Citations

Abstract Background Extramammary Paget disease (EMPD) is a rare cutaneous adenocarcinoma. While localized disease has an excellent prognosis, metastatic EMPD is aggressive with no standardized systemic therapy. This study characterizes the clinicopathologic and molecular landscape of metastatic/recurrent EMPD to identify therapeutic vulnerabilities. Patients and methods We conducted a retrospective cohort study of 26 patients with metastatic or distantly recurrent EMPD treated at Mayo Clinic (2000-2025). Descriptive statistics were used to summarize baseline characteristics. Survival outcomes were estimated using Kaplan–Meier methods, with group comparisons performed using log-rank testing. Continuous variables were compared using nonparametric tests, and categorical variables were analyzed using appropriate comparative tests. Pathway-level genomic correlations with TCGA reference cohorts were assessed using Spearman rank correlation. Treatment responses were retrospectively evaluated from radiologic reports. Results The cohort was predominantly male (73.1%) and White (92.3%), with a median age of 68 (IQR, 53-79). Lymph nodes were the most common metastatic site (61.5%). The median overall survival (OS) was 20.0 months (95% CI, 0-40.4). IHC showed universal CK7 and GATA3 positivity, with 80% androgen receptor (AR) expression. HER2 was positive (3+) or amplified in approximately one-third of cases. NGS revealed frequent mutations in TP53 (68.8%), ERBB2 (50%), and CDKN2A/B (50%). A “triple-hit” deletion at the 9p21 locus (CDKN2A/B/MTAP) was identified in 25% of cases. Genomic profiling showed strong similarity to HER2+ breast and urothelial carcinomas. HER2-directed therapy was associated with longer overall survival (median OS 73.0 months [95% CI, 0-177.9] vs 19.0 months [95% CI, 0-41.4]; HR 0.50 [95% CI, 0.0-177.9], P = .3), although this difference did not reach statistical significance. Conclusion Metastatic EMPD exhibits distinct clinicopathologic and molecular features, including high AR and HER2 expression, TP53 and CDKN2A/B alterations, and similarity to systemic HER2+ malignancies. The observed activity of HER2-directed therapies in this cohort suggests the potential value of further investigating biomarker-guided treatment strategies in metastatic EMPD. NGS-guided profiling may inform precision treatment strategies, including AR-targeted therapy and emerging MTAP-directed approaches.

Abstract Spatial transcriptomics links gene expression to tissue architecture, providing a mechanistic view of cellular organization. Yet existing datasets cover few donors and miss the complexity of human disease. Experimental costs remain prohibitive, and large-scale profiling is impractically slow for population-level studies. Accurate computational methods are urgently needed. Predicting gene expression from standard histology, however, remains an open problem, as current approaches transfer poorly to unseen cohorts and diseases. Here, we present Phoenix, a (latent) flow matching generative model that infers pan-cancer spatially resolved single-cell gene expression with high accuracy. Phoenix analyzes treatment response in silico: Applied to 763 head and neck cancer patients, it identified three new spatial biomarkers that we validated across two cancers (breast cancer, n = 84; ovarian cancer, n = 157) and treatment regimens (platinum, trastuzumab). Phoenix generalizes beyond carcinomas: In a large sarcoma cohort (802 tissue microarray cores), it accurately predicted cell-type-specific signatures in held-out samples and captured chemotherapy-induced immune remodeling. Phoenix also extends across species: In a mouse model, it accurately predicted the expression of pancreatic cancer lineage markers and the mutant mKras^G12D allele in silico. In total, we evaluated Phoenix on over 10,000 patients. Our results establish virtual spatial transcriptomics as a scalable framework for studying tissue organization, therapeutic response, and disease mechanisms.

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.

Abstract Motivation Multiplex immunofluorescence (MxIF) enables the quantification of multiple protein markers at a single-cell level while preserving spatial information, offering a powerful tool for studying tissue microenvironments. However, the flexibility in MxIF panel design poses challenges in standardizing cell phenotyping. Results We present SpaFlow, an efficient, customizable pipeline for unsupervised clustering and classification of MxIF data, implemented using Nextflow. SpaFlow performs quality control, clustering, and postclustering analysis on segmented and quantified MxIF data, facilitating reproducible and scalable analyses across various computing platforms. The SpaFlow pipeline integrates three clustering and classification packages—Seurat, SCIMAP, and CELESTA—each providing unique methodologies for identifying cell types based on phenotypic markers. A novel “meta-clustering” approach condenses clusters across multiple regions of interest into common meta-clusters, streamlining the cell-type identification process in large datasets. SpaFlow’s robust quality control steps, including signal summation and cell density filtering, mitigate artifacts that may impact clustering accuracy. We demonstrate the utility of SpaFlow in a case study involving 297 ovarian tumor cores, where SpaFlow successfully identified biologically meaningful cell populations, including tumor-infiltrating lymphocytes, efficiently and rapidly. Additionally, SpaFlow’s reproducibility is validated using serial tonsil sections, confirming its capability to consistently identify distinctive cell populations across matched ROIs. Availability and implementation SpaFlow is freely available with detailed documentation and examples at https://github.com/dimi-lab/SpaFlow.

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.

Abstract Ewing sarcoma is the second most common primary bone tumor in children. Typical Ewing sarcoma most frequently occurs in long bones and within the pelvis. ALES (adamantinoma-like Ewing sarcoma) is a rare subtype of Ewing sarcoma that is characterized by epithelial differentiation in addition to small round blue cells. Unlike typical Ewing sarcoma, ALES has been described in several cases in the head and neck. Herein, we describe a case of a 9-year-old boy with ALES of the mandible evaluated on 18F-FDG PET/CT with correlative MRI scans.