IntroductionMacrophages are highly plastic innate immune cells that have a broad range of phenotypic and functional roles in the body. The Wnt/β-catenin signaling pathway is known to play important roles in regulating the immune system, but the literature contains contradictory evidence for how Wnt impacts macrophages. Given the plasticity of macrophages, as well as the growing interest in utilizing Wnt inhibitors therapeutically, there is a need to better understand how Wnt signaling affects macrophage phenotype and function.MethodsWe treated murine bone marrow derived macrophages with Wnt3a, LPS/IFN-γ, or IL-4 and measured gene/protein expression with bulk RNA sequencing, RT-qPCR, flow cytometry, and immunofluorescence to assess macrophage phenotype.ResultsRNA sequencing of macrophages treated continually for 5 days with Wnt3a demonstrated upregulation in genes associated with chemotaxis, cytokine activity, and both pro- and anti-inflammatory phenotypes. A time-course of Wnt3a treatment revealed acute upregulation of the inflammatory cytokines Il6, Tnf, and Il12b. Later timepoints showed upregulation of regulatory markers, such as Il10. Finally, re-treating with classic inflammatory cytokines revealed a Wnt-induced tolerant phenotype.DiscussionIn this study, we expanded upon past work to show that acute stimulation by Wnt3a induces inflammatory activation of macrophages in a time-dependent manner. Chronic stimulation with Wnt3a, as may be expected in a Wnt-ligand rich tissue microenvironment, caused macrophages to become tolerant to additional inflammatory stimuli and to upregulate markers of an anti-inflammatory phenotype. This study highlights the importance of considering time-dependent plasticity and regulatory feedback mechanisms in understanding macrophage phenotypes.

Distribution of PRECISE scores based on (A) overall stage and (B) driver mutation status.

Development of effective second-line treatment options for patients with BRAFwtNRASwt or BRAFwtNRASmut melanoma resistant to immune checkpoint blockade (ICB) is crucial. While systemic delivery of agonist CD40 (aCD40) plus anti-PD1 (αPD1) showed activity in patients with ICB-resistant melanoma, the objective response rate was modest (15

Importance:Older age at diagnosis is associated with worse outcomes in papillary thyroid carcinoma (PTC). The current American Joint Committee on Cancer tumor-node-metastasis staging system incorporates an age of 55 years or older as staging criteria. TERT promoter variants are also associated with aggressive disease. Objective:To evaluate the interplay between age, TERT promoter variants, and survival outcomes in PTC. Design, Setting, and Participants:This multi-institutional retrospective cohort study was conducted from 1993 to 2020 at 3 academic centers in the US, with integration of publicly available cohorts from The Cancer Genome Atlas (TCGA) and American Association of Cancer Research's Project Genomics Evidence Neoplasia Information Exchange (AACR GENIE). The cohort included 169 patients with PTC from 3 US academic centers, who were integrated with TCGA and AACR GENIE, yielding a combined analytic population of 1555 patients with PTC. The association between age and TERT was examined across all patients. Within the multi-institutional cohort with long-term follow-up (n = 169), the association between TERT and survival was further examined. Exposures:Age at diagnosis and TERT promoter status. Main Outcomes and Measures:The primary outcome was disease-specific survival (DSS), and the secondary outcome was progression-free survival (PFS). Results:Across all cohorts (N = 1555; 966 female individuals [62.1%] and 589 male individuals [37.9%]), the prevalence of TERT promoter variants increased progressively with age (r = 0.59; 95% CI, 0.54-0.63), being rare in patients younger than 30 years (<2%) and reaching 32% to 70% in those older than 65 years. For survival analyses, 169 patients were included (median follow-up, 13.7 years; 95% CI, 10.4-15.9 years). Among patients younger than 55 years without a TERT promoter variant, 10-year DSS was 100% and 10-year PFS was 83%. Among those 55 years or older, 10-year DSS was 91% for TERT wild-type tumors and 51% for TERT-variant tumors (91% vs 51%, respectively; difference, 40%; 95% CI, 4-68). A similar trend was observed for PFS (10-year PFS of 75% for TERT wild-type vs 23% for TERT variant; difference, 52%; 95% CI, 9-82). In multivariable analysis, TERT promoter variants remained independently associated with worse DSS after adjustment for age and pathologic tumor, node, and metastasis categories. Conclusions and Relevance:The findings of this cohort study suggest that the high prevalence of TERT promoter variants in older patients largely explains the association between age and worse prognosis in PTC. TERT promoter status offers more biologically grounded prognostic information than age and may improve risk stratification.

