Evaluating User Experience and DNA Yield from Self-Collection Devices.

BACKGROUND: The COVID-19 pandemic emphasized an urgent need for devices used in the self-collection of biospecimens in an evolving patient care system. The mailing of biospecimen self-collection kits to patients, with samples returned via mail, provides a more convenient testing regimen, but could also impart patient sampling variabilities. User compliance with device directions is central to downstream testing of collected biospecimens and clear instructions are central to this goal. METHODS: Here, we performed an evaluation of 10 oral DNA collection devices involving either swab or saliva self-collection and analyzed ease of use and comfort level with a device, as well as DNA recovery quantity/quality and sample stability. RESULTS: We show that while these DNA quality/quantity metrics are comparable between devices, users prefer direct saliva collection over swab-based devices. CONCLUSIONS: This information is useful in guiding future experiments including their use in human RNA, microbial, or viral sample collection/recovery and their use in clinical testing.

Functional reorganization of North American wintering avifauna.

Wintering birds serve as vital climate sentinels, yet they are often overlooked in studies of avian diversity change. Here, we provide a continental-scale characterization of change in multifaceted wintering avifauna and examine the effects of climate change on these dynamics. We reveal a strong functional reorganization of wintering bird communities marked by a north-south gradient in functional diversity change, along with a superimposed mild east-west gradient in trait composition change. Assemblages in the northern United States saw contractions of the functional space and increases in functional evenness and originality, while the southern United States saw smaller contractions of the functional space and stasis in evenness and originality. Shifts in functional diversity were underlined by significant reshuffling in trait composition, particularly pronounced in the western and northern United States. Finally, we find strong contributions of climate change to this functional reorganization, underscoring the importance of wintering birds in tracking climate change impacts on biodiversity.

Epigenomics may begin to explain in vitro differential response to hypomethylating agents in MMR-D hypermethylated endometrial cancer.

This work examines differences in chromatin accessibility, methylation, and response to DNA hypomethylating agents between mismatch repair-deficient and non-mismatch repair-deficient endometrial cancer. Next-generation sequencing of a stage 1B, grade 2 endometrioid endometrial cancer tumor revealed microsatellite instability and a variant of unknown significance in POLE along with global and MLH1 hypermethylation. Inhibition of viability by decitabine in the study and comparison tumors was minimal, as shown by an inhibitory effect of 0 and 17.9, respectively. Conversely, the inhibitory effect of azacitidine on the study tumor was more pronounced, at 72.8 versus 41.2. In vitro, mismatch repair-deficient endometrial cancer with MLH1 hypermethylation respond better to DNA methyltransferase inhibition by azacytidine (DNA/RNA inhibition), than to decitabine (DNA-only inhibition). Additional large studies are needed to substantiate our findings.

Frequency of inter-specialty consensus decisions and adherence to advice following discussion at a weekly neurovascular multidisciplinary meeting.

BACKGROUND/AIMS: Data are limited on the frequency of 'consensus decisions' between sub-specialists attending a neurovascular multidisciplinary meeting (MDM) regarding management of patients with extracranial carotid/vertebral stenoses and post-MDM 'adherence' to such advice. METHODS: This prospective audit/quality improvement project collated prospectively-recorded data from a weekly Neurovascular/Stroke Centre MDM documenting the proportion of extracranial carotid/vertebral stenosis patients in whom 'consensus management decisions' were reached by neurologists, vascular surgeons, stroke physicians-geriatricians and neuroradiologists. Adherence to MDM advice was analysed in asymptomatic carotid stenosis (ACS), symptomatic carotid stenosis (SCS), 'indeterminate symptomatic status stenosis' (ISS) and vertebral artery stenosis (VAS) patients, including intervals between index event to MDM + / - intervention. RESULTS: One hundred fifteen patients were discussed: 108 with carotid stenosis and 7 with VAS. Consensus regarding management was noted in 96.5% (111/115): 100% with ACS and VAS, 96.2% with SCS and 92.9% with ISS. Adherence to MDM management advice was 96.4% (107/111): 100% in ACS, ISS and VAS patients; 92% (46/50) in SCS patients. The median interval from index symptoms to revascularisation in 50-99% SCS patients was 12.5 days (IQR: 9-18.3 days; N = 26), with a median interval from MDM to revascularisation of 5.5 days (IQR: 1-7 days). Thirty patients underwent revascularisation. Two out of twenty-nine patients (6.9%) with either SCS or ISS had a peri-procedural ipsilateral ischaemic stroke, with no further strokes/deaths during 3-months follow-up. CONCLUSIONS: The high frequency of inter-specialty consensus regarding management and adherence to proposed treatment supports a collaborative/multidisciplinary model of care in patients with extracranial arterial stenoses. Service development should aim to shorten times between MDM discussion-intervention and optimise prevention of stroke/death.

