BACKGROUND:The induction of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α; encoded by PPARGC1A) expression and activation of oxidative phosphorylation (OXPHOS) is associated with disease progression and treatment resistance in patients with cutaneous melanoma. Loss of the TET2 (ten-eleven translocation 2)/5-hydroxymethylcytosine (5-hmC) epigenetic pathway is linked to melanoma aggressiveness, although the underlying mechanisms remain unclear. OBJECTIVES:To explore a relationship between TET2-mediated DNA hydroxymethylation and the induction of PPARGC1A/PGC-1α expression and activation of OXPHOS in melanoma. METHODS:RNA sequencing data from 368 melanoma metastases and 102 primary melanoma tumours were analysed, with tumours categorized as 'TET2-low' or 'TET2-high', based on TET2 gene expression. Differential gene expression and gene set enrichment analyses were done, with further validation using a tissue microarray comprised of 33 clinical specimens and a publicly available gene expression dataset from 209 primary tumours. Confirmatory in vitro and in vivo studies were performed using melanoma cell lines with altered TET2 and PGC-1α expression. 5-hmC and 5-methylcytosine levels at PPARGC1A were assessed using hydroxymethylated (hMeDIPseq) and methylated DNA immunoprecipitation sequencing. RESULTS:PGC-1α expression and activation of OXPHOS were significantly upregulated in TET2-low metastases and primary tumours. Tissue microarray analysis and gene expression studies showed an inverse relationship between TET2/5-hmC and PPARGC1A/PGC-1α expression and OXPHOS. In vitro and in vivo, PPARGC1A/PGC-1α expression and activation of OXPHOS was higher in TET2-low cells. hMeDIPseq identified significantly lower 5-hmC levels at an upstream PPARGC1A active enhancer in melanoma compared with naevi, in TET2-low compared with TET2-high cells and in cells expressing catalytically inactive TET2 compared with cells expressing wildtype TET2. Inhibition of PGC-1α expression and activation of OXPHOS mitigated migration and invasion in vitro and metastasis in vivo; TET2 loss was associated with resistance to mitogen-activated protein kinase (MAPK) pathway inhibition and enhanced sensitivity to OXPHOS inhibition. CONCLUSIONS:Loss of TET2 promotes activation of PGC-1α/OXPHOS in melanoma, driving metabolic reprogramming that supports tumour progression and resistance to MAPK inhibition in a subset of tumours. Importantly, this phenotype renders TET2-deficient melanomas selectively vulnerable to OXPHOS inhibition, identifying an actionable therapeutic opportunity. These findings establish an epigenetic-metabolic axis as a critical determinant of melanoma aggressiveness and highlight TET2/5-hmC as a potential biomarker and a targetable pathway.

Computational pathology foundation models (CPathFMs) have emerged as a powerful approach for analyzing histopathological data, leveraging self-supervised learning to extract robust feature representations from unlabeled whole-slide images. These models, categorized into uni-modal and multi-modal frameworks, have demonstrated promise in automating complex pathology tasks such as segmentation, classification, and biomarker discovery. However, the development of CPathFMs presents significant challenges, such as limited data accessibility, high variability across datasets, the necessity for domain-specific adaptation, and the lack of standardized evaluation benchmarks. This survey provides a comprehensive review of CPathFMs in computational pathology, focusing on datasets, adaptation strategies, and evaluation tasks. We analyze key techniques, such as contrastive learning and multi-modal integration, and highlight existing gaps in current research. Finally, we explore future directions from four perspectives for advancing CPathFMs. This survey serves as a valuable resource for researchers, clinicians, and AI practitioners, guiding the advancement of CPathFMs toward robust and clinically applicable AI-driven pathology solutions.

