Trial Design

We conducted a phase 3, investigator-initiated, multicenter, randomized, placebo-controlled trial involving patients with acute ischemic stroke, in whom the assigned intervention was initiated between 4.5 and 9.0 hours after the onset of stroke or on awakening with stroke symptoms. The trial methods have been published previously,11 and the protocol is available with the full text of this article at NEJM.org.

The design of the trial, the analysis and collection of the data, and the writing of the manuscript were performed by the members of the executive committee and by the investigators at the trial sites listed in the Supplementary Appendix, available at NEJM.org. All the authors vouch for the accuracy and completeness of the data, for the fidelity of the trial to the protocol, and for the reporting of adverse events. Boehringer Ingelheim provided the alteplase and matching placebo used in this trial. A research version of RAPID software was provided free of charge to the trial sites by iSchemaView. Neither company was involved in the design, conduct, or reporting of the trial. No confidentiality agreements were in place between the authors and either commercial entity.

After the results of the Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke (WAKE-UP) trial were published in May 2018,12 the data and safety monitoring board recommended the discontinuation of the trial because of loss of equipoise. Recruitment was suspended on June 6, 2018.

Patients

Patients were eligible for inclusion if they were at least 18 years of age; had excellent functional status before enrollment (defined by a score of <2 on the modified Rankin scale, on which scores range from 0 [no neurologic deficit] to 6 [death]); had a stroke with a clinical severity score at presentation of 4 to 26 on the National Institutes of Health Stroke Scale (NIHSS), on which scores range from 0 to 42, with higher scores indicating greater deficit; and had hypoperfused but salvageable regions of brain detected on automated perfusion imaging. Occlusion of a large cerebral vessel was not a prerequisite for inclusion. Imaging techniques included CT perfusion imaging or perfusion-diffusion MRI, and images were processed with the use of a research version of RAPID automated software (Stanford University and iSchemaView).

In estimating the volume of irreversibly injured ischemic-core tissue, we used a threshold for relative cerebral blood flow of less than 30% of that in normal brain regions13 or we used diffusion-weighted MRI.14 Critically hypoperfused brain was measured on perfusion MRI or CT perfusion imaging according to a delayed arrival of an injected tracer agent (time to maximum of the residue function exceeding 6 seconds).15-17 Perfusion lesion–ischemic core mismatch was defined as a ratio greater than 1.2 between the volume of hypoperfusion and the volume of the ischemic core, an absolute difference in volume greater than 10 ml, and an ischemic-core volume of less than 70 ml.5,18 Patients were not eligible if the investigator was considering the use of endovascular thrombectomy at the time of enrollment.

The trial was approved by the institutional ethics committee at each participating site. Written informed consent was obtained from all the patients or their legal representatives before enrollment. Additional details of the inclusion and exclusion criteria are provided in the Supplementary Appendix.

Trial Interventions

The patients were randomly assigned, in a 1:1 ratio, to receive either alteplase (0.9 mg per kilogram of body weight [maximum, 90 mg], administered intravenously as a 10% bolus and 90% infusion over 1 hour) or matching placebo. Randomization was performed through a centralized website, with stratification according to geographic region (Australia, New Zealand, and Finland vs. Taiwan) and time of intervention (>4.5 to 6.0 hours after stroke onset, >6.0 to 9.0 hours after stroke onset, or on awakening with stroke symptoms). Guideline-based care for acute stroke was recommended for all patients.1,2 From 2010 through February 2018, the guidelines did not include the use of endovascular thrombectomy in extended time windows.