Spatial and temporal non-stationarity in long-term population dynamics of over-wintering birds of North America.

Understanding population changes across long time scales and at fine spatiotemporal resolutions is important for confronting a broad suite of conservation challenges. However, this task is hampered by a lack of quality long-term census data for multiple species collected across large geographic regions. Here, we used century-long (1919-2018) data from the Audubon Christmas Bird Count (CBC) survey to assess population changes in over 300 avian species in North America and evaluate their temporal non-stationarity. To estimate population sizes across the entire century, we employed a Bayesian hierarchical model that accounts for species detection probabilities, variable sampling effort, and missing data. We evaluated population trends using generalized additive models (GAMs) and assessed temporal non-stationarity in the rate of population change by extracting the first derivatives from the fitted GAM functions. We then summarized the population dynamics across species, space, and time using a non-parametric clustering algorithm that categorized individual population trends into four distinct trend clusters. We found that species varied widely in their population trajectories, with over 90% of species showing a considerable degree of spatial and/or temporal non-stationarity, and many showing strong shifts in the direction and magnitude of population trends throughout the past century. Species were roughly equally distributed across the four clusters of population trajectories, although grassland, forest, and desert specialists more commonly showed declining trends. Interestingly, for many species, region-wide population trends often differed from those observed at individual sites, suggesting that conservation decisions need to be tailored to fine spatial scales. Together, our results highlight the importance of considering spatial and temporal non-stationarity when assessing long-term population changes. More generally, we demonstrate the promise of novel statistical techniques for improving the utility and extending the temporal scope of existing citizen science datasets.

Identification of Caveolae-Associated Protein 4 Autoantibodies as a Biomarker of Immune-Mediated Rippling Muscle Disease in Adults.

Importance: Immune-mediated rippling muscle disease (iRMD) is a rare myopathy characterized by wavelike muscle contractions (rippling) and percussion- or stretch-induced muscle mounding. A serological biomarker of this disease is lacking. Objective: To describe a novel autoantibody biomarker of iRMD and report associated clinicopathological characteristics. Design, Setting, and Participants: This retrospective cohort study evaluated archived sera from 10 adult patients at tertiary care centers at the Mayo Clinic, Rochester, Minnesota, and Brigham & Women's Hospital, Boston, Massachusetts, who were diagnosed with iRMD by neuromuscular specialists in 2000 and 2021, based on the presence of electrically silent percussion- or stretch-induced muscle rippling and percussion-induced rapid muscle contraction with or without muscle mounding and an autoimmune basis. Sera were evaluated for a common biomarker using phage immunoprecipitation sequencing. Myopathology consistent with iRMD was documented in most patients. The median (range) follow-up was 18 (1-30) months. Exposures: Diagnosis of iRMD. Main Outcomes and Measures: Detection of a common autoantibody in serum of patients sharing similar clinical and myopathological features. Results: Seven male individuals and 3 female individuals with iRMD were identified (median [range] age at onset, 60 [18-76] years). An IgG autoantibody specific for caveolae-associated protein 4 (cavin-4) was identified in serum of patients with iRMD using human proteome phage immunoprecipitation sequencing. Immunoassays using recombinant cavin-4 confirmed cavin-4 IgG seropositivity in 8 of 10 patients with iRMD. Results for healthy and disease-control individuals (n = 241, including myasthenia gravis and immune-mediated myopathies) were cavin-4 IgG seronegative. Six of the 8 individuals with cavin-4 IgG were male, and the median (range) age was 60 (18-76) years. Initial symptoms included rippling of lower limb muscles in 5 of 8 individuals or all limb muscles in 2 of 8 sparing bulbar muscles, fatigue in 9 of 10, mild proximal weakness in 3 of 8, and isolated myalgia in 1 of 8, followed by development of diffuse rippling. All patients had percussion-induced muscle rippling and half had percussion- or stretch-induced muscle mounding. Four of the 10 patients had proximal weakness. Plasma creatine kinase was elevated in all but 1 patient. Six of the 10 patients underwent malignancy screening; cancer was detected prospectively in only 1. Muscle biopsy was performed in 7 of the 8 patients with cavin-4 IgG; 6 of 6 specimens analyzed immunohistochemically revealed a mosaic pattern of sarcolemmal cavin-4 immunoreactivity. Three of 6 patients whose results were seropositive and who received immunotherapy had complete resolution of symptoms, 1 had mild improvement, and 2 had no change. Conclusions and Relevance: The findings indicate that cavin-4 IgG may be the first specific serological autoantibody biomarker identified in iRMD. Depletion of cavin-4 expression in muscle biopsies of patients with iRMD suggests the potential role of this autoantigen in disease pathogenesis.

