Citations

Abstract Spatial transcriptomics links gene expression to tissue architecture, but detecting spatially variable genes at atlas scale remains difficult because biologically relevant patterns are multiscale, sparse, and often non-parametric. Here we show that FlashS, a frequency-domain kernel test using random Fourier features, sparse sketching, and a kurtosis-corrected null, retains Gaussian-kernel flexibility at near-linear per-gene cost without permutation. Across 50 benchmark datasets spanning 9 spatial transcriptomics platforms, F lash S achieves the highest ranking accuracy among 14 compared methods while maintaining calibrated inference under simulation and full-atlas permutation. In human heart tissue, it recovers a mitochondrial biogenesis program that co-localizes with ventricular cardiomyocytes. On the Allen Brain MERFISH atlas containing 3.94 million cells, F lash S completes in minutes on a single workstation and separates biological signal from negative-control barcodes even when p-values saturate.

Abstract The role of DNA topoisomerase II beta (TOP2B) in cardiomyocyte differentiation is poorly understood. To address this, Human induced pluripotent stem cells (hiPSC) were differentiated into cardiomyocytes (CM) that are wildtype or contain a genomic deletion of Topoisomerase 2B (BKO). Both WT and BKO hiPSC could be induced to differentiate into sheets of beating cardiomyocytes. BKO hiPSC take slightly longer to differentiate into sheets of beating CM than WT iPSC. RNA was prepared from both undifferentiated and differentiated WT and BKO hiPSC. RNA seq was used to examine gene expression changes when the WT and BKO hiPSC were differentiated into CM. Gene expression changes following differentiation of BKO cells were largely similar to those in WT cells. In addition, the differentiated WT CM were treated with dexrazoxane (ICRF-187), a TOP2 catalytic inhibitor that targets both TOP2A and TOP2B, or topobexin, a new TOP2B selective catalytic inhibitor. Topobexin inhibition partially phenocopied a TOP2B deletion and thus providing an alternative to TOP2B gene knockout in many cell lines. In future, hiPSC derived CM with and without TOP2B and inhibition by topobexin ex vivo CM could be used to study anthracycline-induced cardiotoxicity and to screen for cardioprotectants. Highlights Used CRISPR-Cas9 to delete TOP2B from hiPSC Produced beating cardiomyocytes from both WT and TOP2B null hiPSC Transcriptome analysis of WT and TOP2B null hiPSC and derived cardiomyocytes RNA seq showed he specific TOP2B inhibitor topobexin largely phenocopies TOP2B gene inactivation in iPSC derived cardiomyocytes. Topobexin inhibition could be used as an alternative to a TOP2B gene knockout in many different cell types, speeding up the analysis of the function of TOP2B.

Abstract Rare Mendelian disorders affect 300-400 million people globally. Although genetic testing has become widely adopted, gene-specific evidence for tailored variant interpretation remains scattered across resources. We present Gene Portals, a framework for gene-centered multimodal knowledge bases that co-localize expert-harmonized clinical data, functional assays, population variation, structural annotations and gene-specific ACMG/AMP specifications within a single resource. A modular interface integrates this unified evidence with VCEP-refined ACMG specifications to enable automated gene-specific variant classification, infer molecular mechanisms, and support cross-gene analyses. We demonstrate the framework’s utility across five Gene portals spanning eleven neurodevelopmental disorder-associated genes, integrating data from 4,423 individuals with 2,838 unique variants, 36,149 ClinVar submissions, and 1,044 expert-curated molecular readouts. By organizing evidence that is otherwise dispersed across multiple sources into a unified, queryable framework, the SCN, GRIN, CACNA1A, SATB2 and SLC6A1 Gene Portals became widely used community resources and provide an extensible template for standardized rare-disease variant interpretation and mechanism-aware discovery.

