Translational Oncology

Hepatocellular carcinoma (HCC) is particularly aggressive and difficult to treat. Due to the lack of early clinical diagnosis and the unsatisfactory clinical treatment effect, it is particularly important to identify novel markers that can predict tumor behavior in HCC. biogenesis of ribosomes BRX1 (BRIX1) is abundant in various tissues of the human body. However, the regulatory mechanisms and its role in various tissues are not fully understood. Here, we analyzed the expression pattern of BRIX1 in HCC from public gene expression databases and tissue samples from clinical HCC. We confirmed that BRIX1 was upregulated in both HCC cell lines and HCC paraffin section samples. BRIX1 depletion significantly dicreased the capacity of cells to grow and migrate in vitro, and knockdown BRIX1 suppressed tumor growth in xenograft tumor model. Mechanistically, BRIX1 depletion suppressed the MAPK/ERK pathway, as reflected by reduced phosphorylated ERK (p-ERK) levels. In summary, we provide a rational clue for the further investigation of BRIX1 as an invaluable biological marker for diagnosing and predicting prognosis of patients with HCC.

BCL2L13 is a mitochondrial BCL2 family protein linked to mitophagy and ceramide metabolism, but its role in NSCLC metastatic plasticity remains unclear. Human lung cancer Tissue Microarray and matched patient specimens showed subtype and site dependent BCL2L13 expression, with higher cytoplasmic granular staining in primary NSCLC and reduced, heterogeneous staining in lymph node metastases, most evident in adenocarcinoma and squamous cell carcinoma. Because Epithelial mesenchymal transition and anoikis resistance are central requirements for metastatic dissemination, this primary to node attenuation provided the rationale to test BCL2L13 knockdown and overexpression in metastasis relevant NSCLC models. In A549 and LLC cell lines. TGF beta 1 induced coordinated mitophagy and EMT with mitochondrial enrichment of BCL2L13. BCL2L13 knockdown impaired TGF beta 1 and carbonyl cyanide m chlorophenyl hydrazone associated mitophagy, reducing LC3 beta mitochondria colocalization, TOMM20, LAMP1 overlap and mitochondrial LC3 II, p62, TOMM20 turnover; BNIP3 and NIX redistribution did not compensate. BCL2L13 loss enhanced EMT marker switching and migration, whereas overexpression partially opposed these changes. During detachment, BCL2L13 knockdown reduced anoikis associated apoptosis despite preserved mitochondrial recruitment of BAX, BAK, BNIP3,NIX, altered BID processing, non parallel caspase activity and shifted FAK phosphorylation. Pharmacological autophagy modulation did not reverse this anoikis phenotype. Lipidomics identified adhesion state dependent ceramide synthases CerS2, CerS6 linked sphingolipid remodeling: BCL2L13 knockdown increased C24 linked sphingolipid species in attached cells but reduced C16, C24 ceramide related profiles during anoikis. These findings identify BCL2L13 downregulation as a metastasis associated mitochondrial-lipid state that limits mitophagic quality control while favoring EMT and detachment survival in NSCLC adenocarcinoma.