A juxtamembrane basolateral targeting motif regulates signaling through a TGF-ß pathway receptor in Drosophila.

In polarized epithelial cells, receptor-ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. We explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-ß family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. We find the Dpp Type II receptor Punt, but not the Type I receptor Tkv, is enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. We also show that apicalized Punt does not produce an ectopic signal, indicating that the apical pool of Dpp is not a significant signaling source even when presented with Punt. Instead, we find that basolateral presentation of Punt is required for optimal signaling. Finally, we present evidence that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.

Platelet Biomarkers in Patients with Atherosclerotic Extracranial Carotid Artery Stenosis: A Systematic Review.

OBJECTIVE: The aim was to enhance understanding of the role of platelet biomarkers in the pathogenesis of vascular events and risk stratifying patients with asymptomatic or symptomatic atherosclerotic carotid stenosis. DATA SOURCES: Systematic review conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. REVIEW METHODS: A systematic review collated data from 1975 to 2020 on ex vivo platelet activation and platelet function/reactivity in patients with atherosclerotic carotid stenosis. RESULTS: Forty-three studies met the inclusion criteria; the majority included patients on antiplatelet therapy. Five studies showed increased platelet biomarkers in patients with ≥ 30% asymptomatic carotid stenosis (ACS) vs. controls, with one neutral study. Preliminary data from one study suggested that quantification of "coated platelets" in combination with stenosis severity may aid risk stratification in patients with ≥ 50% - 99% ACS. Platelets were excessively activated in patients with ≥ 30% symptomatic carotid stenosis (SCS) vs. controls (≥ 11 positive studies and one neutral study). Antiplatelet-High on Treatment Platelet Reactivity (HTPR), previously called "antiplatelet resistance", was observed in 23% - 57% of patients on aspirin, with clopidogrel-HTPR in 25% - 100% of patients with ≥ 50% - 99% ACS. Aspirin-HTPR was noted in 9.5% - 64% and clopidogrel-HTPR in 0 - 83% of patients with ≥ 50% SCS. However, the data do not currently support the use of ex vivo platelet function/reactivity testing to tailor antiplatelet therapy outside of a research setting. Platelets are excessively activated (n = 5), with increased platelet counts (n = 3) in recently symptomatic vs. asymptomatic patients, including those without micro-emboli on transcranial Doppler (TCD) monitoring (n = 2). Most available studies (n = 7) showed that platelets become more reactive or activated following carotid endarterectomy or stenting, either as an acute phase response to intervention or peri-procedural treatment. CONCLUSION: Platelets are excessively activated in patients with carotid stenosis vs. controls, in recently symptomatic vs. asymptomatic patients, and may become activated/hyper-reactive following carotid interventions despite commonly prescribed antiplatelet regimens. Further prospective multicentre studies are required to determine whether models combining clinical, neurovascular imaging, and platelet biomarker data can facilitate optimised antiplatelet therapy in individual patients with carotid stenosis.

Personalized tumor-specific DNA junctions to detect circulating tumor in patients with endometrial cancer.