TPS290 Background: Landmark randomized trials established the survival benefit of adding D to ADT in mCSPC. Building on this foundation, subsequent trials (PEACE-1 and ARASENS) demonstrated further survival improvements when androgen receptor pathway inhibitors (ARPI) were added to the ADT + D backbone. However, the independent contribution of D within the triplet regimen remains unclear given lack of prospective evaluation. Additionally, tumor suppressor gene (TSG) alterations involving TP53, PTEN, and RB1 are frequently observed in PC and are associated with aggressive biology and poor outcomes, providing an opportunity to enhance prognostic accuracy and optimize selection of candidates for treatment intensification. We hypothesize that adding D to ADT plus A will improve survival compared to ADT + A in mCSPC and stratification by TSG status may identify different treatment effects across molecular subgroups. Methods: ASPIRE is an open-label, two-arm, randomized phase 3 study to assess the benefit of D added to ADT plus A for patients with mCSPC. Eligible patients include those with confirmed adenocarcinoma of the prostate with evidence of distant metastatic disease on conventional imaging and are appropriate to receive D. Patients with metachronous low-volume disease or PSMA-PET only disease are excluded. Candidates must have next-generation sequencing (NGS) results from a CLIA-certified tissue test available for TSG status stratification at registration. Eligible patients will receive ADT plus A (240 mg PO daily) +/- D for up to 6 cycles until progression of disease by PCWG3/RECIST version 1.1 criteria. Prior therapy with ADT (with or without ARPI) initiated within 120 days before registration is permitted. The primary endpoint is overall survival (OS). Secondary endpoints include OS by TSG status; radiographic progression-free survival (rPFS); time to castration resistance; symptomatic skeletal event–free survival; time to worsening of disease-related symptoms (NCCN-FACT FPSI-17); safety and tolerability (CTCAE v5.0); PSA90 response at 6 weeks and 6 months; time to PSA progression; and objective response rate in patients with measurable disease. The study will enroll 1200 patients and is currently actively accruing patients. Clinical trial information: NCT06931340 .

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mitochondrial DNA (mtDNA) and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro . Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci as an example of near-complete induction of mtDNA heteroplasmy in vivo . Gene editing specificity as precise as a single nucleotide was observed for a protein-coding gene. Nondestructive genotyping enables single-animal mtDNA analyses for downstream biological functional genomic applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

Modulation of the gut microbiota represents a promising approach to counteract diet-induced metabolic alterations, with microalgae emerging as potential interventions. Building on our previous in vivo evidence that dietary supplementation with the marine microalga Tisochrysis lutea F&M-M36 (T. lutea) positively modulates selected metabolic alterations under high-fat feeding, the present study aimed to identify potential associations between these metabolic changes and coordinated modifications of the gut microbiota. Animals were fed normal-fat (NF), high-fat (HF), or HF supplemented with 5% T. lutea (HFTiso) diets for three months. Gut microbial profiles were analyzed by 16S rRNA sequencing and correlated with plasma lipids, glucose, blood pressure, fecal lipid excretion, and adiponectin levels. T. lutea supplementation was associated with significant modulation of selected metabolic parameters and coherent alterations in gut microbial communities. Multivariate analyses revealed treatment-dependent clustering of metabolic profiles, with HFTiso forming an intermediate group between HF and NF diets. Beta-diversity analyses showed marked treatment-specific shifts, while alpha-diversity remained stable. Linear discriminant analysis identified 31 discriminative genera, with the HFTiso group enriched in taxa associated with fermentative metabolism and lipid-related metabolic pathways including Anaerotruncus, Marvinbryantia, and Eubacterium coprostanoligenes, while the HF group was linked to Clostridium sensu stricto 1 and Terrisporobacter. Positive correlations between HFTiso-associated taxa and adiponectin levels were consistent with microbiota-associated metabolic signatures. In parallel, T. lutea supplementation was associated with downregulation of colonic Niemann-Pick C1-like 1 (NPC1L1) mRNA expression, a key mediator of intestinal cholesterol uptake. The bioactivity of T. lutea likely reflects its content of polyunsaturated fatty acids, oleic acid, phytosterols, and fucoxanthin; however, whether these components act synergistically or whether specific bioactive compounds are primarily responsible remains to be clarified. Together, these findings indicate that T. lutea supplementation is associated with coordinated changes in gut microbiota composition and transcriptional modulation of the intestinal cholesterol transporter NPC1L1 in the context of selected early-stage metabolic alterations under high-fat feeding. While direct extrapolation to humans remains limited, these results suggest potential translational relevance of T. lutea as a nutraceutical approach targeting early-stage metabolic dysregulation. Future studies will be required to determine the mechanistic contribution of individual bioactive components and to assess whether microbiota- and gene expression-associated changes play a causal role in mediating the observed metabolic outcomes, thereby informing the rational development of T. lutea-derived interventions.