Abstract Background: Cytolytic activity (CYT), a widely used transcriptomic surrogate of anti-tumor immune cytotoxicity derived from GZMA (granzyme A) and PRF1 (Perforin 1) expression, is associated with clinical outcomes across cancers. MicroRNAs (miRNAs) are key post-transcriptional regulators of tumor immunity, yet their pan-cancer roles in modulating cytolytic activity remain incompletely understood. Objective: This study aimed to identify conserved miRNA regulators of tumor cytolytic activity and their downstream gene-mediated networks across diverse cancer types, while evaluating their clinical and therapeutic relevance. Methods: Matched miRNA and mRNA expression profiles from 9,288 primary tumors across 31 TCGA cancer types were analyzed. A multi-stage framework was applied: per-cancer Spearman correlations (|{rho}| >= 0.30, FDR < 0.05) identified recurrent CYT-associated miRNAs (at least 3 cancer types); these were integrated with TargetScan-predicted targets and subjected to pan-cancer and cross-cancer triple filtering (miRNA-gene and gene-CYT associations). All associations underwent tumor purity adjustment using Consensus Purity Estimate (CPE), with LUMP (Leukocytes Unmethylation for Purity) as sensitivity analysis. Candidates were further prioritized by random forest modeling with bootstrap stability, cancer-type-adjusted Cox regression, mediation analysis, immune cell deconvolution, k-means molecular subtyping, pathway enrichment, and DGIdb-based drug-target prioritization. Results: The analysis converged on 38 high-confidence miRNA-gene-CYT regulatory triplets involving 9 conserved miRNAs and 31 target genes after stringent purity adjustment and multi-layer validation. All nine miRNAs exhibited complete bootstrap stability. Mediation analysis confirmed significant gene-level mediation in 37 of 38 triplets (FDR < 0.01), with mediated proportions up to 94%. The final miRNA signature defined two distinct pan-cancer immune subtypes (immune-hot vs. immune-cold) with significantly different cytolytic activity and overall survival (OS) (HR = 0.754, FDR = 1.12 x 10^-4). The network was enriched for T-cell activation and lymphocyte differentiation pathways and highlighted multiple druggable targets, including CTLA4 and CD274 (PD-L1), nominating 124 candidate compounds. Conclusions: In conclusion, this tumor purity-adjusted pan-cancer study defines a compact, reproducible, and clinically relevant miRNA network that regulates cytolytic activity across diverse malignancies. By linking miRNA biology to immune subtyping and actionable therapeutic targets, the present work provides a valuable foundation for advancing precision immuno-oncology.

Background: Immunochemotherapy (ICT) is considered standard in regards to care for small-cell lung cancer (SCLC) in extensive stages, yet reliable biomarkers for treatment response remain elusive. While previous univariate analyses suggest specific peripheral lymphocyte subsets correlate with survival, the systemic immune response involves complex, multivariate interactions that require advanced analytical approaches. Methods: This paper analysed high-dimensional flow cytometry data from 32 patients with stage IV SCLC treated with carboplatin, etoposide, and atezolizumab. Peripheral blood was analysed at baseline (V0) and longitudinally during treatment. To identify potential early predictive biomarkers and mitigate sample attrition in later cycles, we focused on baseline and measurements after two cycles of ICT (V1). We employed a rigorous machine learning framework utilising nested cross-validation, bootstrapping, and permutation-based statistical testing to evaluate eleven different regression and survival models. Results: Under model-appropriate metrics, regressors did not generalise (R2 <0); conversely, censoring-aware Random Survival Forests (RSF) successfully extracted robust prognostic signatures. Baseline immune profiles (V0) achieved a concordance index (C-index) of 0.66 (p= 0.015), while dynamic changes from V0 to V1 ({triangleup}V) achieved a C-index of 0.65 (p= 0.022). Crucially, absolute values measured after two cycles of ICT (V1) yielded no significant signal (p= 0.445). Feature importance analysis confirmed the prognostic value of Th17 normalisation and identified Naive Regulatory T cells and Memory B cells as candidate components. Conclusion: Machine learning validation confirms a predictive signal in the peripheral immune profile of SCLC patients. Early dynamic shifts in the balance between regulatory and effector immune arms are associated with prognosis, contrasting with the lack of signal in absolute counts after two cycles of ICT. These findings establish a proof of concept for multivariate liquid biopsy immune profiling, warranting confirmation in larger cohorts and highlighting the necessity of integrating systemic and tumour-intrinsic data.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by high metastatic dissemination, therapy resistance, and poor clinical outcome. Inhibitor of differentiation 1 or ID1, is frequently overexpressed in PDAC and is associated with tumour progression and adverse clinical outcome. However, the mechanisms governing its post-transcriptional regulation remain insufficiently characterized. Here, we identify tumour-suppressive miR-615-5p as a regulator of ID1 expression in PDAC. Integrative in-silico target prediction prioritized miR-615-5p based on seed complementarity and thermodynamic stability with the ID1 3' -UTR. Expression analysis of available PDAC clinical datasets revealed reduced miR-615-5p expression associated with increased ID1 expression. Direct association was validated using luciferase reporter assays, where miR-615-5p suppressed 3' -UTR reporter activity of ID1 in a sequence dependent manner, while mutation of the predicted binding site attenuated this effect. Further biotinylated-RIP and AGO2-RIP assays demonstrated the co-enrichment of ID1 transcripts and miR-615-5p with AGO2 associated RISC complexes, while AntimiR mediated inhibition of miR-615-5p perturbs association between miR/ID1 to AGO2, supporting interaction specificity. Functionally, modulation of miR-615-5p altered ID1 expression and impacted PDAC cell migration in vitro. Mechanistic analyses further indicated that the miR-615-5p/ID1 axis influences autophagic flux where miR-615-5p mediated inhibition of autophagy suppresses ID1 dependent cellular migration. Collectively, these findings define a previously uncharacterized miRNA-dependent regulation of ID1 expression and link this axis to autophagy-associated migratory responses in PDAC cells. The study expands the post-transcriptional regulatory landscape of ID1 and provides a possible mechanism where suppression of miR-615-5p leads to ID1 overexpression and subsequent poor clinical outcome in PDAC cells.