Cutaneous melanoma develops from precursor cells that progress to melanoma in situ (MIS) and eventually to invasive disease. In metastatic tumors, cells switch from a melanocytic to an undifferentiated, and neural crest (NC)-like state, which is associated with metastasis and therapeutic resistance. However, it is unclear whether these cell states or gene expression changes occur at the premetastatic stage or if they are associated with features of the local tumor microenvironment (TME). We analyzed a cohort of 62 patients with primary cutaneous melanoma (stages 0-III), using cyclic multiplex immunofluorescence imaging (CyCIF), and spatial transcriptomics to assess the molecular and TME changes that occur during the transitions from normal to precursor lesions, MIS and invasive melanoma. The loss of MART1 was associated with increased NGFR expression and elevated proliferation (KI67) in thicker tumors compared to thin (<1mm) lesions. Various tumor cell states coexisted in close proximity within early-stage, untreated melanomas. These tumor states were not arranged by depth of invasion but were associated with specific immune and stromal cell niches. Using CyCIF, we quantified the spatial heterogeneity of tumor cell states at whole-slide, microregion, and single-cell levels by calculating spatial entropy. We found that samples with a range of spatial entropy values contained both regions of high and low local neighborhood Shannon entropy at the single cell level. High spatial heterogeneity at the protein level was associated with enrichment of interferon gamma and alpha pathway transcripts. We also identified that transcriptomic heterogeneity within a single primary melanoma sample could be just as varied as across the entire cohort, with microregions showing distinct morphologic and spatial features. To understand the gene expression changes leading to intratumoral heterogeneity, we applied a self-organizing map to the spatial transcriptomic data to determine a developmental trajectory from melanocytic atypia to invasion. We discovered that tumor cells in invasive regions followed two developmental branches, characterized by epithelial-to-mesenchymal transition, angiogenesis, interferon response pathways, and dedifferentiation signatures. These developmental branches also showed differences in the local TME composition and the depth of invasion. Our multi-omic analyses reveal that a spectrum of tumor states exists even in early-stage primary cutaneous melanomas, suggesting an association between these tumor states and TME niches. Tuulia Vallius, Yingxiao Shi, Edward Novikov, Ajit Nirmal Johnson, Roxanne Pelletier, Shishir Pant, Zoltan Maliga, David Liu, Guihong Wan, Yevgeniy Semenov, Christine Lian, George Murphy, Sandro Santagata, Peter Sorger. Spatial heterogeneity of tumor cell states in primary cutaneous melanoma [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 158.

Tumor heterogeneity remains an obstacle in the treatment of melanoma, with unique tumor microenvironments contributing to therapy resistance and differential tumor growth. Dedifferentiated melanoma states have been associated with resistance to targeted and immunotherapy. AXL and NGFR are dedifferentiation markers linked to these states; however, the mechanisms by which they interact or result in distinct tumor programs remain unclear. Both AXL and NGFR have been linked to therapy resistance. AXL has been associated with cell survival, proliferation and immune suppression and NGFR with tumor progression, invasion, metastasis and therapy resistance. Understanding these interactions is critical for identifying therapeutic strategies and improving patient outcomes. In a melanoma patient with longitudinal tumor samples, we used highly multiplexed cyclic immunofluorescence (CyCIF) and matched H&E to study the tumor and immune states associated with progression. Spatial transcriptomic profiling was performed using GeoMX on selected regions of interest to further study tumor states characterized by different morphology and proteomic differentiation markers. In the primary and in-transit metastasis, we observed intratumoral heterogeneity, with histologically distinct tumor nodules. CyCIF of the in-transit sample showed upregulation of AXL in one nodule and NGFR in the other. The NGFR nodule was characterized by densely packed tumor cells, while the AXL nodule featured an inner cleft region encased by dense peripheral tumor cells. In the NGFR nodule, differential expression analysis (DEA) revealed upregulation of EPAS1, HIF1A, ERBB3 and VEGFA suggesting hypoxic stress, cell signaling, active angiogenesis and cell migration. Transcription factor analysis (TFA) showed upregulation of HDGF, TGFB1I1, HOXA7, IRF6 and SRSF2, TFs associated with proliferation, differentiation and increased translational activity. Downregulated HIP2K and PAWR suggest apoptosis inhibition, further facilitating tumor survival and expansion. In the AXL nodule, DEA revealed increased CDKN1A, indicating stress response and altered cell cycle regulation. Gene set enrichment analysis (GSEA) highlighted WNT and Hedgehog signaling, hormonal responses (e.g., estrogen signaling), and metabolic alterations (bile acid metabolism, myogenesis). EMT upregulation suggested a mesenchymal-like phenotype associated with resistance. TFA showed elevated KLF8 and TGFB1I1 driving EMT and metastasis, while SRSF2, HSF4, CXXC1, and NR1H2 suggested stress resilience, apoptosis regulation, and ER stress mitigation. Distinct signaling in AXL and NGFR regions suggests NGFR drives metabolic activity and proliferation, while AXL promotes resilience via stress and hormonal signaling. These findings offer insights into therapies targeting melanoma heterogeneity and resistance. Mark Woodnorth, Giuseppe Tarantino, Anne Zaremba, Tuulia Vallius, Florian Rambow, Lisa Zimmer, Antje Sucker, Elisabeth Livingstone, Eva Hadaschik, Roxanne Pelletier, Yingxiao Shi, Mariana Lopez, Samira Makhzami, Christine Lian, George Murphy, Genevieve Boland, Peter Sorger, Dirk Schadendorf, David Liu. Tumor heterogeneity and differential pathway activation in melanoma: insights from spatial profiling of AXL and NGFR upregulated regions [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 169.