Outcomes

The primary outcome was a score of 0 or 1 on the modified Rankin scale at 90 days (indicating an excellent functional outcome with a return to all usual activities). The risk ratio for the primary outcome was adjusted for age and clinical severity of stroke (NIHSS score) at baseline. The secondary clinical outcomes were the score (0 to 6) on the modified Rankin scale at 90 days (with the distribution of scores in each trial group used in an ordinal analysis to assess functional improvement); a score of 0 to 2 on the modified Rankin scale at 90 days (indicating functional independence); and percentages of reperfusion of at least 50% and of at least 90% at 24 hours after the intervention (defined as ≥50% and ≥90% reductions, respectively, in the volume of the perfusion lesion in which there had been a delayed arrival of an injected tracer agent exceeding 6 seconds).19 The prespecified tertiary outcomes were recanalization at 24 hours after stroke (defined as a score of 2 or 3 on the Arterial Occlusive Lesion scale [range, 0 to 3], indicating partial or complete opening of the artery, respectively, with the presence of distal blood flow)20 in the patients who had occlusion of a cerebral vessel detected on CT or MR angiography at baseline; and major neurologic improvement (defined as a reduction in the NIHSS score of ≥8 points or a score of 0 or 1 within 24 hours, 72 hours, and 90 days after the intervention). Safety outcomes were death within 90 days after the intervention and symptomatic intracranial hemorrhage, which was adjudicated in a blinded manner by a central panel of stroke neurologists and neuroradiologists as parenchymal hematoma type 2 (confluent blood clot occupying >30% of the infarct with substantial mass effect) within 36 hours after intervention, accompanied by an increase of at least 4 points in the NIHSS score from baseline.21

Statistical Analysis

We originally estimated that a sample size of 400 patients would provide the trial 80% power to detect a between-group difference of 15 percentage points in the primary outcome (36% in the alteplase group and 21% in placebo group) at a two-sided significance level of P=0.05, with allowance for 90 patients to be lost to follow-up or have data that could not be evaluated.11 The effect size was based on the results of our previous trial (Echoplanar Imaging Thrombolytic Evaluation Trial [EPITHET]) that compared alteplase with placebo administered 3 to 6 hours after the onset of stroke in patients who had a mismatch in perfusion-weighted MRI and diffusion-weighted MRI.5 After a blinded review of the observed number of patients who had data that could not be evaluated and the number who were lost to follow-up, the sample size was revised to 310 patients (see the final protocol). A prespecified adaptive sample-size reestimation was also performed in a blinded manner with the use of data from the first 200 patients.22 This analysis confirmed a final intended sample size of 310 patients.23

The statistical analysis plan, available in the protocol, was finalized before the database was locked.23 Statistical analyses were performed with the use of Stata software, version 13 (StataCorp). We used covariate-adjusted modified Poisson regression with robust error estimation24 to compare the trial groups in the prespecified primary outcome analysis (adjusted for age and NIHSS score at baseline) and in the analyses of the dichotomous secondary efficacy outcomes and the dichotomous safety outcomes.23 For consistency with the original protocol, we also analyzed the results using logistic-regression modeling and report them in the Supplementary Appendix. The proportional-odds assumption was valid according to the Brant test and the approximate likelihood-ratio test. Therefore, an ordinal logistic-regression model with adjustment for age and NIHSS score at baseline was used to compare the trial groups across the full range of scores on the modified Rankin scale (data were combined for scores of 5 and 6), with the effect estimate for an improvement of at least 1 point in the score presented as a common odds ratio with a 95% confidence interval. The primary analysis for all clinical outcomes was prespecified to be adjusted for baseline prognostic factors of age and NIHSS score; we report both covariate-adjusted and unadjusted results. Treatment effects for secondary outcomes are presented as adjusted risk ratios with 95% confidence intervals.

Because the analyses of the secondary or tertiary outcomes did not include adjustment for multiple comparisons, the results are reported as point estimates with unadjusted 95% confidence intervals. The handling of missing data and sensitivity analyses are described in the statistical analysis plan (see the protocol).

Preplanned subgroup analyses explored the effects of age (<75 vs. ≥75 years and <80 vs. ≥80 years), severity of stroke at baseline (NIHSS score <10 or ≥10), time to intervention (>4.5 to 6.0 hours, >6.0 to 9.0 hours, or on awakening with stroke), geographic region (Australia, New Zealand, and Finland vs. Taiwan), and presence of any large-vessel occlusion. Large-vessel occlusion was defined as occlusion of the internal carotid or middle cerebral artery (M1 or proximal [retrievable] M2 segments or both) and was independently graded on MR or CT angiography in a blinded manner by two assessors who then reached a consensus.