Abstract Background: Tertiary lymphoid structures (TLS), which are organized ectopic lymphoid aggregates resembling secondary lymphoid organs, have been associated with improved outcomes across various cancer types. However, limited data exist on the prognostic relevance of TLS in HER2+ BC pts treated with trastuzumab. In our prior work, we demonstrated that the presence of functional TLS, defined by lymphoid aggregates with elevated expression of BCL6 or IL21R, correlated with improved outcomes in the NCCTG N9831 trial. In this study, we evaluated TLS-related gene and outcomes in the real-world database. Methods: 1344 HER2+ (HR-HER2+: 560; HR+HER2+: 784) BC samples were analyzed via NGS (592-gene panel, NextSeq; WES/WTS, NovaSeq; Caris Life Sciences). Above (high) or below (low) the 50th percentile expression levels of TLS-related genes were used to calculate overall survival (OS). IL21R-high (H) and -low (L) tumors were classified by RNA expression above or below the 50th percentile. Real-world OS was derived from insurance claims and calculated from date of biopsy to last contact using Kaplan-Meier. Statistical significance was assessed using chi-square and Mann-Whitney U with multiple comparison adjustments (q < 0.05). Results: Among pts with HR-HER2+ BC, high expression of IL21R, CXCL9, CD38, and CXCL10 were associated with improved OS (Table). In contrast, among pts with HR+HER2+ BC, high expression of CXCR3, PDCD1, TBX21, CD200 and IL21R, were associated with improved OS. In both HR-HER2+ and HR+HER2+, IL21R-H groups had 1) higher infiltration of B cells (HR-HER2+: 4.4% vs 2.9%; HR+HER2+: 5.6% vs 4.2%), M1 MΦ (HR-HER2+: 3.9% vs 2.6%; HR+HER2+: 3% vs 2.1%), M2 MΦ (HR-HER2+: 4% vs 3.2%; HR+HER2+: 5.7% vs 4.4%), NK cells (HR-HER2+: 2.8% vs 2.5%; HR+HER2+: 2.7% vs 2.4%), CD8+ T cell (HR-HER2+: 0.9% vs 0%; HR+HER2+: 0.7% vs 0%), Tregs (HR-HER2+: 2.9% vs 1.4%; HR+HER2+: 2.8% vs 1.5%), all q<.05; 2) higher T cell inflamed score (HR-HER2+: 87.5 vs -95; HR+HER2+: 71 vs -86), IFNy score (HR-HER2+: -0.19 vs -0.48; HR+HER2+: -0.27 vs -0.49), all q<.05; 3) higher expression levels of MHC class I genes (HLA-A, HLA-B, HLA-C, HLA-E, FC: 1.6-2.3; all q<.05), MHC class II genes (HLA-DQA1, HLA-DOA, HLA-DPA1, HLA-DMA, FC: 2.2-3.2; all q<.05) compared to IL21R-L group. Conclusion: Consistent with our prior findings, overexpression of TLS-related genes was associated with improved outcomes in HER2+ BC in real-world data. Notably, the prognostic impact of TLS-related genes differed by hormone receptor (HR) status. Among these, IL21R emerged as the only gene significantly associated with clinical outcomes across both datasets and HR subgroups. Higher IL21R expression is associated with more B cell, M1 MΦ, CD8 T cell and NK cells. These findings underscore the potential importance of the IL21/IL21R axis in HER2+ BC and warrant further investigation to validate its role and therapeutic relevance. Citation Format: S. Chumsri, S. Deshmukh, Y. Liu, S. Wu, J. Xiu, N. Norton, S. Shipra Gandhi, E. Torres, M. Lustberg, E. Perez, A. Nassar, G. Sledge, E. Thompson, K. Knutson. Tertiary lymphoid structure (TLS)-related gene expression and outcomes in HER2-Positive (HER2+) breast cancer (BC) in the Real-World Database [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-09-08.

