Atherton, Chathu L.

Abstract Circumscribed meningeal melanocytic neoplasms (CMMNs) represent a spectrum of rare central nervous system (CNS) tumors. Knowledge of their clinical behavior and molecular correlates remains limited. In this single‐institution retrospective study, we analyzed 31 patients (14 male, 17 female; median age, 59 years) diagnosed with CMMN between 2004 and 2025. In this patient cohort, based on the current World Health Organization Central Nervous System Tumor Classification (WHO CNS5) criteria, the majority of cases (18, 58.1%) fell into the intermediate‐grade melanocytic tumor (IMT) category based on histology, with either increased mitotic activity, CNS invasion, or both; 7 (22.6%) were melanocytomas, 5 (16.1%) melanomas, and 1 (3.2%) indeterminate case because of limited tissue. By next‐generation sequencing, mutations were identified in GNAQ/GNA11 in 21/24 cases, BAP1 in 5/23 (2 IMT, 3 melanoma), EIF1AX in 9/21 (1 melanocytoma, 8 IMT), and SF3B1 in 3/23 (2 IMT, 1 melanoma). Primary sites were spinal (20), posterior fossa (6), and supratentorial (5). During follow‐up, progression was observed in 5/6 melanocytomas, 9/13 IMTs, and 4/5 melanomas, suggesting a high frequency of progression across all groups. Two‐year progression‐free survival was 83.3% (95% confidence interval [CI], 53.5%–100.0%) for melanocytoma, 55.9% (95% CI, 26.7%–85.2%) for IMT, and 25.0% (95% CI, 0.0%–67.4%) for melanoma. Five‐year overall survival (OS) was 80.0% (95% CI, 44.9%–100.0%) for melanocytoma, 65.3% (95% CI, 37.0%–93.6%) for IMT, and 0.0% for melanoma. OS was significantly worse in melanoma compared with melanocytoma ( p = 0.002). BAP1 mutation correlated with worse OS (hazard ratio 8.73, p = 0.006). Upfront maximal safe resection appeared to be associated with improved outcomes in selected patients, whereas radiotherapy and systemic therapy did not demonstrate clear additional benefit in this cohort. In summary, our study shows that all CMMNs, irrespective of grade, have a high propensity to recur and melanoma histology and BAP1 mutation are associated with significantly worse OS.

Abstract The sentinel lymph node (SLN) is often the first site of melanoma spread and a critical immune checkpoint that determines local tumor dissemination to systemic metastasis. Although lymph nodes are central to antitumor immunity, the mechanisms that render them permissive to metastasis remain poorly understood. This study used spatial imaging to characterize immune and stromal phenotypic alteration in tumor-negative SLNs (SLN-) and tumor-positive SLNs (SLN+) from patients with cutaneous melanoma, compared to non-melanoma-associated normal lymph nodes (NLN). We performed multiplex immunofluorescence (MxIF) analysis of formalin-fixed, paraffin-embedded lymph node tissues from patients with cutaneous melanoma and control subjects, including two subjects per group (NLN, SLN-, and SLN+). Furthermore, quantitative single-cell segmentation and classification using QuPath generated median fluorescence intensities for immune, stromal, and tumor markers across LN, SLN- and SLN+ regions of interest. In addition to the initial analysis, we employed spatial neighborhood analysis to characterize markers expression profiles surrounding target immune population. This approach quantified local microenvironment based on marker normalized intensity-based co-expression pattern and stromal remodeling signatures. We grouped these markers based on the functional panels representing T-cell activation and exhaustion, myeloid polarization, tumor interaction, stromal remodeling, and apoptosis pathways providing insights into how prometastatic niche evolve within SLN. Normalized data, via centered log ratio (CLR) transformation and Gaussian Mixture Model (GMM) gating was analyzed using principal component analysis, UMAP/tSNE embedding, and hierarchical clustering in in python. Progressive immune remodeling was observed from normal NLN to SLN+, including expansion of regulatory T cells, enrichment of exhausted T cells as well as myeloid-derived suppressor cells (MDSC), and transition toward an M2-like macrophage phenotype. The spatial data from SLN- samples showed an early sign of immune suppression, with reduced cytotoxic capacity. These results show that SLN gets features of peripheral immune tolerance for melanoma early, prior to pathological dissection of metastatic spread supporting the existence of a pre-metastatic niche that facilitates lymphatic colonization of tumor cells. This work provides insights into early immune escape and may inform strategies for immunotherapy and early intervention in melanoma. Citation Format: Shankar Suman, Wendy K. Nevala, Noah A. Stueven, Raymond M. Moore, Chathu L. Atherton, Jeffrey E. Johnson, Ray Guo, James W. Jakub, Svetomir N. Markovic. Spatial remodeling of immune and stromal microenvironments in sentinel lymph nodes during melanoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6193.