Background: Spatial organization is increasingly recognized as a key determinant of tumor-immune interactions in head and neck squamous cell carcinoma (HNSCC). The GSE300147 Xenium spatial transcriptomic resource generated by McCord and colleagues established a framework for mapping spatially coordinated T-cell states in HNSCC. However, how tumor-enriched epithelial immune states relate to metabolic, redox, and stress-adaptive transcript programs remains incompletely defined. Methods: A secondary, data-driven reanalysis of GSE300147 was performed, focusing on 17 confirmed HNSCC Xenium sections after exclusion of a non-HNSCC ameloblastoma specimen. A total of 1,148,244 cells were analyzed, including 558,867 EpCAM+ tumor-enriched epithelial cells. Tumor-enriched epithelial cells were classified into Hot, Intermediate, and Cold states using a Composite Hotness framework integrating T-cell inflammatory signature score, checkpoint-associated signaling, CD274 expression, IFN/antigen-presentation signature score (IFN/AP), and tumor-immune proximity. Six metabolic ecosystem states, neighborhood profiling, spatial permutation testing, and an integrated Immune-Metabolic-Redox Ecosystem Score (IMRES) were then applied. Results: Immune activation was spatially heterogeneous across HNSCC sections. Immune-hot tumor-enriched epithelial regions showed not only inflammatory, checkpoint-associated, and antigen-presentation signature scores, but also coordinated metabolic, oxidative-redox, and stress-response transcript programs. IMRES, derived from available immune, metabolic, redox, and stress-response transcript components represented in the Xenium panel, increased progressively from Cold to Intermediate to Hot tumor-enriched epithelial states and was associated with NFE2L2, GDF15, HLA-DRA, CD274, KEAP1, and MDM2. Integrating IMRES with Composite Hotness identified a distinct Hot+IMREShigh ecosystem comprising 106,874 tumor-enriched epithelial cells. This state showed the strongest immune-active and stress-adaptive features and was positioned closer to immune populations than expected by random assignment. An alternative rank-based robustness analysis reproduced the IMRES-associated ecosystem axis and correlated with the original module-based score (Spearman r = 0.597). Conclusions: This secondary reanalysis extends the original spatial T-cell framework by defining a complementary tumor-centered immune-metabolic-redox ecosystem in HNSCC. IMRES provides a transcript-derived framework for identifying Hot+IMREShigh neighborhoods where immune activation, checkpoint signaling, metabolic remodeling, and stress adaptation converge, providing a hypothesis-generating framework for studying immune resistance and therapeutic vulnerability.