Diseases like cancer involve alterations in in cell proportions, states, and local interactions as well as complex changes in 3D tissue architecture. However, disease diagnosis and most multiplexed spatial profiling studies rely on inspecting thin (4-5 micron) tissue specimens. Here, we use confocal microscopy and cyclic immunofluorescence (3D CyCIF) to show that few if any cells are intact in these thin sections; this reduces the accuracy of cell phenotyping and interaction analysis. In contrast, high-plex 3D CyCIF imaging of intact cells in thick tissue sections enables accurate quantification of marker proteins and detailed analysis of intracellular structures and organelles. Precise imaging of cell membranes also makes it possible to detect juxtacrine signalling among interacting tumour and immune cells and reveals the formation of spatially-restricted cytokine niches. Thus, 3D CyCIF provides insights into cell states and morphologies in preserved human tissues at a level of detail previously limited to cultured cells.

Several studies have outlined whole genome-doubled (WGD) tumor tissue characterized by elevated genome ploidy as an important biomarker positively associated with immunotherapy response and overall survival of cutaneous melanoma patients (Liu NatMed2019, Tarantino BioRXiv2022), as well as conferring therapeutic vulnerabilities (Quinton Nat2021). A robust and cost-effective method for detecting this biomarker would significantly improve patient risk assessment and treatment optimization. Unfortunately, current approaches depend on whole-exome sequencing of both tumor and normal tissue - a costly process rarely performed in standard clinical practice. We focus on H&E-stained whole-slide images (WSIs) routinely collected to diagnose cancer patients. We use publicly available melanoma WSIs (TCGA SKCM) and ploidy calls (Conway NatGen2020) to train a machine learning pipeline for classifying WSIs as diploid or WGD. After splitting the WSI into tiles we apply a wide array of recently released deep-learning foundation models that have been pre-trained on vast amounts of WSIs and were shown to be effective at extracting biologically meaningful features. To demonstrate the utility of these features for predicting WGD, we use tile-level features to train several types of slide-level classifiers. To evaluate the prognostication potential of H&E-inferred WGD, we use it to fit a univariate Cox survival model using publicly available survival data (PanCanAtlas). We perform a cluster analysis on tile-level features and model-assigned importance scores leveraging the help of a board-certified pathologist to identify visually interpretable morphological features significantly correlated with WGD. We performed both an internal cross-validation using our training cohort and an external validation cohort collected at Dana Farber Cancer Institute. We evaluate 100s of deep learning pipelines, each characterized by a unique combination of foundation model, slide classifier, and various other hyperparameters. Best-performing pipelines predict WGD with AUC >0.7 on both internal and external validation cohorts, demonstrating a robust association between tissue morphology and ploidy. Furthermore, in univariate Cox survival analyses, best-performing pipelines achieve a hazard ratio <0.7 with a p-value <0.05, underlining the ability of WGD to serve as a visually identifiable biomarker for predicting melanoma outcomes. Our study demonstrates the potential of H&E-based deep learning pipelines to robustly predict WGD in melanoma tissue with reliable performance across internal and external cohorts. Furthermore, WGD inferred from tissue morphology shows significant prognostic value for overall survival, offering a cost-effective and clinically feasible biomarker for personalized treatment strategies. Bojan Karlaš, Marc Glettig, Giuseppe Tarantino, Jiajia Chen, Guihong Wan, Tyler J. Aprati, Haitham A. Elmarakeby, Christine G. Lian, Yevgeniy R. Semenov, David Liu. Automated detection of genome-doubled tumors in H&E whole-slide images: towards a novel visual biomarker for predicting overall survival [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 2432.