Abstract Background: The tumor microenvironment (TME) plays a pivotal role in ovarian cancer (OC) progression and prognosis, while also contributing to biological differences between primary and metastatic tumors. This study examines OC TMEs to uncover associations with clinical prognosis and tumor biology by analyzing paired primary and metastatic tumors from the same patients. Methods: This study focused on 57 stage-IIIC/IV OC patients treated at the Mayo Clinic, all of whom underwent surgical debulking and adjuvant therapies. All patients provided informed consent, and the study received IRB approval. A tissue microarray (TMA) was specifically designed and constructed, containing paired primary and metastatic tissue cores from the same patients. 147 tissue cores from the constructed TMA were profiled using a 35-antibody panel, with 17 antibodies for cell phenotyping and 18 for functional state characterization. Profiling was performed using Cell DIVE, a multiplexed imaging solution at the single-cell level developed by Leica Microsystems. After cell segmentations and classifications, detailed spatial niche and regional analyses were performed at the single-cell level. Immune cell populations, as well as their TME distributions, were characterized to conduct prognostic associations and contrast primary and metastasis differences. Results: A total of 1,292,988 total cells (after excluding low-density signals and ambiguous classifications) were analyzed, with a median of 8,033 cells per core. Using spatial neighborhood analysis, 12 distinct spatial niches were identified with specific cell types and/or unique functional states. Among these, a macrophage- and fibroblast-enriched niche at the tumor-stroma interface was associated with improved overall survival (p=0.008). Another rare Treg-enriched niche, along with other immune cells, demonstrated protective effects, particularly in patients with residual macroscopic tumors after debulking surgery (p=0.05), while no significant effects were observed in those without visible residual disease (p=0.8). When comparing primary and metastatic tumors, metastatic lesions exhibited significantly increased tumor mass (p=2e-4) and a notable reduction in helper T cells (p=0.04) in tumor areas, as well as significantly increased fibroblast cells in tumor-stroma interfacing regions (p=0.002), reflecting distinct immunosuppressive and tumor-promoting shifts. Conclusion: This study reveals spatial and cellular differences in primary and metastatic OC TMEs, distribution differences linked to prognosis and surgical outcomes. These findings emphasize the potential of spatially resolved TME profiling to refine adjuvant and neoadjuvant therapeutic strategies. Citation Format: {Authors}. {Abstract title} [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 6266.

CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To understand the role of CD36 in melanoma, we first analyzed the SKCM dataset for clinical prognosis, evaluated the percentage of CD36 in lymphatic fluid-derived EVs (LEVs), and tested whether melanoma-derived EVs increase CD36 expression and induce M2-macrophage-like characteristics. Furthermore, we performed a multiplex immunofluorescence (MxIF) imaging analysis to evaluate the CD36 expression and its colocalization with various other cells in the lymph node (LN) of patients and control subjects. Our findings show that cutaneous melanoma patients have a worse clinical prognosis with high CD36 levels, and a higher percentage of CD36 in total LEVs were found at baseline in melanoma patients compared to control. We also found that monocytic and endothelial cells treated with melanoma EVs expressed more CD36 than untreated cells. Furthermore, melanoma-derived EVs can regulate immunosuppressive macrophage-like characteristics by upregulating CD36. The spatial imaging data show that cells in tumor-involved sentinel LNs exhibit a higher probability of CD36 expression than cells from control LNs, but this was not statistically significant. Conclusively, our findings demonstrated that CD36 plays a vital role in controlling the immunosuppressive microenvironment in the LN, which can promote the formation of a protumorigenic niche.