Abstract Uptake of tamoxifen is limited to 5-15% of all eligible women due to concerns regarding medication side effects combined with the lack of ability to predict which women will benefit from the therapies. We used a unique retrospective sample of n=92 high-risk women with paired benign biopsies obtained before (PreTam) and during tamoxifen (OnTam) to discover tissue gene expression biomarkers of biologic response, defined as change (Δ) in lobular Ki67. Ki67 was digitally quantified in normal lobules from PreTam and OnTam benign tissue samples and samples with PreTam Ki67 = 0 were excluded. Data were analyzed from 51 women with both PreTam and OnTam Ki67 (median age = 49 years and median BMI = 25.5 kg/m2 at PreTam biopsy). OnTam biopsies were collected a median of 11.5 months after PreTam biopsy (median 6.5 months after starting tamoxifen; dose 20 mg/day in 86%). Median Ki67 significantly decreased overall (Δ Ki67 = -0.29%, p=0.003), but the effect was restricted to women with baseline Ki67 ≥ 0.5% (n=38, Δ Ki67 = -0.78%, p=0.0002); no significant change occurred with baseline Ki67 < 0.5 (Δ Ki67 = 0.19%, p=0.23). Δ Ki67 was inversely proportional to baseline Ki67. RNA-seq was successful for 171 benign tissue samples (including 53 PreTam-OnTam pairs). After edgeR normalization (>14,600 genes), PreTam vs OnTam comparison across 33 pairs with baseline Ki67 ≥ 0.5% yielded 66 differentially expressed genes (fold-change > 2 and p<0.01). At p<0.01, ranked gene set enrichment analysis revealed 27 gene sets enriched for reduced expression with Tam (with top-ranked gene set related to steroid hormone biosynthesis) and 59 gene sets enriched for increased expression with Tam (with top-ranking gene sets related to B cell and T cell receptor signaling, regulation of cell cycle, and other immune-related pathways). To identify predictors of response, we correlated baseline PreTam gene expression with Δ Ki67 in 37 women with available baseline PreTam RNAseq and Δ Ki67 data. Partial correlation analysis (via ‘ppcor’ package, adjusted for library qubit) revealed 73 genes with significant positive correlation (rho > 0.50) and 14 genes with significant negative correlation (rho < -0.50) with Δ Ki67 (p<0.01). At p<0.01, ranked gene set enrichment analysis yielded 67 gene sets enriched with negative correlation (i.e. increased expression associated with a reduction in Ki67), including pathways related to ribosome biogenesis, DNA recombination, and nucleic acid metabolic processes; there were 62 gene sets enriched for positive correlation (i.e. decreased expression associated with a reduction in Ki67), including pathways such as VEGF and ERBB signaling. Our data support (i) baseline Ki67 ≥ 0.5 % and (ii) an 87-gene baseline signature as complementary biomarkers of tamoxifen response. Prospective validation of this combined assay could target tamoxifen to the women most likely to benefit, potentially overcoming a major barrier to chemoprevention uptake. Citation Format: S. J. Winham, C. Wang, Y. Liu, B. M. McCauley, D. J. Tabrizi, D. L. Gehling, J. L. Fischer, L. R. Seymour, M. H. Solanki, M. E. Sherman, K. R. Lohani, T. L. Hoskin, D. C. Radisky, A. C. Degnim. Gene expression signatures of beneficial response to tamoxifen for breast cancer prevention [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-01-18.