Distribution of PRECISE score in the MDACC (A), VUMC (B), and TCGA (C) cohorts.

Reverse Kaplan-Meier curves for (A) progression-free and (B) disease-specific survival, illustrating censoring patterns across cohorts.

Abstract Purpose: Papillary thyroid carcinoma (PTC) exhibits heterogeneous behavior. There is a need for effective biomarkers and improved risk stratification methods in PTC. Gene signatures derived from bulk RNA sequencing capture composite transcriptional profiles that are confounded by tumor microenvironment cells. We developed a thyroid follicular cell-derived gene signature, Prognostic RNA Expression Cell-specific Integrated SignaturE (PRECISE), using single-cell and nucleus RNA sequencing techniques and evaluated its prognostic utility across independent cohorts. Experimental Procedures: PRECISE was developed using a discovery cohort of PTC patients (MDACC n=109) with a median follow-up of 14 years. Within the cohort, 11 PTC tumors and 4 normal thyroid samples were successfully sequenced using single-nucleus RNA sequencing. Genes that were significantly downregulated in malignant cells compared to normal thyroid follicular cells were integrated with previously identified differentially expressed genes from single-cell RNA sequencing. Prognostic significance was assessed using bulk RNA sequencing in the discovery cohort and validated in 2 additional cohorts (VUMC n=65; TCGA n=370). A rank-based single-sample method was used for score calculation. Associations between PRECISE score and progression-free (PFS) and disease-specific survival (DSS) were evaluated using multivariate Cox proportional hazards models, and predictive models were compared using Harrell’s C-statistic and ANOVA. Results: PRECISE comprised of 41 genes downregulated in PTC tumor cells and upregulated in normal follicular cells, capturing dysregulation of thyroid function and metabolism pathways. Higher PRECISE score was significantly associated with shorter PFS across all 3 cohorts (MDACC HR=1.64, P=0.002; VUMC HR=2.54, P<0.001; TCGA HR=1.63, P=0.012) and remained significant after TNM stage adjustment in two cohorts with ≥5 years follow-up (MDACC aHR=1.42, P=0.038; VUMC aHR=2.12, P=0.024). PRECISE score was also associated with DSS in these cohorts (MDACC HR=4.16, P<0.001; VUMC HR=2.23, P=0.01), independent of TNM stage in the MDACC cohort (aHR=2.83, P=0.015). Incorporating PRECISE significantly improved predictive performance for PFS (MDACC P=0.031; VUMC P=0.024) and DSS (MDACC P=0.008) beyond stage-based models. Conclusions: PRECISE is a thyroid epithelial gene-derived signature with independent prognostic value in PTC. Citation Format: Sophie Li, Chia C. Wu, Vicente R. Marczyk, Matthew A. Loberg, Aatish Thennavan, Maxime Tarabichi, Li Xu, Ying C. Henderson, Tuan M. Tran, Quanhu Sheng, George J. Xu, Eric C. Huang, Marie-Claude Hofmann, Xiao Zhao, Stephen Y. Lai, Michelle D. Williams, Wenyi Wang, Sarah Hamidi, Mark E. Zafereo, Maria E. Cabanillas, Nicholas E. Navin, Vivian L. Weiss, Jennifer R. Wang. PRECISE: A prognostic thyroid follicular cell-derived gene signature for papillary thyroid carcinoma [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 2709.

Distribution of PRECISE score in the FNA samples based on T stage.