IMPORTANCE: A recent meta-analysis showed that chemoimmunotherapy was associated with improved overall survival (OS) compared with immune checkpoint inhibitor (ICI) monotherapy for programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) [&ge;] 50% advanced non-small-cell lung cancer (NSCLC). However, whether this benefit reflects chemotherapy effect or ICI heterogeneity remains unclear. OBJECTIVE: To reassess the survival benefit of adding chemotherapy to ICI monotherapy using agent-stratified comparisons anchored to chemotherapy. DATA SOURCES: The 24 phase 3 randomized clinical trials included in the original meta-analysis (search date, August 3, 2025). DATA EXTRACTION AND SYNTHESIS: Hazard ratios (HRs) for OS and progression-free survival (PFS) were extracted from each trial in the original meta-analysis. Two analytic frameworks were used: within-agent comparisons (same ICI in both chemoimmunotherapy and monotherapy) and across-agent comparisons (ICI in one treatment strategy only). For within-agent comparisons, a two-stage random-effects meta-analysis was conducted. In stage 1, ICI-specific HRs for chemoimmunotherapy and ICI monotherapy versus chemotherapy were pooled and their ratio was calculated (RHR = HRchemoimmuno/HRmono; RHR < 1 favors chemoimmunotherapy). The RHRs were pooled in stage 2. For across-agent comparisons, RHR was derived from pooled HRs by treatment strategy. MAIN OUTCOMES AND MEASURES: Endpoints were OS and PFS. RESULTS: In within-agent comparisons (4 ICIs; 13 trials; N = 3252), pooled RHR was 0.94 (95% CI, 0.78-1.13; P = .48; I2 = 0.0%) for OS and 0.85 (95% CI, 0.68-1.06; P = .14; I2 = 0.0%) for PFS. In across-agent comparisons (7 ICIs; 11 trials; N = 2231), RHR favored chemoimmunotherapy for OS (0.68; 95% CI, 0.50-0.92; P = .01) and PFS (0.46; 95% CI, 0.37-0.58; P < .001). In a sensitivity analysis restricted to trials of NCCN-recommended regimens, pooled RHR was 1.02 (95% CI, 0.81-1.28; P = .87) for OS. CONCLUSIONS AND RELEVANCE: In the within-agent comparisons, adding chemotherapy to ICI monotherapy did not improve OS or PFS in patients with PD-L1 TPS [&ge;] 50% advanced NSCLC. The benefit in the original meta-analysis appears driven by across-ICI heterogeneity. These findings are consistent with ICI monotherapy as a standard first-line option and underscore the need for agent-level stratification in across-trial comparisons.

Lung squamous cell carcinoma (LUSC) is an aggressive malignancy characterized by high cellular plasticity and few targeted treatment options. TNIK overexpression is common in LUSC and promotes tumor growth, with TNIK inhibition sensitizing LUSC to radiotherapy, though the underlying mechanisms are not well defined. Through transcriptomic analyses and functional assays, we identified TNIK as a regulator of a MYC-dependent transcriptional network that coordinates epithelial-mesenchymal plasticity and cell proliferation in LUSC. Depletion of TNIK reprogrammed LUSC cells from a hybrid epithelial/mesenchymal state towards an epithelial, senescent-like state characterized by reduced cell migration, invasion, reduced DNA synthesis, and enhanced {beta}-galactosidase activity. Using a small-molecule screen approach, we found that TNIK inhibitors cooperated with agents suppressing the histone methyltransferase and MYC binding partner EZH2, which further suppressed partial epithelial-to-mesenchymal transition (pEMT). Mechanistically, we identified MYC as a key downstream TNIK effector in LUSC cells: MYC depletion phenocopied the effects of TNIK loss on pEMT and senescence, and restoring MYC expression bypassed the effects of TNIK depletion. Collectively, these results implicate TNIK in the mechanisms linking epithelial-mesenchymal plasticity with proliferation and evasion of senescence and provide insights into future strategies for the clinical deployment of TNIK inhibitors in LUSC and other TNIK-dependent malignancies.

