Wang, Chen

Background: Discovering risk biomarkers in small benign breast disease (BBD) biopsies is constrained by scarce tissue and microanatomic heterogeneity of terminal duct lobular units (TDLUs). We tested whether tissue-sparing tissue microarray (TMA)–based Digital Spatial Profiling (DSP) can deliver reproducible, biologically coherent protein measurements across benign lobules and breast cancers (BCs), and how well DSP aligns with standard immunoassays. Methods: We performed a pilot using tissues from the Mayo Clinic BBD cohort using TMAs representing four contexts: terminal duct lobular units (TDLUs) from BBD biopsies preceding BC and matched BBD-controls, subsequent BCs, and BC-associated TDLUs. We profiled 79 proteins by DSP (37 retained after QC) and benchmarked against chromogenic IHC and OPAL immunofluorescence. Reproducibility was evaluated using intraclass correlation coefficients (ICCs), cross-platform agreement (weighted kappa), marker correlations, and mixed-effects models with false-discovery-rate (FDR) control. Results: We analyzed 368 BBD-TDLU cores (88 cases; 88 controls), 204 BC cores and 110 BC-associated TDLU cores. ICCs were highest in BC tissues, and lower in BC-associated TDLUs and BBD-TDLUs. Agreement was slight–to-fair in TDLUs but moderate (ER/PR) to substantial (BCL2) in BC. DSP recapitulated expected immunologic correlations (CD45 with T-cell, B-cell, and macrophage markers) and tissue-type gradients (BC > BC-associated TDLUs > BBD-TDLUs). Exploratory case–control differences in BBD-TDLUs did not persist after FDR control. Conclusions: TMA-based DSP is feasible in archival breast tissues and yields biologically coherent, cross-platform-benchmarked profiles that are particularly robust in BC, while conserving scarce TDLUS and clarifying current limits of single-marker risk stratification from benign lobules. These data provide a foundation for refined sampling and expanded panels in future TDLU-focused studies.

Abstract The highest risk for liver allograft rejection is within the first weeks to months after a liver transplant; however, while later-stage mechanisms are well described, the initial window at the moment of alloantigen influx (e.g., ~ 1 h post-reperfusion) remains unmapped. T-cell priming is initiated in lymph nodes, yet these sites are rarely profiled in humans and their relationship to rejection risk is unclear. To address this gap, we profiled paired pre- and ~ 1 h post-reperfusion hepatic draining lymph nodes from liver transplant recipients using multiplexed immunofluorescence and GeoMx whole-transcriptome profiling to identify features that predict rejection versus non-rejection. Early after reperfusion, CD4 + and CD8 + T cells became proximal to macrophages in rejection compared to dendritic cells in non-rejection, which also displayed an immunoregulatory-leaning profile marked by PD-1, IDO1, and CD39, suggesting that macrophages and dendritic cells differentially influence early T-cell alloimmunity. These results provide nodal targets for precise immune modulation.

Lacking effective therapeutics, cholangiocarcinoma (CCA) remains a deadly malignancy of the biliary tract. The Hippo pathway effector protein Yes-associated protein (YAP) is implicated in CCA pathogenesis and chemotherapeutic resistance; however, the oncogenic mechanisms underlying YAP regulation remain incompletely understood. An enhanced understanding of YAP and its role in CCA may uncover novel therapeutic targets and better define resistance pathways. Human CCA cells and murine syngeneic CCA models were utilized to explore the molecular relationship of YAP and protein tyrosine phosphatase 1B (PTP1B). Previous work in CCA has demonstrated that YAP interacts with multiple protein tyrosine phosphatases, including SHP2 and PTP1B. We observed that PTP1B pharmacologic inhibition was associated with increased cell proliferation and YAP target gene expression, while genetically enforced overexpression of PTP1B was associated with a decrease in YAP activation. Treatment of CCA cells in vitro and syngeneic, orthotopically implanted CCA murine tumors in vivo with standard cytotoxic chemotherapy, gemcitabine/cisplatin, had enhanced efficacy in the setting of PTP1B overexpression. These findings demonstrate that pYAPY357 can be modulated through protein tyrosine 1B phosphatase activity, and reducing pYAPY357 through enhanced phosphatase levels can sensitize CCA to chemotherapy.

Abstract BRCA-associated homologous recombination deficiency (HRD) is present in ~ 50% of high-grade serous carcinomas (HGSC) and predicts sensitivity to platinum-based therapy. However, there is little understanding of why some patients with BRCA-deficient tumors experience unexpectedly poor outcomes. We profiled 154 tumors, enriched for patients with BRCA-deficient tumors that experienced short overall survival (≤ 3 years, n = 42), using whole-genome, transcriptome, and methylation analyses. All but one BRCA-deficient tumor exceeded an accepted HRD genomic scarring threshold. However, patients with BRCA1-deficient HGSC with a more elevated HRD score survived significantly longer. Patients with BRCA2-deficient HGSC and loss of NF1 survived twice as long as those without NF1 loss, whereas PIK3CA or RAD21 amplification defined BRCA2-deficient HGSC with exceptionally short survival. BRCA1-deficient tumors in short survivors had evidence of immunosuppressive c-kit signaling and EMT. In a large HGSC cohort (n = 1,389) including 282 individuals with pathogenic germline BRCA variants (gBRCApv), the location of the mutation within functional domains stratified clinical outcomes. Notably, residual disease after primary surgery had limited prognostic effect in gBRCApv-carriers compared to non-carriers. Our findings indicate that tumor HR proficiency in the context of therapy response and survival is not a binary property, and highlight genomic and immune modifiers of outcomes in BRCA-deficient HGSC.