Abstract Metastatic melanoma remains a significant clinical challenge despite recent advances with immune checkpoint inhibitors (ICIs) like anti-programmed cell death-1 (PD-1), which show variable efficacy across metastatic organ sites. The lung is a frequent site of metastasis, and while complete responses to ICIs occur more often in pulmonary lesions than in other organs, up to 60% of patients with lung metastases fail to respond. Pulmonary metastases are also a leading cause of death in patients with metastatic melanoma. This suggests that we will need to comprehensively understand the tumor immune microenvironments of pulmonary metastatic melanoma including the expression of alternative immune checkpoint targets. V-domain immunoglobulin suppressor of T cell activation (VISTA) is an emerging checkpoint molecule predominantly expressed in myeloid cells that suppresses immune activation. We found that VISTA was highly expressed in human pulmonary metastatic melanoma while PD-L1 expression was not significantly elevated. To investigate the role of VISTA in pulmonary metastatic melanoma, we utilized a variety of mouse models with B16-F10 murine melanoma. VISTA knock-out (KO) mice develop significantly fewer lung metastases than wild-type (WT) controls (lung-to-body weight ratio: VISTA WT = 0.05 ± 0.018, VISTA KO = 0.02 ± 0.011; p = 0.0012), with no difference in subcutaneous tumor growth (p = 0.1625). We assessed the immune cell infiltration in these mice. VISTA KO mice had significantly more proinflammatory monocytes (2.78% ± 1.50 vs. 0.52% ± 0.54; p = 0.0003) and macrophages (14.27% ± 6.18 vs. 6.75% ± 4.27; p = 0.006) in the metastatic lung tissue compared to WT mice. These data suggest that myeloid cell VISTA promotes lung metastasis of melanoma. We also demonstrated that Rag1-/- mice, which lack T and B lymphocytes, showed increased melanoma lung metastases compared to Rag1-/- VISTA-/- mice, suggesting that the metastatic phenotype is independent of T and B lymphocytes (Rag1-/- = 0.03 ± 0.008, Rag1-/- VISTA-/- = 0.01 ± 0.002; p = 0.0008). Mechanistically, VISTA-deficient bone marrow-derived macrophages (BMDMs) produce more TNFα and nitric oxide compared to WT BMDMs, indicating that VISTA suppresses macrophage activation as a mechanism of immune suppression (TNFα: 1.2-fold-change, 1166 vs. 946.3 pg/ml; p = 0.0001; nitric oxide: 3.2-fold-change, 29.42 vs. 9.089 µmol/L). Our findings establish VISTA as a critical regulator of immune suppression in pulmonary melanoma metastases. Ongoing mechanistic studies will clarify its potential as a therapeutic target, leading to improved treatment strategies and patient outcomes. Citation Format: Carly M. Fielder, Qianni Hu, Hyundong Yoon, Raymond Zhang, Anjali Raman, Megan L. Tigue, Chi Yan, Vivian L. Weiss, Douglas B. Johnson, Jin Chen, Tae Kon Kim. The role of VISTA in the lung tumor microenvironment of metastatic 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 6556.

Although well-differentiated thyroid carcinoma (WDTC) is characterized by a robust treatment response, aggressive subtypes, such as anaplastic thyroid carcinoma (ATC), remain highly lethal. To understand thyroid cancer evolution in both children and adults, we analyzed single-cell transcriptomes of 423,733 cells from 81 samples and spatially resolved key tumor and microenvironment populations across 28 tumors with spatial transcriptomics, including rare and unique composite WDTC/ATC tumors and pediatric diffuse sclerosing thyroid carcinomas. Additionally, we identified gene signatures of stromal cell populations in 5 large thyroid cancer bulk RNA-sequencing cohorts. Through this multi-institutional effort, we defined a population of POSTN+myofibroblast cancer-associated fibroblasts (myCAFs) that are intimately associated with invasive tumor cells and correlate with poor prognosis, lymph node metastasis, and disease progression in thyroid carcinoma. We also revealed a population of inflammatory CAFs that are distant to tumor cells and are found in the inflammatory stromal microenvironment of autoimmune thyroiditis. Together, our study provides spatial profiling of thyroid cancer evolution in samples with mixed WDTC/ATC histopathology and identifies a prognostic myCAF subtype with potential clinical utility in predicting aggressive disease in both children and adults.

Receiver operating characteristic (ROC) curves for 177 genes cited in at least 3 publications related to aggressive PTC and 328 genes from KEGG cancer pathways.

Kaplan-Meier analysis of low- vs high-risk PRECISE score in (A-C) progression-free survival and (D-F) disease-specific survival in the MDACC, VUMC, and TCGA cohorts, respectively.

Network of PRECISE genes (red nodes) and their direct interactors (blue).