INTRODUCTION: There are no reliable blood biomarkers for monitoring endometrial cancer patients in the current clinical practice. Circulating tumor DNA (ctDNA) is emerging as a promising non-invasive method to measure tumor burden, define prognosis and monitor disease status in many solid cancers. In this pilot study, we investigated if unique tumor-specific DNA junctions can be used to detect ctDNA levels in patients with endometrial cancer. METHODS: Chromosomal rearrangements in primary tumors of eleven patients with high-grade or advanced stage endometrial cancer were determined by whole-genome Mate-Pair sequencing. Identified unique tumor-specific junctions were evaluated in pre- and six-week post-surgery patient plasma using individualized quantitative polymerase chain reaction (qPCR) assays. The relationship between clinicopathological features and detection of ctDNA was investigated. RESULTS: CtDNA was detected in 60% (6/10) of cases pre-surgery and in 27% (3/11) post-surgery. The detection of ctDNA pre-surgery was consistent with clinical indicators of aggressive disease such as advanced stage (80% - 4/5), lymphatic spread of disease (100% - 3/3), serous histology (80% - 4/5), deep myometrial invasion (100% - 3/3), lympho-vascular space invasion (75% - 3/4). All patients in which ctDNA was detected post-surgically had type II endometrial cancer. DISCUSSION: This pilot study demonstrates the feasibility of using personalized tumor-specific junction panels for detecting ctDNA in the plasma of endometrial cancer patients. Larger studies and longer follow-up are needed to validate the potential association between pre-surgical ctDNA detection and the presence of cancers with aggressive pathologic tumor characteristics or advanced stage observed in this study.

Assessment of isochromosome 12p and 12p abnormalities in germ cell tumors using fluorescence in situ hybridization, single-nucleotide polymorphism arrays, and next-generation sequencing/mate-pair sequencing.

The identification of isochromosome 12p [i(12p)] and 12p gains have significant clinical utility in the diagnosis of germ cell tumors (GCTs). We have summarized the results of fluorescence in situ hybridization (FISH) assays to identify i(12p), performed in a Clinical Laboratory Improvement Amendments (CLIA)-validated setting for 536 specimens. In addition, the American Association for Cancer Research (AACR) Project GENIE registry and The Cancer Genome Atlas (TCGA) data sets were evaluated for chromosome 12p gains, and a limited number of cases were concurrently evaluated using FISH, single-nucleotide polymorphism (SNP) arrays and next-generation sequencing (NGS; including mate-pair sequencing). Specimens submitted for FISH testing were frequently from potential sites of metastases (male: 70.9% and female: 69.3%), and polysomy of chromosome 12 with or without concurrent i(12p) was a frequent finding, seen in 3% (16/536) and 35% (186/536) of cases, respectively. Our analysis suggests that 12p gains are likely to be present in approximately 73% of male GCT and in 32% of female GCT (AACR GENIE, n = 555). When comparing TCGA cases of testicular GCT (n = 149) to combined cases of sarcoma, colorectal, prostate, and urothelial carcinoma (n = 1754), 12p gains had a sensitivity of 77.2% and specificity of 97.3% for GCT. Some advantages of FISH over SNP arrays/NGS include relatively lower cost, rapid turnaround time, the ability to analyze biopsy material with a limited number of tumor cells (50 cells), and the ability to distinguish i(12p) from polysomy. The ability to spatially restrict the analysis to cells of interest is critical, as specimens submitted for testing often have low tumor purity. Disadvantages include false negative results due to an inability to detect segmental gains due to FISH probe design. With the availability of numerous testing modalities, including FISH, SNP arrays, and NGS-based assays, a nuanced understanding of the advantages and disadvantages of each methodology, as has been presented in this study, may inform appropriate testing strategies.

Theragnostic chromosomal rearrangements in treatment-naive pancreatic ductal adenocarcinomas obtained via endoscopic ultrasound.

A crucial mutational mechanism in malignancy is structural variation, in which chromosomal rearrangements alter gene functions that drive cancer progression. Herein, the presence and pattern of structural variations were investigated in twelve prospectively acquired treatment-naïve pancreatic cancers specimens obtained via endoscopic ultrasound (EUS). In many patients, this diagnostic biopsy procedure and specimen is the only opportunity to identify somatic clinically relevant actionable alterations that may impact their care and outcome. Specialized mate pair sequencing (MPseq) provided genome-wide structural variance analysis (SVA) with a view to identifying prognostic markers and possible therapeutic targets. MPseq was successfully performed on all specimens, identifying highly rearranged genomes with complete SVA on all specimens with > 20% tumour content. SVA identified chimeric fusion proteins and potentially immunogenic readthrough transcripts, change of function truncations, gains and losses of key genes linked to tumour progression. Complex localized rearrangements, termed chromoanagenesis, with broad pattern heterogeneity were observed in 10 (83%) specimens, impacting multiple genes with diverse cellular functions that could influence theragnostic evaluation and responsiveness to immunotherapy regimens. This study indicates that genome-wide MPseq can be successfully performed on very limited clinically EUS obtained specimens for chromosomal rearrangement detection and potential theragnostic targets.