Abstract Background: Neuropilin 2 (NRP2) is a co-receptor that enhances signaling of growth factor and cytokine receptors and is expressed in tumor and microenvironment (TME) cells in melanoma and other tumors. Neuropilin 2 expression in tumor cells is associated with more aggressive tumors. Expression in tumor associated macrophages enhances efferocytosis and immunologically silent tumor cell death. Methods: Whole excisional lymph node biopsies were obtained from 10 treatment naïve patients with metastatic cutaneous melanoma who subsequently received anti-PD1 treatment. A single 5mm formalin-fixed paraffin embedded tissue section was used to assess a panel of 39 analytes by cyclic multiplex immunofluorescence (MxIF). Fields of view (~1mm2, n=288) were selected from pathologist-annotated regions of the tumor core, tumor-immune interface and adjacent lymphoid tissue. Pixel-based single cell segmentation and a supervised classifier approach was applied to resolve 12 distinct tumor, stromal and immune cell phenotypes and functional states (NRP2 positive or negative) in 1.5 million cells. Results: Tumor recurrence was observed in 6 patients (relapsed), whereas 4 patients were recurrence-free (non-relapsed) during follow up. Among the different cell types, NRP2 expression was more frequent in macrophages (44%), tumor cells (40%), dendritic cells (40%), followed by Tregs (31%) and T helper cells (29%), and less frequent in neutrophils (24%), cytotoxic T cells (23%) and B cells (15%). In colocalization permutation analysis, NRP2 expressing macrophages clustered away from tumor cells in non-relapsed patients while a random spatial distribution of NRP2 expressing macrophages with respect to tumor cells was observed in relapsed patients. In comparing the odds of tumor cell interactions with NRP2-expressing macrophages to those with NRP2 non-expressing macrophages, we found in the tumor:stromal interface (10% tumor cells) that cells from both non-relapsed and relapsed patients were more likely to interact with NRP2 non-expressing macrophages (OR = 0.73, 95% CI = [0.71, 0.75] and OR = 0.56, 95% CI = [0.55, 0.58] for non-relapsed and relapsed patients, respectively). However, in the tumor core (90% tumor cells), tumor cells from relapsed patients had significantly higher odds of interacting with NRP2 expressing macrophages, (OR = 1.15, 95% CI = [1.12, 1.18]), and this was not seen for non-relapsed patients (OR = 0.68, 95% CI = [0.66, 0.71]). In FOVs dominated by tumor cells (75% tumor), tumor cells from relapsed patients had 4.97 times the odds of expressing NRP2 compared to tumor cells from non-relapsed patients (95% CI = [2.23, 11.09], p = 0.0001). Conclusions: These spatial analytics suggest complex interactions among NRP2 expression in tumor and TME of melanoma determining risk for relapse with anti-PD1 treatment. Citation Format: Anastasios Dimou, Raymond M. Moore, Caitlin Ward, Alexey Leontovich, Ruifeng Guo, Chen Wang, Shankar Suman, Betty Dicke, Jill M. Schimke, Noah A. Stueven, Chathu A. Atherton, Wendy Nevala, Svetomir N. Markovic. Spatial analysis of neuropilin 2 expression in the microenvironment of melanoma [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 2905.

Abstract Spread to regional lymph nodes is the most common site of metastasis from melanoma and almost all solid tumors. Lymphatic metastases begin in the first lymph nodes draining from the primary tumor, i.e., the sentinel lymph nodes (SLN), preceded by the development of the premetastatic niche (PMN) initiated by primary cutaneous melanoma. Melanoma mediators are known to play a critical role in developing the PMN through the immune subversion in SLN. The DCs are a vital component of LN immunity that directly relates to eliciting T-cell functions. Our multiplex immunofluorescence (MxIF) analysis of SLNs indicates significantly decreased levels of DCs and cytotoxic-T cells in both core and interface areas of tumors in recurred patients treated with immunotherapy compared to non-recurred melanoma patients. The goal of this study is to determine whether the lymphatic extracellular vesicles (L-EVs) of melanoma patients regulate DC tolerization in SLN in order to stimulate the development of PMN. To identify whether L-EVs stimulate DC suppression, we extracted L-EVs from the lymphatic fluid of patients with melanoma downstream of primary cutaneous melanomas, compared it with controls (non-malignant post-operative fluid collected from the lymph node dissection field, and evaluated their effect on DC maturation by challenging these L-EVs in monocytic (CD14+) and hematopoietic stem cells (CD34+) cells. We found that L-EVs derived from melanoma patients suppress differentiation of CD11c+HLA-DR+ DCs and their maturation (defined by CD83 and CD86) compared to control L-EVs in CD14+ based DC maturation assay. We also found a reduction of conventional type 1 DC (cDC1) in CD34+-based DC maturation assay those challenged with patients’ L-EVs compared to control. To understand the mechanism of DC suppression in patients' L-EVs, we also conducted a total RNA sequencing analysis of subsets of DCs from maturation experiments to evaluate differently regulated transcriptomic profiles. The gene ontology analysis indicates that patient-derived L-EVs interfere with tRNA processing, oncostatin M & IL-10 signaling, and other immunoregulatory pathways to alter the hematopoietic cell lineage pathways in the DC maturation. Furthermore, the proteomic analysis of L-EVs also indicated several differentially regulated cargo-identified proteins in patient L-EVs, which are associated with immunomodulation, including regulation of DC function compared to control L-EVs. In conclusion, our study revealed that the patient's L-EVs carry differential proteomic cargoes secreted by cutaneous melanoma, which may be involved in regulating DC differentiation and maturation to diminish their functions and support the developing PMN in SLN. Citation Format: Shankar Suman, Wendy K. Nevala, James W. Jakub, Ray Guo, Alexey A. Leontovich, Raymond M. Moore, Chathu L. Atherton, Liyi Geng, Jeffrey E. Johnson, Sarah A. McLaughlin, Svetomir N. Markovic. Melanoma-derived lymphatic extracellular vesicles suppress dendritic cell differentiation and maturation in the sentinel lymph node [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 1503.