Abstract Background: Thyroid cancer incidence and mortality are rising in the U.S., as is metabolic disease. Some studies have shown in predominantly Non-Hispanic White populations that obesity and diabetes are positively associated with thyroid cancer risk while physical activity is inversely associated. However, comparatively little is known about metabolic diseases as risk factors for thyroid cancer among Black Americans and low-income Americans, despite a high prevalence of these factors within these populations. Methods: The Southern Community Cohort Study is a prospective cohort study of >84,000 participants enrolled between 2002 to 2009 designed to evaluate the etiology of cancer and other health conditions in the southeastern U.S. states including for Black individuals (∼2/3) and individuals with low income (∼1/2). Self-reported body mass index (BMI), diabetes diagnosis, and physical activity levels were collected at baseline. Thyroid cancer incidence was ascertained through state cancer registry linkages. BMI was categorized as normal/underweight: <25 kg/m2, overweight: 25–<30 kg/m2, and obese: ≥30 kg/m2. Participants with missing data were excluded, leaving 68,532 participants and 135 incident cases. We used Cox proportional hazards models adjusted for potential confounders to evaluate risk factors for thyroid cancer overall and stratified by sex, self-reported race, and income. Results: The mean enrollment age was 51.8 years, with a median follow-up of 12.6 years. In comparison to normal/underweight, being overweight (HR: 2.39; 95% CI: 1.30-4.40) or having obesity (HR: 2.56; 95% CI: 1.44-4.58) were both associated with increased thyroid cancer risk (Ptrend: <0.01). These associations were also observed for female participants (HR: 2.55; 95% CI: 1.26-5.20 and HR: 2.55; 95% CI: 1.30-5.00, respectively; Ptrend: 0.02). Obesity was also associated with increased risk for Non-Hispanic Black participants (HR: 2.81; 95% CI: 1.19-6.63; Ptrend: 0.02) and for those with household income <$15,000 (HR: 5.42; 95% CI: 1.92-15.3; Ptrend: <0.01). No statistically significant association was observed for obesity among those with higher income although being overweight was associated with a two-fold increased risk in comparison to normal/underweight. Neither overweight nor obesity was associated with risk for male or Non-Hispanic White participants, possibly due to a smaller sample size. History of diabetes and physical activity were not independently associated with thyroid cancer risk. Conclusions: Thyroid cancer risk is associated with obesity in a predominantly Black and low-income population, in line with previous findings in other populations. However, neither diabetes nor physical activity were associated. These findings suggest that obesity prevention and treatment should be evaluated as potential prevention strategies for thyroid cancer risk. Citation Format: Jessica Rampy, Xijing Han, Xiangzhu Zhu, Xiao-Ou Shu, Vivian L. Weiss, Martha J. Shrubsole, . Metabolic disease and risk for thyroid cancer in a predominantly Black and low-income cohort [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 5056.

PURPOSE:Loss of thyroid differentiation underlies the aggressive behavior of a subset of papillary thyroid carcinomas (PTCs). The Thyroid Differentiation Score (TDS), introduced by The Cancer Genome Atlas (TCGA), quantifies tumor differentiation but has limited reproducibility and clinical applicability. We developed and validated a revised score (xTDS) that enables reproducible assessment of tumor differentiation with prognostic relevance across cohorts. METHODS:We analyzed RNA sequencing data from 570 patients with PTC across three independent cohorts: a discovery cohort from the MD Anderson Cancer Center (n = 111) and two external validation cohorts from Vanderbilt University (n = 69) and TCGA (n = 390). xTDS was evaluated for correlation with the original TDS, association with oncogenic drivers, and prognostic value for disease-specific survival (DSS) and progression-free survival (PFS). RESULTS:xTDS showed near-perfect correlation with the original TDS (Spearman ρ = 0.98) and recapitulated known biological patterns, with BRAF V600E-driven tumors exhibiting the lowest differentiation scores and RAS-driven tumors the highest across all cohorts (P < .001). Low xTDS was associated with worse DSS in the MD Anderson (P < .001) and Vanderbilt (P = .005) cohorts and showed a similar numerical trend in TCGA, without statistical significance because of limited events. Low xTDS was consistently associated with worse PFS across all three cohorts (P = .048, <0.001, and 0.007, respectively). CONCLUSION:xTDS is a reproducible measure of thyroid differentiation that preserves the biological and prognostic relevance of the original TDS while overcoming technical constraints affecting reproducibility.

