Advancing a Prognostic and Monitoring Biomarker of Neutrophil-driven Secondary Brain Injury in Spontaneous Intracerebral Hemorrhage for Neurotherapeutics Development

Summary: Spontaneous intracerebral hemorrhage (sICH) is the stroke subtype with the highest mortality and disability, and remains without effective pharmacotherapy to improve outcomes. To advance sICH neurotherapeutic development, peripherally accessible biomarkers are needed that are 1] prognostic for progressive non-resolving neutrophil-driven secondary brain injury (ND-2°BI), and/or 2] able to monitor increase or decrease of ND-2°BI in sICH patients. However, to date, no neutrophil biomarker has been validated. Addressing this knowledge gap, we have identified a “rogue” CD11b+ neutrophil subset, expressing the pro-survival dual endothelin-1/signal peptide receptor, DEspR+CD11b+ neutrophil or DEspR+[N] subset, whose levels correlate with perihematomal edema (PHE) volume and 90d modified Rankin Scale (mRS) score. Data validate biomarker ‘context of use’ that DEspR+[N] levels are a potential prognostic biomarker in sICH to better stratify patients in clinical trials, and/or a potential monitoring biomarker for tracking ongoing ND-2°BI. In response to PAR-22-089, we propose testing “Fit-for-purpose” with FDA-required biomarker milestones in a primarily underserved and diverse patient cohort of hypertensive, supratentorial deep sICH, with no anticoagulant therapy or immunological deficiencies. R61- Aim1. To define and validate a standardized ‘universal protocol’ to detect DEspR+[N] levels as potential biomarker in sICH patient samples by flow cytometry, we will determine the best sampling procedure and flow cytometry detection requirements, and validate flow cytometry results with quantitative immunofluorescence- cytology results. The ‘universal protocol’ should attain 1] <10% intra-assay variation, and 2] <10% false positive and false negative rates. R61-Aim 2. To validate biomarker performance as a systemic indicator of ND-2BI, we will A] test if the DEspR+[N] subset comprises the majority of pro-injury NET-forming [Ns], anti-resolution PDL1+ neutrophils, and PDL1+ NET-forming neutrophils in sICH patient whole blood samples, and B] test association with cell-injury markers: S100A8/A9 (BBB endothelial injury), GFAP (BBB astrocyte endfeet injury), Nfl (white matter injury). R61-Aim 3. To ascertain robustness of DEspR+[N] as a biomarker of ND-2BI, we will identify the optimal unit measure by comparing 3 units for correlation with peak PHE volume in sICH patients: a] actual DEspR+[N] levels, b] DEspR+[N]: summation delta increase/decrease in DEspR+[N], and c] DEspR+[N]/IPH- vol or ICH score ratio: DEspR+[N] level normalized to d1 IPH vol or ICH score. Significant largest effect size correlation and robust high/low threshold levels will define the best unit. R33-Aim 1. To obtain proof-of-concept (POC) as a prognostic biomarker in sICH patients (n=72), we will assess the high/low thresholds for 30d mortality of DEspR+[N] levels for sensitivity, specificity and receiver operating curve area under the curve. R33-Aim 2. To obtain POC as a monitoring biomarker, we will determine whether best unit measure of DEspR+[N] levels 1] predicts 90d mRS, and 2] mediates worse 90d mRS in high ICH score patients. Impact: Completion will identify a universal protocol for measuring, and qualify DEspR+[N] levels for Analytics Validation as ND-2°BI biomarker.