Associations of Clonal Hematopoiesis with Recurrent Vascular Events and Death in Patients with Incident Ischemic Stroke

Clonal hematopoiesis (CH), defined by the presence of somatic mutations in hematopoietic stem cells of individuals without hematologic disease, is common among older people and associated with an increased risk of atherosclerosis, inflammation, and shorter overall survival. The mechanism leading to cardiovascular disease is best understood for TET2, one of the most frequently mutated genes, for which accelerated development of atherosclerosis driven by an altered function of the NLRP3/IL-1β-inflammasome in mutated macrophages was reported in preclinical models. CH has recently been associated with a higher risk of stroke in large patient series, however its role with respect to secondary vascular risk and death after ischemic stroke remains unknown.

To fill this gap, we investigated CH in peripheral blood DNA from 585 patients with first-ever ischemic stroke from the Prospective Cohort with Incident Stroke-Berlin study (PROSCIS-B) using error-corrected targeted sequencing of 45 genes recurrently mutated in CH. The primary composite endpoint (CEP) consisted of recurrent stroke, myocardial infarction, and all-cause mortality.

We identified 355 somatic mutations with a variant allele frequency (VAF) ≥ 1% in 240/585 patients (41%). While 158 patients harbored a single mutation, 82 patients harbored multiple (up to five) mutations. The most frequently mutated genes were DNMT3A, TET2, ASXL1 and PPM1D. In terms of the different etiological subtypes of ischemic stroke, CH was associated with large-artery atherosclerosis stroke (odds ratio 1.60, 95%-CI 1.07 - 2.44, P = 0.024) in logistic regression after correcting for age, sex and cardiovascular risk factors. White matter lesion load as quantified by the Wahlund score was significantly higher in CH-positive patients in baseline MRI compared to CH-negative patients (median Wahlund score 7, IQR 4 - 11 vs. 4, IQR 2 - 8, P < 0.001).

To determine whether CH was associated with systemic inflammation, we measured serum levels of pro-inflammatory cytokines shortly after stroke. CH-positive patients showed increased serum levels of IL-6, hsCRP, and VCAM-1 as compared to CH-negative patients (Fig. 1), supporting the current concept that CH mutations alter the inflammatory properties of myeloid cells.

With a median follow-up time of 36.2 months, primary endpoint data were available for all 585 patients with a total of 98 events. In univariable Kaplan-Meier analysis, CH was associated with a higher risk for recurrent vascular events and death (hazard ratio [HR] 1.55, 95%-CI 1.05 - 2.31, P = 0.02), which was more pronounced in patients with larger clones (Fig. 2). This led us to consider CH clone size as estimated by the VAF as a continuous parameter with respect to the primary endpoint. In fact, clone size remained an independent risk factor for the CEP in multivariable Cox regression (HR 1.21, 95%-CI 1.00 - 1.46, P = 0.046) after adjustment for age, sex, stroke severity, cardiovascular risk factors, atrial fibrillation, and peripheral artery disease. On single gene level, this association was mainly driven by TET2-mutated CH (HR 2.18, 95%-CI 1.11 - 4.26, P = 0.023) and PPM1D-mutated CH (HR 7.00, 95%-CI 2.27 - 21.73, P < 0.001).

Our data indicate that CH is associated with large-artery atherosclerosis and an increased risk of recurrent vascular events and death in patients with ischemic stroke, in particular in patients with larger, and TET2- or PPM1D-mutated clones. The CH mutation profile is accompanied by a systemic pro-inflammatory profile, potentially opening new avenues for preventive precision medicine approaches to resolve the self-perpetuating cycle of inflammation and clonal expansion.

Bullinger:Astellas: Honoraria; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer Oncology: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Endres:Bayer: Other: fees paid to Charité Universitätsmedizin Berlin, Research Funding; AstraZeneca: Other: fees paid to Charité Universitätsmedizin Berlin; Boehringer Ingelheim: Other: fees paid to Charité Universitätsmedizin Berlin; BMS: Other: fees paid to Charité Universitätsmedizin Berlin; Daiichi Sankyo: Other: fees paid to Charité Universitätsmedizin Berlin; Amgen: Other: fees paid to Charité Universitätsmedizin Berlin; GSK: Other: fees paid to Charité Universitätsmedizin Berlin; Sanofi: Other: fees paid to Charité Universitätsmedizin Berlin; Covidien: Other: fees paid to Charité Universitätsmedizin Berlin; Novartis: Other: fees paid to Charité Universitätsmedizin Berlin; Pfizer: Other: fees paid to Charité Universitätsmedizin Berlin. Damm:Astra Zeneca: Honoraria; AbbVie: Honoraria; Novartis: Honoraria; Roche: Honoraria; Gilead: Honoraria; Incyte: Honoraria.