Abstract 4879: Delineating lymphocyte aggregates from tertiary lymphoid structures using spatial transcriptomics

Abstract Tertiary Lymphoid Structures (TLSs) are ectopic lymphoid structures that form in chronically inflamed non-lymphoid tissue. The presence of TLSs in tumors is largely associated with favorable outcomes among patients and also exhibit positive associations with response to immune checkpoint blockade. Although mainly comprised of lymphocytes (B cells and T cells), they are considered a separate entity from lymphocyte aggregates within tissue. Lymphocyte aggregates are fairly routine to identify in H&E stained tissue sections, but delineating TLSs from simple lymphocyte aggregates is subjective and not clearly defined. Additionally, the definition of TLSs is only recently being elucidated, so a clear molecular definition of both TLSs and lymphocyte aggregates is needed. To address this problem, we leveraged open-source spatial transcriptomics (ST) data from 17 samples across 7 cancer types. High resolution H&E images were manually evaluated for lymphocyte aggregates by identifying dense pockets of lymphocytic-appearing nuclei. Suspected aggregates were detected in 10 of 17 samples. We then applied a visium spot-level signature scoring method for 4 well published TLS signatures. Of the spots identified, 62% had enhanced signature expression for at least 2 of the signatures, although most of the incorrect annotations came from the lone gastric cancer sample. Differential expression between a TLS-sig- suspected lymphoid aggregate and a TLS-sig+ region not identified by H&E enriched primarily for B cell identity genes, suggesting B-cell-rich TLSs may not always appear as lymphoid aggregates in histology. [AG1] Strikingly, ST revealed strongly positive enhancements in the TLS signatures, where no annotations were made, and no obvious lymphoid aggregation was present, suggesting manual annotation from H&E is inadequate for identifying TLSs. Together, these results demonstrate that literature derived TLS signatures can correctly identify lymphoid aggregates as TLSs and that cellular and molecular differences between standard lymphoid aggregates and TLSs may be smaller than is currently accepted. Citation Format: Jordan Krull, Anjali Byappanahalli, Yi Jiang, Andrew Gunderson, Qin Ma. Delineating lymphocyte aggregates from tertiary lymphoid structures using spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4879.