Abstract Background: We have identified two potentially predictive signatures of palbociclib resistance: the RBsig, composed of E2F1/E2F2 dependent genes, which is associated with genetic loss of RB1, and the ratio between the gene expression levels of CCNE1 to RB1 (CCNE1/RB1). Both signatures have been previously analyzed in vitro and in neoadjuvant and metastatic studies with palbociclib. The present analysis aims to explore the role of RBsig and CCNE1/RB1 in the phase III adjuvant PALLAS trial, which randomized patients to palbociclib plus endocrine therapy, versus endocrine therapy alone. Materials and methods: Gene expression data from PALLAS were generated using the HTG EdgeSeq Oncology BM Panel. Of the 87 genes composing RBsig, 46 were available within the EdgeSeq dataset and were used for the analyses; CCNE1 and RB1 were both available. RBsig was calculated as the mean of the Z-score scaled gene expression (log) of the 46 genes; CCNE1/RB1 was computed as the log ratio between the mRNA expression of CCNE1 and RB1. The prognostic/predictive effect of the continuous signatures in terms of invasive disease-free survival (IDFS), distant recurrence-free survival (DRFS), and overall survival (OS) was evaluated using Cox proportional hazard models. Results: HTG data were available from 2,587 of 5,748 patients from the entire PALLAS intention-to-treat (ITT) cohort. As RBsig and CCNE1/RB1 levels are affected by pre-surgical treatments, we limited the analyses to 1612 pre-treatment samples (530 core biopsies and 1082 surgical specimens) (analysis population). Compared with the entire PALLAS ITT cohort, the analysis population was significantly enriched for patients with low clinical risk (41.3% vs 59.4%) who did not receive prior chemotherapy (17.5% vs 55.9%). In keeping with the PALLAS ITT cohort, no significant benefit from the addition of palbociclib was observed in the analysis population. Higher baseline RBsig or CCNE/RB1 values were significantly and independently associated with worse IDFS (hazard ratio [HR] 1.63, 95% confidence interval [CI] 1.3-2.0; HR 1.22, 95%CI 1.1-1.3, respectively), DRFS (HR 1.94, 95%CI 1.5-2.5; HR 1.26, 95%CI 1.1-1.4, respectively) and OS (HR 1.79, 95% CI 1.3-2.4; HR 1.26, 95%CI 1.1-1.4, respectively) in the analysis population. No predictive effect of palbociclib benefit was observed for either signature in the analysis population, nor according to clinical risk or chemotherapy receipt. Interestingly, higher RBsig was strongly prognostic in patients who did not receive adjuvant chemotherapy (HR 1.83, 95%CI 1.4-2.3) but not in those who received it (HR 1.01, 95%CI 0.6-1.6) (interaction p=0.02) suggesting a potential predictive role of RBsig for chemotherapy benefit. Conclusions: In the PALLAS trial, higher baseline RBsig or CCNE1/RB1 values are associated with worse prognosis, but are not predictive of benefit from palbociclib in the adjuvant setting. RBsig might be associated with differential benefit from chemotherapy. Further study of these biomarkers may strengthen their role in risk stratification.https://acknowledgments.alliancefound.org Citation Format: L. Malorni, D. Hlauschek, E. Mayer, M. Benelli, L. Biganzoli, I. Migliaccio, A. Kermanidis, G. Pfeiler, E. P. Mamounas, A. M. Brufsky, M. Bellet-Ezquerra, K. Clifton, G. Rubovszky, T. Foukakis, M. Goetz, S. Lee, M. Ruiz-Borrego, W. Symmans, K. V. Ballman, F. Liu, J. Machacek-Link, C. Denkert, A. DeMichele, M. Gnant. Prognostic and predictive role of RBsig and CCNE1/RB1 gene-expression signatures for patients with early breast cancer treated with endocrine therapy with or without palbociclib in the PALLAS trial (ABCSG-42, AFT-05, BIG 14-03) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS2-07-13.