Heterogeneity of PD-L1 expression between invasive and lepidic components of lung adenocarcinomas.

The dynamics of PD-L1 expression are poorly understood over the development of lung adenocarcinomas from pre-invasive lesions to fully invasive carcinomas. Given the importance of PD-L1 expression for the selection of patients to receive immunotherapy in the metastatic setting and possibly in the neoadjuvant setting, we sought to evaluate the agreement of PD-L1 expression in invasive and lepidic components of resected tumor specimens. We stained 86 adenocarcinomas for PD-L1 using the SP263 clone. We assessed the agreement of PD-L1 expression by tumor cells and immune cells between lepidic and invasive components. When both lepidic and invasive components were considered, PD-L1 positive immune cells and tumor cells were observed in 50 (58.1%) and 18 (20.9%) samples, respectively, using a ≥ 1% PD-L1 expression cutoff. Using a ≥ 1% cutoff for PD-L1 expression, positively stained tumor cells were observed in 11 (13%) lepidic and 15 (17%) invasive patterns, with agreement in 76 (88%) specimens and disagreement in 10 (12%) specimens (ĸ = 0.549). At ≥ 1% PD-L1 expression cutoff, PD-L1 positive immune cells were observed in 31 (35%) lepidic and 32 (37%) invasive patterns with an agreement of PD-L1 expression in 49 (57%) specimens and disagreement in 37 (43%) specimens (ĸ = 0.073). In our study of early stage adenocarcinomas of the lung, there was poor agreement in PD-L1 expression between paired invasive and lepidic components of tumors. Our data suggest that the non-invasive tumor components may not be as immunostimulatory as the invasive components, resulting in less adaptive expression of PD-L1.

von Willebrand Factor Antigen, von Willebrand Factor Propeptide, and ADAMTS13 in Carotid Stenosis and Their Relationship with Cerebral Microemboli.

BACKGROUND: The relationship between von Willebrand factor antigen (VWF:Ag), VWF propeptide (VWFpp), VWFpp/VWF:Ag ratio, ADAMTS13 activity, and microembolic signal (MES) status in carotid stenosis is unknown. METHODS: This prospective, multicenter study simultaneously assessed plasma VWF:Ag levels, VWFpp levels and ADAMTS13 activity, and their relationship with MES in asymptomatic versus symptomatic moderate-to-severe (≥50-99%) carotid stenosis patients. One-hour transcranial Doppler ultrasound of the middle cerebral arteries classified patients as MES+ve or MES-ve. RESULTS: Data from 34 asymptomatic patients were compared with 43 symptomatic patients in the "early phase" (≤4 weeks) and 37 patients in the "late phase" (≥3 months) after transient ischemic attack (TIA)/ischemic stroke. VWF:Ag levels were higher (p = 0.049) and VWFpp/VWF:Ag ratios lower (p = 0.006) in early symptomatic than in asymptomatic patients overall, and in early symptomatic versus asymptomatic MES-ve subgroups (p ≤0.02). There were no intergroup differences in VWFpp expression or ADAMTS13 activity (p ≥0.05). VWF:Ag levels and ADAMTS13 activity decreased (p ≤ 0.048) and VWFpp/VWF:Ag ratios increased (p = 0.03) in symptomatic patients followed up from the early to late phases after TIA/stroke. Although there were no differences in the proportions of symptomatic and asymptomatic patients with blood group O, a combined analysis of early symptomatic and asymptomatic patients revealed lower median VWF:Ag levels in patients with blood group O versus those without blood group O (9.59 vs. 12.32 µg/mL, p = 0.035). DISCUSSION: VWF:Ag expression, a marker of endothelial ± platelet activation, is enhanced in recently symptomatic versus asymptomatic carotid stenosis patients, including in MES-ve patients, and decreases with ADAMTS13 activity over time following atherosclerotic TIA/ischemic stroke.

Novel strategy for manufacturing autologous dendritic cell/allogeneic tumor lysate vaccines for glioblastoma.