Background and Aims: Cholangiocarcinoma (CCA) is one of the most lethal cancers, characterized by molecular heterogeneity and treatment resistance. To uncover new biological signals and therapeutic opportunities, we employed multiomic characterization combined with machine learning. Approach and Results: We profiled all anatomical CCA subtypes using whole exome sequencing, mRNA sequencing, and proteome/phosphoproteome analysis. Integrative dimensional reduction revealed RNA, protein, and phosphoprotein features driving tumor heterogeneity, enabling clustering. Machine learning algorithms identified molecular features for each cluster and mapped external datasets and patient-derived xenograft (PDX) models onto these clusters. Kinase enrichment analysis highlighted targetable kinases active in each cluster. In vivo validation was performed in cluster-specific PDX models using the selective TNK1 inhibitor, TP-5801. We identified 3 molecular clusters with distinct pathway characterization: immunomodulatory (cluster 1), metabolic (cluster 2), and gene regulation/cellular fate (cluster 3). Cluster assignment was independent of anatomic subtype but correlated with overall survival following curative-intent resection. We also identified multiomic features and pathways linked to overall survival and lymph node metastases, crucial for patient treatment selection. Kinase enrichment analysis pinpointed TNK1 as a highly active kinase in the metabolic cluster. Treatment with TP-5801 significantly reduced tumor growth in a metabolic PDX model, but not in models representing the other clusters. Combining internal data with publicly available datasets, we identified the immunomodulatory cluster as most responsive to gemcitabine/cisplatin therapy, confirmed in vivo using cluster-specific PDX models. Conclusions: Integrated multiomic characterization provides translational insights by defining unique molecular subtypes associated both with therapeutic response and overall clinical outcomes. This approach identified TNK1 as a previously unrecognized therapeutic target in a defined subset of CCA tumors.

Abstract Multiplexed Immunofluorescence (MxIF) enables detailed immune cell phenotyping, providing critical insights into cell behavior within the tumor immune microenvironment (TIME). However, signal integrity can be compromised due to the complex cyclic staining processes inherent to MxIF. Hematoxylin and Eosin (H&E) staining, on the other hand, offers complementary information through its depiction of cell morphology and texture patterns and is often visually cross-referenced with MxIF in clinical settings. In this study, we proposed a novel framework to align H&E and MxIF images for precise cross-modal cell feature validation. Using cell detection outputs from each modality as anchors, we formulated the multimodal image registration problem as point set alignment. Coherent Point Drift (CPD) is employed for initial alignment, followed by Graph Matching (GM) for refinement. Evaluations on ovarian cancer tissue microarrays (TMAs) demonstrate that our method achieves high alignment accuracy, enabling reliable validation of cell-level features across modalities for both restained and serial sections. Our results indicate that restained H&E enhances confidence in findings derived from MxIF. Additionally, we demonstrated the feasibility of generating high-quality virtual H&E images from MxIF data when restained H&E is unavailable, offering a viable alternative for integrated multimodal analysis.

Abstract Transcriptome profiling using RNA sequencing (RNA-seq) of bulk formalin-fixed paraffin-embedded (FFPE) tissue blocks is a standard method in biomedical research. However, when used on tissues with diverse cell type compositions, it yields averaged gene expression profiles, complicating biomarker identification due to variations in cell proportions. To address the need for optimized strategies for defining individual cell type compositions from bulk FFPE samples, we constructed single-cell RNA-seq reference data for breast tissue and tested cell type deconvolution methods. Initial simulation experiments showed similar performances across multiple commonly used deconvolution methods. However, the introduction of FFPE artifacts significantly impacted their performances, with a root mean squared error (RMSE) ranging between 0.04 and 0.17. Scaden, a deep learning-based method, consistently outperformed the others, demonstrating robustness against FFPE artifacts. Testing these methods on our 62-sample RNA-seq benign breast disease cohort in which cell type composition was estimated using digital pathology approaches, we found that pre-filtering of the reference data enhanced the accuracy of most methods, realizing up to a 32% reduction in RMSE. To support further research efforts in this domain, we introduce SCdeconR, an R package designed for streamlined cell type deconvolution assessments and downstream analyses.

Abstract Purpose: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). Experimental Design: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. Results: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial–mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

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 Dysfunctional liver regeneration following surgical resection remains a major cause of postoperative mortality and has no therapeutic options. Without targeted therapies, the current treatment paradigm relies on supportive therapy until homeostasis can be achieved. Pharmacologic acceleration of regeneration represents an alternative therapeutic avenue. Therefore, we aimed to generate a small molecule inhibitor that could accelerate liver regeneration with an emphasis on diseased models, which represent a significant portion of patients who require surgical resection and are often not studied. Utilizing a clinically approved small molecule inhibitor as a parent compound, standard medicinal chemistry approaches were utilized to generate a small molecule inhibitor targeting serine/threonine kinase 4/3 (MST1/2) with reduced off-target effects. This compound, mCLC846, was then applied to preclinical models of murine partial hepatectomy, which included models of diet-induced metabolic dysfunction-associated steatohepatitis (MASH). mCLC846 demonstrated on target inhibition of MST1/2 and reduced epidermal growth factor receptor inhibition. The inhibitory effects resulted in restored pancreatic beta-cell function and survival under diabetogenic conditions. Liver-specific cell-line exposure resulted in Yes-associated protein activation. Oral delivery of mCLC846 perioperatively resulted in accelerated murine liver regeneration and improved survival in diet-induced MASH models. Bulk transcriptional analysis of regenerating liver remnants suggested that mCLC846 enhanced the normal regenerative pathways and induced them following liver resection. Overall, pharmacological acceleration of liver regeneration with mCLC846 was feasible, had an acceptable therapeutic index, and provided a survival benefit in models of diet-induced MASH.