Markovic, Svetomir N.

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 Understanding how immune checkpoint inhibitors (ICIs) reshape human T-cell responses requires models that move beyond static transcriptomic snapshots and discrete cell-state classifications. Here, we present an integrative AI-enabled virtual cell framework that represents human CD8⁺ T-cell responses as dynamic and computable systems during ICI therapy. By integrating single-cell RNA sequencing with paired T-cell receptor sequencing within the C2S-scale foundation model, we construct a virtual representation of individual T cells, in which each cell is encoded by a unique functional identity that captures its transcriptional, signaling, and clonal characteristics. Using this framework, we identify a previously unrecognized dynamic latent effector state of CD8⁺ T cells characterized by intermediate expression of effector genes, distinct signaling activity, and ongoing clonal expansion. Across independent patient cohorts, the virtual cell model consistently indicates that ICI therapy mainly acts by unmasking pre-existing effector potential rather than inducing de novo effector differentiation. Notably, this latent effector population remains transcriptionally restrained despite active signaling and clonal expansion, revealing a hidden reservoir of antitumor immune capacity. More broadly, our study demonstrates how AI-enabled virtual cell modeling can reconstruct latent cellular states and their dynamic transitions from multidimensional single-cell data. By incorporating functional identity into virtual cell model, this framework uncovers biologically meaningful yet non-obvious T-cell effector program during cancer immunotherapy and provides a generalizable approach for studying immune dynamics in human disease.

Abstract Background Multiplexed immunofluorescence (MxIF) enables high-dimensional immune cell phenotyping and detailed characterization of the tumor immune microenvironment (TIME), but complex cyclic staining can introduce signal degradation and uncertainty in cell-level measurements. In routine practice, pathologists rely on hematoxylin and eosin (H&E) staining as the primary reference for tissue morphology and frequently cross-reference H&E to interpret MxIF findings. Methods We present a framework for aligning H&E and MxIF images at the tissue microarray (TMA) core level to support cross-modal analysis. Cell nucleus detections from each modality are used as anchor points, formulating the task as a point-set alignment problem. Coherent Point Drift (CPD) is applied for global alignment, followed by graph-matching–based refinement to improve local consistency. Evaluation on ovarian TMAs demonstrates consistent alignment performance for both restained and serial sections. We further explore the use of restained H&E as a reference for MxIF interpretation and investigate virtual H&E generation from MxIF data as a complementary approach when restained H&E is unavailable. Results The aligned images enable quantitative assessment of cross-modal feature concordance between MxIF-derived measurements and H&E morphology. The generated virtual H&E demonstrated similar cell population compared to the real H&E. Conclusions Cell-centric alignment can facilitate integrative multimodal histopathology analysis for TMA cores in spatial co-localization, cell feature validation, and virtual H&E generation.

Background/Objectives: Tumor-infiltrating lymphocyte (TIL) therapy is an important option for patients with metastatic melanoma progressing after standard systemic therapy, but real-world data on treatment delivery, toxicity monitoring, and immune recovery remain limited. We evaluated clinical outcomes, treatment tolerance, immune reconstitution, and cardiac biomarker dynamics across three Mayo Clinic sites. Methods: We retrospectively analyzed adults with metastatic melanoma who received lymphodepleting chemotherapy followed by TIL infusion and high-dose interleukin-2 (IL-2) between April 2024 and December 2025. Clinical outcomes, treatment delivery, and adverse events were assessed. Longitudinal immune monitoring included CD4 and CD8 T-cell counts, CD4:CD8 ratio, and immunoglobulin G (IgG) at baseline and follow-up. In a prespecified cardiac sub-cohort, high-sensitivity troponin (hs-Tn) was measured during IL-2 administration to evaluate associations with cardiac events and IL-2 interruption. Results: Thirty-six patients underwent TIL infusion. The objective response rate was 50.0%, including complete responses in 13.9%, and the disease control rate was 72.2%. Median progression-free survival was 3.61 months, and median overall survival was 12.94 months. M1d disease was associated with inferior overall survival on univariable analysis (HR 6.55, 95% CI 2.03–21.17; p = 0.002), with attenuation after multivariable adjustment. Receipt of ≥3 IL-2 doses was associated with longer overall survival on univariable analysis (HR 0.20, 95% CI 0.06–0.64; p = 0.007), but this association also attenuated after adjustment. Longitudinal immune monitoring demonstrated persistent CD4 lymphopenia through 6 months, sustained inversion of the CD4:CD8 ratio, and declining IgG at months 3 and 6. In the cardiac sub-cohort (24 patients; 87 IL-2 doses), post-dose hs-Tn ≥15 ng/L was associated with clinically significant cardiac events (OR 9.6, 95% CI 1.5–60.6; p = 0.016) and IL-2 interruption (OR 3.4, 95% CI 1.1–10.7; p = 0.036). For cardiac events, hs-Tn ≥15 ng/L had 100% sensitivity and 100% negative predictive value. Conclusions: In routine practice, TIL therapy was feasible and active in metastatic melanoma. M1d disease identified a subgroup with poor survival, peri-dose hs-Tn showed promise as a tool to support safer IL-2 delivery, and prolonged CD4 suppression with IgG decline suggests that recovery after TIL therapy extends beyond initial hematologic reconstitution. These findings support prospective validation of biomarker-guided IL-2 monitoring and extended post-treatment immune surveillance.

