Abstract 4734: Alteration of tumor immune environment in metastatic and non-metastatic papillary thyroid microcarcinoma

Abstract Introduction: Papillary thyroid microcarcinomas (PTMC), defined as tumors ≤1 cm in size, are mostly indolent and warrant conservative management. However, up to 10% of PTMC present with clinically significant lymph node metastases or very rarely with distant metastases (M1). Deciphering molecular signatures of metastases-prone PTMCs may help strategize management options. Therefore, the goal of this study was to analyze the transcriptome profiles and tumor microenvironment in metastatic and non-metastatic PTMC. Methods: Patients diagnosed with indolent non-metastatic (N0/Nx) PTMCs and with tumors metastatic to the lateral neck lymph nodes or distant sites (N1b/M1) were selected by reviewing medical records and pathology slides. RNA sequencing was done on 85 tumor samples and adjacent normal tissues. An R package pathfinder was used to do pathway enrichment analysis utilizing active subnetworks. A subset of samples (n=9) was subjected to multiplex immunofluorescence (MxIF) staining using CD8a and CD20 antibodies. Image analysis of the whole tissue, tumor, and three 400 µm regions around the tumor to count cytotoxic T cells, B-cells, and macrophages was done in a single-blinded manner using HALO (Indica Labs). Differential spatial expression was tested using t-test for normally distributed data and Mann-Whitney test for skewed data, with two-sided p≤0.05 rendered statistically significant. Results: The study cohort consisted of 17 patients from N0/Nx group and 19 presenting with N1b/M1 disease. No difference in the clinical characteristics, viz. age (39y vs 46y, p=0.64), gender (female 82.4% vs. 94.7%, p=0.24), median tumor size (0.6cm vs 0.8cm, p=0.27) was found between the groups. There was a significant downregulation of pathways involved in the regulation of B cell activation (FDR-adjusted p-value<0.001), activation of immune response (p<0.001), antigen receptor-mediated signaling pathway (p<0.001), humoral immune response (p<0.001), B cell receptor signaling pathway (p<0.001), pathways activating and regulating cell surface receptor and signal transduction (p<0.001) in primary tumors derived from the N1b/M1 compared to N0/Nx group. However, MxIF revealed no difference in B cell (CD20+/CD8a) infiltration in the primary tumors (p=0.43) nor in the tumor rims (p=0.92) between the groups. In contrast, there was a significantly higher number of cytotoxic T cells (CD8a+/CD20-) in the rims of the primary tumors from N1b/M1 group compared to N0/Nx (p=0.007). Conclusion: Metastasis-prone PTMC is characterized by a different immune landscape as compared with the classic indolent variant, with increased cytotoxic T cell infiltration in tumor edges and decreased activation of infiltrating B-cells. GeoMx DSP experiments are undergoing to identify immune cells and their crosstalk with the cancer cells that might be involved in transducing metastatic potential for a subset of PTMCs. Citation Format: Rhitajit Sarkar, Shilpa Thakur, Gus Fridell, Noemi Kedei, Maria Hernandez, Sonam Kumari, Maria Merino, Joanna Klubo-Gwiezdzinska. Alteration of tumor immune environment in metastatic and non-metastatic papillary thyroid microcarcinoma [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 4734.