Impact of tertiary lymphoid structures in response to immune checkpoint blockade in patients with head and neck squamous cell carcinoma.

Abstract

e18021 Background: Immune checkpoint blockade (ICB) is the standard of care for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), yetefficacy remains low. Current assessment for tumor response relies on a single proportional biomarker expressed in immune and tumor cells (Combined Positive Score, CPS) without differentiation by cell type, potentially explaining its limited predictive value. Tertiary Lymphoid Structures (TLS) have shown a stronger association with improved ICB response than PD-L1. However, their exact composition, size, and spatial biology in HNSCC remain understudied, and there is a need for a deeper examination of TLS biology in HNSCC and its link with clinical response to ICB. Methods: Pre-ICB tumor tissue sections (formalin-fixed, paraffin-embedded) were obtained from 10 responders (complete response, partial response, or stable disease) and 10 non-responders to ICB (progressive disease). A custom multi-immunofluorescence (mIF) staining assay was designed, optimized, and applied to characterize tumor cells (pan-cytokeratin), T cells (CD4, CD8), B cells (CD19, CD20), myeloid cells (CD16, CD56, CD163), dendritic cells (LAMP3), fibroblast (alpha Smooth Muscle Actin), proliferative status (Ki67) and immunoregulatory molecules (PD1). Following imaging and tissue classification using machine learning techniques, spatial profiling was performed, including proximity, density, and infiltration analysis. Response status was assessed via RECIST 1.1. Spatial metrics were compared among groups based on response to ICB. An unpaired t-test was used to compare mean densities. Multivariable logistic regression was used to measure the effect of TLS size on achieving response to ICB (CR, PR or SD). Results: A higher density of dendritic cells (LAMP3) was found in responders compared to non-responders to ICB (p=0.0415). A positive correlation was observed between mIF and pathologist identification of TLS (r2=0.51; p=0.0039). TLS trended toward more prevalent in responders to ICB (p=0.0906). There was no association of high CPS (>20) with improved PFS (HR=0.91; p-value=0.8538 C.I.= 0.3538-2.369). Multivariate logistic regression analysis showed that total TLS area > 5.8 (mm²) (mean TLS size in this cohort) in pretreatment tissue biopsies was associated with 2.4 increased odds of achieving objective response (OR= 2.4, p=0.05, 95% C.I.=1.2-275.1) independent of CPS score. Conclusions: Tertiary Lymphoid Structures play a critical role in the immune response to HNSCC, are quantifiable through histopathologic assessment by a pathologist, and may potentially outperform CPS as a predictor of ICB response.