OC39 - Exploring tumor-associated macrophages (TAM) polarisation in adrenocortical adenomas

Background/Introduction: Macrophages polarisation toward M2 has been described in normal adrenal gland (NAG) and adrenocortical carcinoma (ACA), however their role in ACA remains poorly characterized.

Purpose: To characterize tumor-associated macrophages (TAM) in ACA and investigate associations with clinicopathological characteristics.

Methods: We performed immunohistochemistry for CD163 and CD206 (M2-specific markers), CD68 (pan-macrophage marker), and CD16 (immune-infiltrate marker) on 102 ACA and 16 adjacent-NAG. Immunostaining was quantified using QuPath-0.5.1 pixel classification and expressed as mean percentage of positive pixels. Pathogenic variants in CTNNB1, PRKACA, and GNAS were assessed by Sanger sequencing in all matched-ACA frozen tissues. TAM associations with clinical, hormonal parameters and circulating monocytes were analyzed. Myeloid profile was further explored in single-nucleus RNA sequencing (snRNA-Seq, DOI:10.1002/ctm2.1798) using Seurat.

Results: In NAG, median CD16 (8.23%) and CD206 (6.58%) expression were higher than CD68 (5.59%; p=0.006 and p=0.02), while CD163 expression was similar. In ACA, CD16 (6.80%) and CD163 (6.86%) were more expressed than CD206 and CD68 (1.60% and 1.69%, all p <0.001), with CD68 positively correlating with CD163 (ρ=0.41) and CD16 (ρ=0.34) (p <0.001).

Matched ACA-NAG comparisons revealed in ACA reduced CD206 expression at both IHC and snRNA-Seq levels (p <0.001). Consistently, CD163/CD206 ratio was elevated in ACA (3.11 vs 0.84, p <0.001) indicating a shift toward CD163 polarization. In ACA, CD16 expression correlated with circulating monocytes (ρ=0.29, p=0.02), but not with other markers. Cortisol excess was associated with lower CD68 and CD16, and higher CD163/CD68 and CD163/CD206 ratios (all p <0.05). In women, CD163/CD206 positively correlated with cortisol after 1 mg-DST (ρ=0.27, p=0.04), and CD206/CD16 was lower in cortisol-producing ACA (CPA) (p=0.01), suggesting sex-specific immune modulation. ACA with PRKACA showed lower CD206 and CD16 (p <0.01), and higher CD163/CD206 ratio (17.42 vs 3.21, p=0.002) compared to those without. These associations remained significant within CPA and were confirmed by multivariate logistic regression, supporting a PRKACA-driven effect.

Conclusion(s): TAM in ACA exhibit a distinctive polarization pattern characterized by an increased CD163/CD206 ratio. This immunophenotype, which could be modulated by cortisol levels, sex, and PRKACA, highlights the intricate heterogeneity of TAM in ACA and underscores the importance of deeper characterization to better define their clinical and biological impact.