Introduction Uveal melanoma has a marked tropism for the liver where immune tolerance facilitates metastatic progression and resistance to immunotherapy. Impaired CD8 + T cell immunosurveillance is a central determinant of disease progression in uveal melanoma, yet the underlying mechanisms driving liver-mediated immune subpression and hepatic metastasis are not fully understood. Here, we investigate molecular pathways that regulated hepatic CD8 + T cell function and evaluate clinical outcomes associated with liver-directed therapy. Methods Expression of autotaxin (ATX), the expression responsible for generating lysophosphatidic acid (LPA), was evaluated in hepatic resident cell populations. ATX transcript levels were assessed in CD8 + T cells isolated from hepatic metastases and compared with matched peripheral blood and healthy donor control. Antigen-specific effector CD8 + T cells were used to characterize downstream signaling responses to LPA stimulation. Functional studies evaluating cytokine production during chronic antigen exposure were performed using CD8 + T cells from LPA receptor 5-deficient ( Lpar5 -/- ) mice. Results We identify the autotaxin-lysophosphatidic acid (ATX-LPA) axis as a prominent immunoregulatory pathway in the metastatic hepatic tumor microenvironment. ATX, the enzyme responsible for LPA production, is constitutively expressed by hepatic resident cells, is detectable on Kupffer cells and is associated with increased immunosubpressive markers. CD8 + T cells isolated from hepatic metastasis in a patient with uveal melanoma display markedly elevated ATX transcripts compared to matched peripheral blood (>5 log 2 fold change) and healthy controls. Using antigen-specific effector CD8 + T cells, we demonstrate LPA signaling induces low level, persistent ERK phosphorylation distinct from canonical antigen-induced TCR signaling. Genetic deletion of the LPA receptor 5 ( Lpar5 -/- ) on CD8 + T cells restores CD8 T cell percentages and cytokine function during persistent antigen stimulation in the presence of LPA. Discussion These findings define an ATX-LPA mechanisms of dysfunctional ERK signaling that may contribute to hepatic immune subpression in uveal melanoma. We highlight this pathway as a rational therapeutic target alongside liver-directed clinical interventions.

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 Circulating tumor DNA (ctDNA) as a liquid biopsy is an emerging tool used for diagnosis and disease monitoring, yet not all tumors shed DNA equally, thus currently limiting the clinical applicability of this tool. Our institution has developed and validated a droplet digital Polymerase Chain Reaction (ddPCR) ctDNA test for BRAF V600 mutation. We collected data on all BRAF mutated melanoma patients tested with the ddPCR BRAF assay at our institution over the years 2018–2024 and correlated the test results with clinical and radiological baseline parameters, with a focus on Positron Emission Tomography‐Computed Tomography (PET‐CT) data, incorporating both location of lesions and volume of disease. Linear and logistic regression modelling was used to assess possible associations between the baseline parameters and the ctDNA results. We identified 71 BRAF mutated melanoma patients, of which 65 had active disease on imaging studies and 43 had PET‐CT data available for analysis. Multivariate modelling has shown that patients were more likely to have a positive ctDNA result if they had elevated serum lactate dehydrogenase (LDH) (OR = 9.9), had a higher number of lesions on imaging studies (> 10 lesions: OR = 11.3), and if they were younger than 65 (OR = 11.8), with an AUC of 0.84. The PET‐CT calculated total metabolic tumor volume correlated with the quantitative ctDNA result with a Pearson r factor of 0.49. In conclusion, ctDNA ddPCR BRAF testing is easily obtained and might be especially beneficial for patients diagnosed with a clinically aggressive disease, based on elevated LDH, higher PET‐CT metabolic tumor volume, and younger age.