Diseases such as cancer involve alterations in cell proportions, states and interactions, as well as complex changes in tissue morphology and architecture. Histopathological diagnosis of disease and most multiplexed spatial profiling relies on inspecting thin (4–5 µm) specimens. Here we describe a high-plex cyclic immunofluorescence method for three-dimensional tissue imaging and use it to show that few, if any, cells are intact in conventional thin tissue sections, reducing the accuracy of cell phenotyping and interaction analysis. However, three-dimensional cyclic immunofluorescence of sections eightfold to tenfold thicker enables accurate morphological assessment of diverse protein markers in intact tumor, immune and stromal cells. Moreover, the high resolution of this confocal approach generates images of cells in a preserved tissue environment at a level of detail previously limited to cell culture. Precise imaging of cell membranes also makes it possible to detect and map cell–cell contacts and juxtracrine signaling complexes in immune cell niches. Confocal microscopy enables high-resolution, high-plex 3D cyclic immunofluorescence of 30- to 50-µm-thick tissue sections. The approach allows for rich phenotypic assessments of intact cells and intercellular interactions with subcellular resolution.

Early detection of melanoma through skin surveillance is critical for preventing metastatic progression. Primary cutaneous melanomas at early stage offer a unique opportunity to uncover fundamental mechanisms of tumor initiation, progression, and immune surveillance, but detailed spatial profiling of early disease remains limited. Here we integrate high-plex cyclic immunofluorescence (CyCIF) imaging, spatial transcriptomics, and conventional histology to identify factors associated with de-differentiation and dermal invasion in early-stage melanomas. We demonstrate a high level of variability from one primary cancer to the next, from one 100-300 cell microregion to the next within a single cancer, and from one cell to the next within a microregion. Intra-tumoral heterogeneity is influenced by local features of the microenvironment including proximity to T and myeloid cells and to perivascular environments. Thus, tumor plasticity and spatial heterogeneity arise early in melanoma development, potentially allowing for competition among multiple tumor states during the emergence of invasive disease.

Preferentially expressed Antigen in Melanoma (PRAME) and Ten-Eleven Translocation (TET) dioxygenaseemediated 5-hydroxymethylcytosine (5hmC) are emerging melanoma biomarkers. We observed an inverse correlation between PRAME expression and 5hmC levels in benign nevi, melanoma in situ, primary invasive melanoma, and metastatic melanomas via immunohistochemistry and multiplex immunofluorescence: nevi exhibited high 5hmC and low PRAME, whereas melanomas showed the opposite pattern. Single-cell multiplex imaging of melanoma precursors revealed that diminished 5hmC coincides with PRAME upregulation in premalignant cells. Analysis of The Cancer Genome Atlas and Genotype-Tissue Expression databases confirmed negative relationship between TET2 and PRAME messenger RNA expression in melanoma. Additionally, 5hmC levels were reduced at the PRAME 5 & backprime; promoter in melanoma compared with nevi, suggesting a role for 5hmC in PRAME transcription. Restoring 5hmC levels via TET2 overexpression notably reduced PRAME expression in melanoma cell lines. These findings establish a function of TET2-mediated DNA hydroxymethylation in regulating PRAME expression and demonstrate epigenetic reprogramming as pivotal in melanoma tumorigenesis. (c) 2025 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

BackgroundFace transplant rejection is primarily monitored through skin biopsies, but mucosal tissue may detect immune rejection events missed by skin biopsies.MethodsWe retrospectively reviewed 47 paired mucosal and facial skin biopsies and 37 paired facial skin and sentinel flap biopsies from nine face transplant recipients. Rejection was graded using the 2007 Banff classification. Correlation, sensitivity, and specificity metrics were assessed.ResultsMucosa and facial skin rejection grades correlated strongly (r = 0.72, p < 0.0001), with mucosa showing a negative predictive value (NPV) of 0.85 for facial skin rejection. Mucosal biopsies identified rejection in 10 cases missed by facial skin biopsies. Sentinel skin biopsies had high correlation but an NPV of 0.76, missing 24% of rejection cases.ConclusionMucosal biopsies tend to capture the full spectrum of rejection, whereas skin biopsies alone may miss important rejection events occurring in the mucosa. Mucosal biopsies should be integrated into routine monitoring alongside skin biopsies, as they not only sensitively function as sentinel tissue but also provide critical insights into rejection activity that may otherwise go undetected. This dual approach could improve overall transplant surveillance. Inconsistencies in rejection patterns between the two tissues highlight the need for a reworked grading system.