Next-generation sequencing (NGS) for the detection of somatic variants has become the method of choice in a variety of molecular oncology fields and in the clinic. Its use ranges from sequencing entire tumor genomes and transcriptomes to targeted clinical diagnostic gene panels. The NYU Langone Genome PACT (Profiling of Actionable Cancer Targets, LG-PACT) assay is a qualitative in vitro diagnostic test that uses targeted next generation sequencing (NGS) of formalin-fixed paraffin-embedded (FFPE) tumor tissue matched with normal specimens from patients to detect gene alterations in a targeted panel covering 606 genes and the TERT promoter. Indications for testing are cancer (solid tumors and hematological malignancies) where a mutational profile from multiple genes would be informative for disease stratification, prognosis, or treatment options including targeted therapies and eligibility for clinical trials. The test is intended to provide information on somatic mutations including point mutations, small insertions/deletions (indels), and copy number aberrations for diagnostic and treatment decisions. LG-PACT is a United States Food and Drug Administration (FDA) cleared diagnostic test (510K: K202304). The clinical interpretation of sequencing data of molecular tumor markers from NGS encompasses automated variant calling tools with human interpretation. This final mostly manual review of data step is intensive, involving highly trained scientists, encompassing literature review, interpretation and clinical tier classification by pathologists, who then provide a complete molecular diagnostic report to the treating oncologists. We provide analysis of 1339 clinical genomic profiles from 31 different cancers and their subtypes, comprising of central nervous system (CNS) 792 (59%) cases (incl. meningioma, glioma and glioblastoma), with 267 (20%) cases predominantly of lung, pancreatic and colorectal and 280 of others (21%). Here, we present the technical challenges of validating an NGS oncological diagnostic targeted assay for clinical grade accuracy and sensitivity for patient care. We show how copy number alterations provide a more comprehensive description of the tumors genomic profile. We then outline the utility of targeted panel sequencing based on certified pathologist selection of reportable variants for our current patient cohort. Where analysis of variant detection has led to 49.4% (661/1339) of our clinical tumor samples containing mutations in known therapy targeted genes, 35.6% (477/1339) with mutation detected in other genes, and 15% (201/1339) cases being negative.

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown great promise in treating bladder cancer and are now part of the standard treatment for advanced disease. However, many patients still fail to respond to treatment and at present many biomarkers are assessed but have yet shown only limited results. Therefore, with the advent of combination treatments and the increase of immune related adverse event, the search for reliable predictive biomarkers is paramount. Using a multiplexed enhanced FRET-FLIM based technique (QF-Pro) we quantified the interaction of PD-1/PD-L1, CTLA-4/CD80 and TIGIT/CD155 immune checkpoints in a pre-treatment TMA of 46 patients treated with atezolizumab. The association between higher PD-1/PD-L1 ICP interaction state and treatment efficacy was demonstrated in the male sample cohort, where it identified patients with better PFS. Conversely, patients exhibiting higher CTLA-4/CD80 engagement had a worse response to atezolizumab. Remarkably, the dual assessment of patients with high PD-1/PD-L1 and low CTLA-4/CD80 allowed to identify the best responders. These results indicate that the monitoring of patients immune profile in urothelial carcinoma might be critical in identifying patients who may benefit from combination therapy.