OBJECTIVE:Pediatric thyroid nodules are uncommon but have a higher malignancy rate than adult nodules. Existing Bethesda risk-of-malignancy (ROM) estimates are not age-stratified and are affected by verification bias. We aimed to generate age-specific ROM, likelihood ratios (LRs), and post-test malignancy probabilities for pediatric and young adult thyroid cytology. METHODS:We analysed 2728 thyroid fine-needle aspirations (FNAs) from patients aged 0-25 years across multiple tertiary centers (2000-2023). All cases were classified or reclassified using the 2023 Bethesda System and grouped into four age bands (0-8, 9-14, 15-18, and 19-25 years). We calculated lower-bound ROM (ROM overall; assuming nonoperated FNAs were benign) and surgery-only upper-bound ROM (ROM surgery; 991 of 2728 cases with histology). Age-specific pretest probabilities and Bethesda-category LRs were used to compute post-test malignancy probabilities with Bayes' theorem. RESULTS:Among operated nodules, malignancy prevalence decreased with age (84.2% at 0-8 years to 64.6% at 19-25 years). Verification bias was substantial: malignancy prevalence was 24.6% (671/2728) under the lower-bound assumption but 67.7% (671/991) among surgical cases. Upper-bound ROM values were 2-10 times higher than lower-bound values across categories. Benign cytology showed low LRs (0.07-0.15), reducing post-test malignancy probability to 0-25%. Indeterminate categories showed age-related variation; for example, a follicular neoplasm diagnosis carried a ∼71% post-test risk in a 9-year-old versus ∼49% in a 24-year-old. AUS subtyping (nuclear vs other atypia) did not consistently separate ROM. CONCLUSION:Age substantially modifies both pretest and post-test malignancy probabilities in pediatric thyroid cytology. An age-stratified LR framework helps quantify verification bias and provides individualized risk estimates to guide decisions about surgery, molecular testing, and follow-up.

β-catenin is a critical effector of the Wnt pathway and a key component of the cadherin complex. Whether Wnt-regulated cytoplasmic β-catenin interacts with the cadherin-associated pool under physiological conditions is unclear. Using a cell line depleted of N- and E-cadherins, we demonstrate that cadherin-depleted cells exhibit lower levels of basal b-catenin that plateau to a similar level as the parental line with Wnt3a stimulation. The cadherin-depleted line exhibits significantly enhanced levels of Wnt signaling by comparison to its parental control; these effects are reversed by wild-type E-cadherin but not E-cadherin with disrupted b-catenin-binding. Enhanced Wnt signaling in the cadherin-depleted line is consistent with a previous study showing that Wnt pathway activation correlates with fold changes in β-catenin levels, rather than the absolute concentration. Our mathematical modeling suggests a mechanism in which β-catenin binding to cadherins acts as a sink to maintain elevated cytoplasmic b-catenin levels in the face of b-catenin destruction complex activity, and also limits pathway response. Our bioinformatic analysis reveals a correlation between elevated Wnt target gene expression and E-cadherin loss in a Wnt-driven model of thyroid cancer. Our results have relevance for tumorigenesis, as cadherin loss is commonly associated with poor prognosis and increased metastatic potential.

Thyroid hormones (THs [T3 and T4] ) are key regulators of metabolic rate and nutrient metabolism. They are controlled centrally and peripherally in a coordinated manner to elegantly match T3-mediated energy expenditure (EE) with energy availability. Hypothyroidism reduces EE and has long been blamed for obesity; however, emerging evidence suggests that, instead, obesity may drive thyroid dysfunction. Thus, we used a mouse model of diet-induced obesity to determine its direct effects on thyroid histopathology and function, deiodinase activity, and T3 action. Strikingly, overnutrition induced hypothyroidism within 3 weeks. Levels of thyroidal THs and their precursor protein thyroglobulin decreased, and ER stress was induced, indicating that thyroid function was directly impaired. We also observed pronounced histological and vascular expansion in the thyroid. Overnutrition additionally suppressed T4 activation, rendering the mice resistant to T4 and reducing EE. Our findings collectively show that overnutrition deals a double strike to TH biosynthesis and action, despite large efforts to adapt - but, fortunately, thyroid dysfunction in mice can be reversed by weight loss. In humans, BMI correlated with thyroidal vascularization, importantly demonstrating preliminary translatability. These studies lay the groundwork for obesity therapies that tackle hypothyroidism, which are much needed, as no current obesity treatment works for everyone.

Heatmap of gene-gene correlations for the PRECISE gene signature in the (A) MDACC, (B) VUMC, and (C) TCGA cohorts.

The Wnt signaling pathway, a highly conserved molecular cascade, orchestrates critical biological processes including embryonic development, cell differentiation, and proliferation across diverse organisms. Despite the pivotal role that Wnt signaling plays in many diseases, most notably cancer, there are still no FDA-approved, efficacious drugs available that inhibit this pathway. Most Wnt inhibitors target upstream components (e.g., Wnt ligand production and receptors) rather than the most commonly mutated downstream proteins in the pathway. Consequently, there is considerable interest in developing drugs that target the downstream effector, β-catenin. This review examines the challenges in targeting β-catenin, current approaches, and insights into overcoming on-target toxicity associated with cadherin-bound β-catenin.