Mucosal melanoma (MM) is a rare subtype of melanoma that responds poorly to immune checkpoint blockade (ICB), exhibiting low response rates and few durable responses. The biological mechanisms underlying ICB response and resistance in MM remain poorly understood, highlighting the need for novel therapeutic targets and biomarkers to improve patient outcomes. While most MM patients respond poorly to ICB, a subset of durable responders (DRs) exists. We hypothesize that DRs have distinct biological and genomic features compared to rapid progressors. We performed multimodal analysis - including whole exome sequencing, bulk transcriptomics, H&E, and multiplexed Cyclic Immunofluorescence (CyCIF) imaging - on pre-treatment and post-progression tumor samples from 126 MM patients, with a focus on a subset of 80 patients treated with ICB to identify features associated with response. Our genomic analysis of 80 MM patients revealed that DRs (PFS ≥ 1 year) had significantly fewer copy number alterations (CNAs) compared to those with progressive diseases (PFS ≤ 6 months). The most common MM driver mutations were identified including SF3B1, KIT, NF1, and NRAS. Notably, SF3B1 mutations were primarily found in anorectal primary tumors (10/17) and had fewer CNAs, a feature also observed in DRs, suggesting it may be associated with better ICB responses in MM. Bulk RNAseq analysis (n = 67) identified a tumor cluster enriched for epithelial-mesenchymal transition and hypoxia stress pathways, suggesting increased invasiveness and potential ICB resistance. These signatures were observed in 37% of our MM primary tumors, underscoring the aggressive nature of MM. CyCIF analysis of primary and metastatic MM lesions revealed increased CD8+ T cells and CD11C+ myeloid cells in metastatic sites. By defining the tumor/stromal border, we found fewer antitumorigenic CD68+ macrophages in the tumor center but more at the tumor border at the metastatic site compared to the primary sites, suggesting immune exclusion. In sinonasal primary tumors, non-metastatic patients had fewer protumorigenic CD163+ macrophages in both tumor center and border compared to those with distant metastases. Multimodal analysis of a longitudinally sampled MM patient treated with ICB showed distinct resistance clones associated with different microenvironments. Two lymph node metastases collected simultaneously after ICB showed clonal differences, with one (harboring a SOX17 mutation) exhibiting lower CD8+ T cell infiltration, suggesting the evolution of diverse resistance mechanisms. Our preliminary findings suggest that distinct genomic features, tumor states, and microenvironments differentiated ICB response and disease progression in MM, highlighting key tumor-intrinsic and microenvironmental factors associated with patient prognosis. Yingxiao Shi, Tuulia Vallius, Aikaterini Dedeilia, Roxanne Pelletier, Jia-Ren Lin, Mariana Lopez Leon, Christine Lian, Shishir Pant, Giuseppe Tarantino, Annette Wang, Jiajia Chen, Elizabeth I. Buchbinder, Peter K. Sorger, David Liu, Genevieve M. Boland. Integrative molecular and spatial analysis to reveal tumor intrinsic and extrinsic drivers of immunotherapy response in mucosal melanoma [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 5014.

Transcription factor complexes integrate diverse signals into discrete physiological outputs and are often aberrantly regulated in malignancies such as squamous cell carcinoma (SCC). In this study, we sought to discover new transcriptional complexes essential for SCC tumor maintenance, which have catalytic activities that can be targeted to provide new therapeutic options for patients with SCC. Comparing expression patterns of SCC tumors with those of non-SCC tumors, we have identified basonuclin 1 as a highly expressed, SCC-specific transcription factor. Analysis of direct transcriptional targets has uncovered an essential role for basonuclin 1 in controlling the proliferation-differentiation-migration axis. Basonuclin 1 activates proliferation genes while repressing a FRA1-dependent promigratory program and IRF6-dependent differentiation program. In addition, basonuclin 1 physically interacts with PRMT1 (protein arginine methyltransferase 1) to activate cell cycle genes. Importantly, proliferation can be blocked in SCC tumors using PRMT1 inhibitors, which has no effect on the repression of promigratory genes. Given the diverse gene expression programs regulated by transcription factors, this work demonstrates that protumorigenic activities can be specifically targeted through the inhibition of cofactors without activating pathways that may lead to tumor progression.