Abstract Background: High-dose interleukin-2 (IL-2) is administered after tumor-infiltrating lymphocyte (TIL) infusion to promote in-vivo expansion and persistence, yet optimal dosing and the clinical significance of out-of-specification (OOS) TIL products remain unclear. We investigated the impact of number of infused IL-2 doses and manufacturing OOS status on outcomes in metastatic melanoma treated with commercial TIL therapy. Methods: We retrospectively analyzed 31 patients treated across three Mayo Clinic sites. Planned IL-2 was 600,000 IU/kg every 12 hours for ≤6 doses; actual dosing varied based on clinical tolerance. Patients were grouped as low (0-2) vs high (≥3) IL-2 doses. TIL products were classified as within-specification or OOS per commercial release criteria. Survival associations were evaluated using Kaplan-Meier and multivariable Cox regression. Results: In our 31-patient cohort, the median age was 59 years (range 17-77), 58% were male, and 84% had ECOG 0. At TIL infusion, M-stage distribution was M1a 6%, M1b 10%, M1c 61%, and M1d 23%. LDH was elevated above the upper limit of normal in 42%. The median number of IL-2 infusions was 4 (range 0-6); 58% of patients received ≥3 doses, while 2 patients (6%) received none. Out-of-specification (OOS) TIL products occurred in 6 patients (19%). Best responses included complete response (26%) and partial response (23%), for an overall response rate of 48% and a disease-control rate of 61% (CR + PR + SD). Among responders, the median duration of response was 84 days (95% CI 73-95). At data cutoff, the median follow-up was 253 days (95% CI 137-370). The most common grade 3/4 toxicities occurring >14 days after TIL infusion were lymphopenia (39%), anemia (16%), and thrombocytopenia (10%). Median progression-free survival (PFS) was 99 days (95% CI 73-125) with 20 progression events, and median overall survival (OS) was 193 days (95% CI 32-354) with 13 deaths. Number of infused IL-2 doses was dependent on patients tolerability. Receiving ≥3 IL-2 doses was associated with improved overall survival (394 vs 101 days; p < 0.001) and a trend toward longer progression-free survival (110 vs 59 days; p = 0.055). OOS products predicted inferior PFS (30 vs 110 days; p = 0.003) and showed a trend toward worse OS (120 vs 394 days; p = 0.079). In multivariate analysis, ≥3 IL-2 doses independently improved PFS (HR 0.20, 95% CI 0.06-0.69; p = 0.011) and OS (HR 0.09, 0.02-0.36; p = 0.001), while OOS status increased risk of progression (HR 8.10, 2.24-29.25; p = 0.001) and death (HR 6.16, 1.35-28.20; p = 0.019). Conclusions: Higher number of infused doses IL-2 and meeting commercial TIL release criteria were independently associated with improved outcomes. Citation Format: Mohamed A. Aboelatta, Jeffrey E. Johnson, Jabra G. Zarka, Matyáš Benada, James W. Jakub, Roxana S. Dronca, Ruqin Chen, Mahesh Seetharam, Deepti Behl, Svetomir N. Markovic, Lisa A. Kottschade, Heather N. Montane, Matthew S. Block, Anastasios Dimou, Robert R. McWilliams, Paula Gill, Yi Lin, Arkadiusz Z. Dudek. Impact of number of IL-2 infusions and product out of specification status on outcomes of tumor infiltrating lymphocyte therapy for melanoma [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 3706.