BACKGROUND: Glioblastoma, the most common primary malignant brain tumor, is nearly universally fatal by 5 years. Dendritic cell vaccines are promising but often limited clinically by antigen choice, dendritic cell potency, and/or manufacturing yield. We optimized vaccine manufacture, generating potent mature autologous dendritic cells pulsed with allogeneic glioblastoma lysates. METHODS: Platelet lysate-based supplement was used to establish human glioblastoma cell lines. Phenotype and genotype were assessed. An improved culture technique to generate mature dendritic cells from glioblastoma patients' monocytes was developed. The ability of T cells stimulated with autologous dendritic cells pulsed with allogeneic glioblastoma cell lysate to kill HLA-A2-matched glioblastoma cells was assessed. RESULTS: Glioblastoma cell lines established with platelet lysate supplement grew faster and expressed more stem-like markers than lines grown in neural stem cell media or in the presence of serum. They expressed a variety of glioma-associated antigens and had genomic abnormalities characteristic of glioblastoma stable up to 15 doublings. Unlike standard culture techniques, our optimized technique produced high levels of mature dendritic cells from glioblastoma patients' monocytes. Autologous T cells stimulated with mature dendritic cells pulsed with allogeneic glioblastoma cell line lysate briskly killed HLA-A2-matched glioblastoma cells. CONCLUSIONS: Our glioblastoma culture method provides a renewable source for a broad spectrum glioblastoma neoantigens while our dendritic cell culture technique results in more mature dendritic cells in glioblastoma patients than standard techniques. This broadly applicable strategy could be easily integrated into patient care.

Optimizing clinical cytology touch preparations for next generation sequencing.

Intraoperative diagnosis is routinely performed on cytology touch preparations (TPs) from core needle biopsies (CNBs). Current interest promotes their utility as an important source of patient tissue for clinical genomic testing. Herein we present whole genome structural variant analysis (SVA) from mate-pair sequencing (MPseq) and whole exome sequencing (WES) mutation calling in DNA directly whole genome amplified (WGA) from TPs. Chromosomal copy changes and somatic DNA junction detection from MPseq of TPs were highly consistent with associated CNBs and bulk resected tissues in all cases. While increased frequency coverage noise from limitations of amplification of limited sample input was significant, this was effectively compensated by natural tumor enrichment during the TP process, which also enhanced variant detection and loss of heterozygosity evaluations from WES. This novel TP methodology enables expanded utility of frequently limited CNB for both clinical and research genomic testing.

Platelet function/reactivity testing and prediction of risk of recurrent vascular events and outcomes after TIA or ischaemic stroke: systematic review and meta-analysis.

BACKGROUND: The prevalence of ex vivo 'high on-treatment platelet reactivity (HTPR)' and its relationship with recurrent vascular events/outcomes in patients with ischaemic cerebrovascular disease (CVD) is unclear. METHODS: A systematic review and meta-analysis was performed in accordance with the PRISMA statement. MEDLINE, EMBASE and Cochrane Library were searched for completed manuscripts until May 2019 on TIA/ischaemic stroke patients, ≥ 18 years, treated with commonly-prescribed antiplatelet therapy, who had platelet function/reactivity testing and prospective follow-up data on recurrent stroke/TIA, myocardial infarction, vascular death or other cerebrovascular outcomes. Data were pooled using random-effects meta-analysis. Primary outcome was the composite risk of recurrent stroke/TIA, myocardial infarction or vascular death. Secondary outcomes were recurrent stroke/TIA, severe stroke (NIHSS > 16) or disability/impairment (modified Rankin scale ≥ 3) during follow-up. RESULTS: Antiplatelet-HTPR prevalence was 3-65% with aspirin, 8-56% with clopidogrel and 1.8-35% with aspirin-clopidogrel therapy. Twenty studies (4989 patients) were included in our meta-analysis. There was a higher risk of the composite primary outcome (OR 2.93, 95% CI 1.90-4.51) and recurrent ischaemic stroke/TIA (OR 2.43, 95% CI 1.51-3.91) in patients with vs. those without 'antiplatelet-HTPR' on any antiplatelet regimen. These risks were also more than twofold higher in patients with vs. those without 'aspirin-HTPR' and 'dual antiplatelet-HTPR', respectively. Clopidogrel-HTPR status did not significantly predict outcomes, but the number of eligible studies was small. The risk of severe stroke was higher in those with vs. without antiplatelet-HTPR (OR 2.65, 95% CI 1.00-7.01). DISCUSSION: Antiplatelet-HTPR may predict risks of recurrent vascular events/outcomes in CVD patients. Given the heterogeneity between studies, further prospective, multi-centre studies are warranted.