In large data sets discovery is often limited to pre-conceived hypotheses and data fishing. Here we tested whether systematic exploration of AI generated hypotheses could uncover clinically meaningful signals in extensively studied data. We deployed AutoDiscovery, a newly launched large language model (LLM) framework designed to search for hypotheses based on surprisal and systematically interrogate complex datasets, on The Cancer Genome Atlas breast cancer cohort. The system did not identify clinically meaningful novel findings without human input. However, a seeded warm-start run with minimal text input from an oncologist revealed multiple interesting and surprising hypotheses. Among these was that a robust immune signature was present across all subtypes of invasive lobular carcinoma (ILC) that exceeded invasive ductal carcinoma (IDC). This observation was independently validated in independent cohorts and confirmed by high-sensitivity multi-immunofluorescence tumor tissue analyses. These results suggest immunotherapy approaches should be tested in ILC including early stage ER+HER2- ILC; these patients are currently excluded from large neoadjuvant immunotherapy trials. They further demonstrate that surprisal-based hypothesis generation frameworks can extract previously unappreciated patterns from deeply interrogated cancer datasets and imply that disease domain experts working with LLMs can derive more meaningful insights from complex data than either could achieve alone.

Background Conversion surgery for stage IV gastric cancer is supported by a pooled overall survival hazard ratio of 0.36 (95% confidence interval 0.32-0.40) and, in the largest international cohort, median survival of 36.7 versus 12.5-13.8 months on chemotherapy. Survival is measured from diagnosis; the median diagnosis-to-gastrectomy interval is 124 days, which patients must survive to be counted surgical. Methods We simulated cohorts of 3,177 stage IV gastric cancer patients from published parameters: background median survival 14.5 months; median diagnosis-to-surgery interval 124 days (category-specific 92-174 days). Surgery had no effect (true hazard ratio 1.00 by construction). Data were analysed as the literature analyses them (exposure fixed at baseline, follow-up from diagnosis), and by time-varying Cox and landmark analysis. Confounding by indication was added in a second scenario. Results Under immortal time bias alone the naive analysis returned a hazard ratio of 0.794 (95% simulation interval 0.743-0.851), median survival 16.8 versus 12.8 months. Time-varying Cox recovered 1.000 and landmark analysis 1.000-1.004. Bias scaled with the interval: 0.849 at 92 days, 0.715 at 174 days. Adding confounding, the naive estimate fell to 0.601 (0.560-0.644) at strength 0.5 and 0.356 (0.323-0.385) at strength 1.5, overlapping the published estimate; median survival 21.9 versus 8.7 months. Correcting immortal time alone left residual bias (hazard ratio 0.439). Conclusions The reported survival advantage of conversion surgery is reproducible where the operation does nothing; published estimates cannot distinguish benefit from bias. Resolving this requires individual patient data analysed with methods that assign person-time correctly, or completion of JCOG2301.

Liver metastases (LM) remain a major cause of death from different cancer types, in particular malignancies of the gastrointestinal tract. Liver metastases predict a poor response to immunotherapy due, among others, to the immunotolerant microenvironment (ME) of the liver and loss of local and systemic cytotoxic T cells. Thus, strategies that can reprogram the immune ME of the liver and restore cytotoxic T cell reactivity are being sought. We previously reported that estrogen signaling blockade impedes the growth of LM by reducing MDSC accumulation and monocyte/macrophage polarization. The aim of this study was to elucidate the underlying mechanism(s) and assess whether estrogen signaling in the myeloid lineage was driving the immunotolerant ME of LM. To this end, we generated mice with conditional myeloid cell-specific deletions of estrogen receptors (ER) or ER{beta} and analyzed in these mice the effect of ER loss on the liver immune ME and the outgrowth of LM. In mice with ER, but not with ER{beta} deletion, we observed a marked reduction in the growth of murine colon carcinoma MC-38 liver metastases as compared to their respective controls. Flow cytometry and immunohistochemistry revealed a decrease in macrophages that were polarized to the pro-tumorigenic M2-like phenotype and a concomitant increase in activated CD8+ T and NK cells relative to controls. Bulk RNAseq analysis performed on hepatic immune cells infiltrating the liver revealed changes in the expression of key cytokines/chemokines mediating immune cell recruitment, activation and polarization, including Ccl5 (upregulated) and Csf1 (downregulated). Taken together, the data suggest that ER signaling in myeloid-derived cells programs the immune landscape and contributes to an immunosuppressive and metastases-growth permissive ME in the liver.

