cryptogenic stroke.
The mechanisms of thromboembolic phenomena associated with aPL are heterogeneous including immune mediated coagulopathy24 and that resulting from left sided cardiac valvular lesions of thickening and Libman Sacks endocarditis18. The cardiac valve thickening and endocarditis may be manifestations of the same pathological process which is a sub endothelial deposition of immunoglobulins including aCL and complements. Krause and colleagues25 found significant associations between cardiac valve thickening and several nervous system manifestations such as epilepsy, migraine and strokes in patients with primary antiphospholipid antibody syndrome. Interestingly, they defined valve thickening to be present to be present if it was more than 5mm, whereas our study defined it to be more than 2 mm.
The study patients were part of the larger WARSS study and were randomized to receive aspirin or warfarin. It is conceivable that the effect of combination of aPL /PFO and aPL/VaT on patient outcome events could have been affected by the treatment. However, we did not find any significant treatment effect when comparing warfarin versus aspirin (p < 0.36 - aPL/ PFO group and p < 0.88 (aPL /VaT group).
This study has an important limitation in that it is a post-hoc, retrospective analysis and thus should only be used for hypothesis generation. The WARSS study did not systematically evaluate patients for hypercoagulable states (for e.g. protein C and S and antithrombin III def). Similarly, the patients were not evaluated for presence of sub-clinical deep venous thrombosis. Nonetheless, although there did seem to be a trend towards increased risk of the predetermined endpoint among the group of patients with combined PFO and aPL as well as the VaT positive/ aPL positive group, this probably did not reach statistical significance because of the small sample size. Had the sample size been twice as large, the difference in endpoints achieved would have been statistically significant. Our study had reasonable power to detect an association for the valve thickening patients but limited power in the PFO subset. In addition, the patient population of WARSS was typically older and included all non-cardiogenic stroke subtypes, a population in whom traditional stroke risk factors such as hypertension and diabetes typically are more at play. PFO3 and to a lesser extent VaT have typically been shown to be associated with cryptogenic stroke patients of younger age. Also, contrary to more recent recommendations 26, the aPL studies in APASS were done only once at baseline and were not systematically repeated again at three months. Finally, we only tested for aPL whereas other hypercoagulable states such as APC resistance, and protein C and S defeciencies may also be important in patients with presumed paradoxical embolism.
In conclusion, the PICSS-APASS cohort study could not demonstrate significant excess risk of ischemic stroke recurrence, TIA or death among ischemic stroke patients due to the presence of PFO and a single baseline aPL positivity either separately or together. Similarly, the presence of left-sided cardiac valve thickening and a single baseline aPL either individually or together, did not increase the risk of stroke recurrence or death in this cohort. Because this study is a post-hoc, retrospective subset analysis, it should only be used for hypothesis generation. Further prospective studies with a larger number of subjects are needed to confirm or refute these observations.
Supported in part by NIH grants R0130896, K2443992 and R01 NS32525