Abstract 172: Spatial profiling of stage III melanoma microenvironment reveals tertiary lymphoid structures as markers of long-term outcomes

Abstract The recent neoadjuvant anti-PD-1 and anti-CTLA4 therapy, such as the PRADO trial (NCT02977052), has shown a high pathological response rate in many melanoma patients. In resectable macroscopic stage III melanoma, PRADO achieved a 72% pathological response rate. However, recurrence remains a challenge, and the biological mechanisms behind durable responses are unclear. Tertiary lymphoid structures (TLSs), immune sites that enhance antigen presentation, cytokine signaling, and antibody release, are present in several cancers, including melanoma. Their role in the efficacy of neoadjuvant therapy, however, is not fully understood. This study quantifies and characterizes TLS formation in melanoma patients treated in the neoadjuvant setting. Using the most recent data with a median follow-up of 55 months, 17 patients involved in this study were grouped into non-recurrence (n=11) and recurrence (n=6) groups. We performed 41-plex PhenoCycler Fusion imaging on 26 formalin-fixed, paraffin-embedded patient index lymph node samples from the PRADO cohort, including 17 baseline samples and nine Week 6 post-treatment samples. Among these, eight patients had paired baseline and Week 6 samples. Bioinformatic analysis was used to quantify cells, characterize TLS molecular features, and analyze interactions within the tumor microenvironment (TME) associated with recurrence-free survival (RFS). The presence of TLSs—consisting of B cells, CD4+ and CD8+ T cells, myeloid cells, and vessels—in baseline samples was associated with longer RFS (p = 0.0498). At Week 6, TLSs were present in 4 out of 5 non-recurrence patient samples and 1 out of 4 recurrence patient samples. Moreover, the TLSs in the recurrence patient were relatively less mature than those in non-recurrence patients. These findings indicate mature TLS presence at 6 weeks post-treatment may be associated with durable outcomes, and quantification of TLSs is underway. Notably, TLSs were observed in some matched baseline and Week 6 samples in non-recurrence patients. The structural organization of immune cells at baseline compared to Week 6 demonstrates patterns of increased immune activation, where the early TLSs evolved into more mature-like structures with increased immune cell diversity. Molecular profiling of these immune structures is underway to characterize the TME changes associated with neoadjuvant treatment and to identify features of durable immune protection in patients with prolonged RFS. This 41-plex spatial imaging study highlights the immune-responsive features of TLSs in melanoma, shedding light on their mechanisms of promoting durable responses to neoadjuvant therapy and providing a foundation for future translational research. Citation Format: Angel Guan, Xinyu Bai, Nurudeen A. Adegoke, Shila Ghazanfar, Nigel G. Maher, Alexander M. Menzies, Ines Pires Silva, Richard A. Scolyer, Georgina V. Long, James S. Wilmott, Camelia Quek. Spatial profiling of stage III melanoma microenvironment reveals tertiary lymphoid structures as markers of long-term outcomes. [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 172.