ZNF217 is an established oncogenic transcription factor that promotes cancer progression and therapeutic resistance; however, the mechanisms regulating ZNF217 protein abundance remain poorly understood. Here, we identify ubiquitin-specific peptidase 15 (USP15) as a critical regulator of ZNF217 stability and define a reciprocal USP15-ZNF217 signaling loop that sustains malignant phenotypes in ovarian cancer. Stable overexpression of ZNF217 in OVCA420 ovarian cancer cells enhanced proliferation, epithelial-mesenchymal transition, migration, invasion, and extracellular matrix adhesion. Notably, ZNF217 overexpression increased USP15 protein abundance without altering USP15 mRNA levels, whereas ZNF217 depletion reduced USP15 protein levels, suggesting post-transcriptional regulation. Conversely, USP15 depletion markedly reduced ZNF217 protein abundance while increasing ZNF217 mRNA levels, indicating that USP15 regulates ZNF217 predominantly at the post-transcriptional level. Proteasome inhibition restored ZNF217 protein levels following USP15 depletion, further demonstrating that USP15 promotes ZNF217 protein stability. Functionally, USP15 depletion in ZNF217-overexpressing ovarian cancer cells suppressed proliferation and multiple metastatic phenotypes, including migration, invasion, extracellular matrix adhesion, anoikis resistance, and multicellular aggregate formation. In vivo, USP15 depletion significantly reduced tumor progression and metastatic burden and prolonged survival in mice bearing ZNF217-driven ovarian tumors. Furthermore, USP15 depletion enhanced the sensitivity of ZNF217-overexpressing cells to carboplatin, paclitaxel, and doxorubicin. Collectively, these findings identify USP15 as an upstream regulator of ZNF217 protein stability and reveal a positive-feedback loop between USP15 and ZNF217 that reinforces oncogenic signaling. Targeting USP15 may therefore represent an indirect therapeutic strategy for suppressing ZNF217-driven ovarian cancer, particularly given the challenges associated with directly targeting oncogenic transcription factors.

Pancreatic ductal adenocarcinoma (PDAC) harbors a distinct intratumoral microbiome. Yet rigorous characterization of its composition is hampered by pervasive environmental and procedural contamination. Sources of contamination have not been thoroughly explored, and the methods of decontamination have not been sufficiently evaluated in a benchmarking manner. We systematically collected >300 negative control (NCT) samples comprising paraffin from formalin-fixed paraffin-embedded (FFPE) samples, lysis buffer and sterile water over a period of four years processed by different laboratory persons (LP). All samples were sequenced using full-length 16S rRNA gene sequencing with Oxford-Nanopore Technologies. We benchmarked four decontamination methods (restrictive filtering, decontam, SCRuB, and the Nejman et al.-derived (Nj) pipeline) against fresh-frozen tumor samples (FF) from LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) mice, using the abovementioned contamination assessment to calculate a composite score for the assessment. Further, we validated those methods via technical replicates. Microbial profiles of NCT samples were significantly determined by control type, LP, year and season reflecting complex batch effects. The 15 most abundant contaminants spanned well-characterized environmental taxa and human commensals from the oral cavity. The LP processing samples left a significant microbial trace highly contributing to the batch effect. Decontamination benchmarking demonstrated that the Nj method consistently outperformed alternatives in both composite score and inter-replicate concordance. Application of Nj to fresh frozen PDAC samples substantially reduced contaminant burden while preserving putative tumor-associated signals in FF but not FFPE samples. Our results support the adoption of the Nj decontamination approach for future intratumoral microbiome studies in fresh frozen tumor samples.

