Abstract 3303: Standardizing peripheral blood mononuclear cells processing for <i>ex vivo</i> evaluations to predict response to immunotherapy

Abstract In many cancers, including head and neck squamous cell carcinoma (HNSCC), expression of programmed death ligand-1 (PD-L1) is upregulated, which inhibits the anti-tumorigenic immune response of cytotoxic T-cells. Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis has greatly enhanced survival rates in these patients, but many progress and/or relapse due to other immunosuppressive factors. Therefore, predicting patient response to current ICIs and identifying new biomarkers is imperative to advance tailored therapeutics. The current "gold standard" for diagnosing and assessing treatment response and tumor progression is tumor biopsy, however, it is not always feasible, safe, or sufficient for additional molecular testing. While research into alternative methods such as circulating tumor cells (CTCs) are ongoing, these methods are expensive and lack standardization. As a result, our laboratory is investigating a liquid biopsy approach utilizing peripheral blood mononuclear cells (PBMCs) as a surrogate for tumor biopsy, as longitudinal peripheral blood sampling of patients is safe and more readily available in patients before, during, and following treatment. Previous work has demonstrated that preanalytical variables affect PBMC viability and suitability for functional assays. The purpose of our work so far has been to elucidate the optimal collection tube condition and timing of blood processing to standardize the collection and mitigate pre-analytical bias of PBMC processing for downstream critical functional analyses. Peripheral blood samples from eligible HNC patients were collected in EDTA or sodium heparin (SH) tubes and either processed for isolation of PBMCs immediately or at delayed timepoints (8- and 24-hours post-collection) at room temperature (22-30°C) to account for same-day and overnight processing delays. Additionally, the delayed (8 and 24 hour) timepoint samples were diluted 1:1 with sterile PBS immediately following collection and compared to matched undiluted specimens to assess the effects of granulocyte contamination on PBMC viability and functionality following cryopreservation. Functional tests included cytotoxicity assays with patient-derived CD8+ T-cells or NK cells that were co-cultured with HN5 target tumor cells at a 1:5 target to effector cell ratio and stained with 7AAD Viability Solution for flow cytometry analysis. NK or T cell-killed HNC cells are calculated by dividing the percent dead (7AAD-positive) in co-culture and the basal cell death. Preliminary findings suggest that dilution enhances cell recovery but may slightly reduce NK cell cytotoxicity, while the blood collection tube type has no significant influence on recovery or cytotoxicity. Although this research is focused on one solid tumor class, our rigorous approach and findings could be readily expanded to other solid tumors. Citation Format: Katelyn Jansen, Farah Sagin, Claire Girten, Joanna Khoury, Tyler Hendrickson, Kelsey Dillehay McKillip, Dalia El-Gamal, Trisha Wise-Draper. Standardizing peripheral blood mononuclear cells processing for ex vivo evaluations to predict response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3303.