Work

1190 Spatial and phenotypic characterization of tertiary lymphoid structures and immune organization in cutaneous squamous cell carcinoma using multiplex immunofluorescence

Year: 2026

Type: conference-abstract

Abstract: Background Tertiary lymphoid structures (TLS) are increasingly recognized as important components of the tumor immune microenvironment, but their biological relevance may depend on their organization, cellular composition, and maturation state. We used multiplex immunofluorescence (mIF) to characterize TLS and broader spatial patterns of immune organization in post-treatment cutaneous squamous cell carcinoma (cSCC).Methods Post-treatment tumor specimens from 10 patients enrolled in a neoadjuvant study of the PD-1 inhibitor pembrolizumab in PD-1–naïve cSCC (ClinicalTrials.gov NCT04808999) were profiled using a 40-marker mIF panel. Marker performance and technical outliers were evaluated through a standardized quality-control process. Image-processing workflows incorporated artifact detection, cell segmentation, phenotyping, and tissue-region annotation. Immune phenotypes were evaluated across tumor, tumor-adjacent, and stromal compartments. TLS candidates were identified through spatial clustering of B cells and classified into three maturity categories according to their cellular composition and organization. Separately, an outcome-agnostic spatial-program analysis was used to identify recurring multicellular tissue states based on local phenotype composition and cell–cell relationships. The distributions of TLS and spatial programs were then explored across tissue compartments and response groups.Results mIF revealed substantial interpatient and intratumoral heterogeneity in immune composition and spatial organization following neoadjuvant pembrolizumab. Exploratory differences between responders and nonresponders were observed in the abundance and regional distribution of selected immune phenotypes. TLS candidates were identified across the cohort and stratified by maturity. Representative responder specimens contained organized CD20 + B-cell aggregates with CD21+ follicular dendritic-cell networks and PNAd-associated vascular features consistent with more mature TLS, whereas candidate aggregates in nonresponder specimens appeared less organized. Outcome-agnostic spatial analysis additionally identified recurring programs of multicellular organization that varied across specimens and tissue compartments. These programs distinguished tissue regions according to cellular context and interactions, revealing organizational heterogeneity not evident from immune-cell abundance summaries alone.Conclusions High-plex spatial profiling enables TLS to be characterized as a structured multicellular phenotype while supporting data-driven identification of broader spatial tissue states. In this small post-treatment cohort, exploratory findings suggest that TLS maturation and immune organization differ across response groups and may provide complementary information beyond conventional immune-cell abundance measurements. Larger cohorts with paired pre- and post-treatment specimens will be required to determine whether these spatial features represent baseline correlates of response, treatment-induced remodeling, or both.Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.1190