BackgroundApart from the skin, little is known about the immunological processes in deeper tissues, which are typically not accessible to biopsy and inspection, of vascularized composite allografts (VCAs). Face transplant patients develop prominent adenopathy shortly after transplantation that resolves over time. The mechanisms underlying this process are not understood.Materials and methodsA retrospective cohort study was conducted on 9 patients who underwent 10 facial VCAs at the Brigham and Women’s Hospital, Boston, MA, between April 2009 and July 2019. Clinical, radiological, and histological data related to lymphadenopathy of the head and neck were reviewed.ResultsPatients who received donor-derived lymph nodes (LNs) developed bilateral lymphadenopathy of the submental or submandibular superficial LNs. Median time of presentation was POD18 (range POD6-POM3). Notably, bilateral adenopathy of the neck was not observed in later stages of follow-up (mean follow-up, 115 months). Histology of 3 LNs showed increased histiocytes and apoptosis, with the features reminiscent of necrotizing histiocytic lymphadenitis, and B and T lymphocytes (mostly CD8 + T) admixed with CD163 + histiocytes and dendritic cells. Molecular chimerism analysis in one case showed the coexistence of donor (81%) and recipient (19%) derived lymphocytes. Granzyme B (GZMB) expression confirmed the presence of increased cytotoxic T cells in this LN sample.ConclusionOur data suggested the involvement of an immunological process within the donor-derived LNs after facial allotransplantation between the recipient and donor cells. GZMB expression suggested LN rejection that can occurred independently of skin rejection. This finding supports the need to better define the role of donor-derived immune cells in the context of allograft rejection.

Waterfall plot: depiction of percent tumor change among evaluable patients.

Supplementary table 3: Adverse Events/toxicities felt to be at least possibly related to drug.

Disorders in the regulatory arm of the adaptive immune system result in autoimmune-mediated diseases. While systemic immunosuppression is the prevailing approach to manage them, it fails to achieve long-lasting remission due to concomitant suppression of the regulatory arm and carries the risk of heightened susceptibility to infections and malignancies. Alopecia areata is a condition characterized by localized hair loss due to autoimmunity. The accessibility of the skin allows local rather than systemic intervention to avoid broad immunosuppression. It is hypothesized that the expansion of endogenous regulatory T cells (T regs ) at the site of antigen encounter can restore the immune balance and generate a long-lasting tolerogenic response. A hydrogel microneedle (MN) patch is therefore utilized for delivery of CCL22, a T reg -chemoattractant, and IL-2, a T reg  survival factor to amplify them. In an immune-mediated murine model of alopecia, local bolstering of T reg  numbers is shown, leading to sustained hair regrowth and attenuation of inflammatory pathways. In a humanized skin transplant mouse model, expansion of T regs within human skin is confirmed without engendering peripheral immunosuppression. The patch offers high-loading capacity and shelf-life stability for prospective clinical translation. By harmonizing immune responses locally, the aim is to reshape the landscape of autoimmune skin disease management.

In this study, we demonstrate the utility of whole-slide CyCIF (tissue-based cyclic immunofluorescence) imaging for characterizing immune cell infiltrates in immune checkpoint inhibitor (ICI)-induced dermatologic adverse events (dAEs). We analyzed six cases of ICI-induced dAEs, including lichenoid, bullous pemphigoid, psoriasis, and eczematous eruptions, comparing immune profiling results obtained using both standard immunohistochemistry (IHC) and CyCIF. Our findings indicate that CyCIF provides more detailed and precise single-cell characterization of immune cell infiltrates than IHC, which relies on semi-quantitative scoring by pathologists. This pilot study highlights the potential of CyCIF to advance our understanding of the immune environment in dAEs by revealing tissue-level spatial patterns of immune cell infiltrates, allowing for more precise phenotypic distinctions and deeper exploration of disease mechanisms. By demonstrating that CyCIF can be performed on friable tissues, such as bullous pemphigoid, we provide a foundation for future studies to examine the drivers of specific dAEs using larger cohorts of phenotyped toxicity and suggest a broader role for highly multiplexed tissue imaging in phenotyping the immune mediated disease that they resemble.