The role of UBE2N in myeloid cell-mediated immune suppression in cancer remains undefined. Here, we examined the function of UBE2N in myeloid cell-mediated tumor progression using a temporally inducible myeloid-specific knockout model (LysMCreERUbe2nfl/fl). Temporally induced deletion of Ube2n in myeloid cells (Ube2nMyeKO) significantly hindered growth of YUMM1.7 melanoma. This was accompanied by reduced myeloid cell burden within the tumor microenvironment. We observed altered abundance of PD-1, PD-L1, and SPP1 in the Ube2nMyeKO tumor microenvironment at the tissue level. In vitro analysis showed that knock-in expression of a catalytically deficient UBE2NC87S mutant in bone marrow-derived macrophages (BMDMs) markedly decreased expression of Spp1. We observed decreased SPP1 secretion in Ube2nMyeKO BMDM-conditioned media (CM). Treatment with Ube2nMyeKO BMDM-CM decreased co-expression of PD-1, TIM-3, and LAG-3 on chronically stimulated T cells. Antibody-mediated neutralization of SPP1 in Ube2nWT BMDM-CM decreased PD-1 expression on CD8+ T cells. Together, these findings suggest a role for myeloid UBE2N in YUMM1.7 progression.

KRAS is one of the most frequently mutated oncoproteins in cancer. Its ability to induce malignant transformation relies on metabolic reprogramming that causes cells to become dependent on aerobic glycolysis as a primary source of energy and for generating biological building blocks. Thus far, the signaling mechanism used by oncogenic KRAS to promote these changes in cancer cell metabolism has not been fully elucidated. However, through studies in pancreatic ductal adenocarcinoma (PDAC) cell lines and patient-derived organoids, we now demonstrate how oncogenic KRAS triggers an increase in glycolytic activity and identify Survivin as a newly discovered and critical KRAS-signaling partner essential for promoting these metabolic changes. We show that oncogenic KRAS potently upregulates the expression of Survivin in PDAC cells and patient-derived organoids undergoing increased glycolysis, whereas depleting Survivin expression inhibits their glycolytic activity and growth. Through a combination of cellular, biochemical, and imaging approaches, we further show that Survivin promotes the formation of unique microtubule-based structures that resemble invadosome rosettes, allowing for the recruitment of the glycolytic enzymes triose phosphate isomerase (TPI) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to these super-structures which drives the increases in glycolysis. These findings demonstrate that by directing the assembly of a microtubule-based complex of metabolic enzymes, Survivin serves as a vital link in a KRAS signaling pathway responsible for promoting the metabolic changes necessary for the accelerated growth of PDAC cells, and thus potentially highlight new therapeutic strategies for treating KRAS-dependent cancers.

Bladder carcinoma in situ (CIS) is a multifocal, non-muscle-invasive disease with a high risk of progression to muscle-invasive cancer. Current management strategies are often guided by genomic profiling of single tumor samples, which incompletely capture tumor heterogeneity and may contribute to treatment failure. In particular, the multifocal nature of CIS raises uncertainty regarding the uniformity of genomic, immunologic, and microenvironmental features across anatomically distinct sites within the same patient. To address this, we performed spatial transcriptomic profiling of CIS-containing tissue from four anatomically distinct sites within a single individual. Unsupervised clustering with marker-based annotation, integrated with metabolic inference, identified epithelial tumor populations alongside stromal, immune, and smooth muscle compartments. While key cellular states were conserved, their spatial organization and relative abundance varied by site. Metabolic analysis further revealed region-specific microenvironments shaped by local cellular architecture. These findings indicate that both cellular composition and metabolic activity are spatially structured. Collectively, these results demonstrate that CIS exhibits significant intra-patient heterogeneity not captured by single-site profiling. These findings require validation in larger cohorts but support multi-region sampling could help improve risk stratification, biomarker development, and prediction of response to intravesical therapies, with potential implications for more personalized treatment strategies.

Purpose/Objective There is paucity of data reporting outcomes of breast cancers with initial internal mammary nodal involvement and no visceral metastases, treated with curative hypofractionated radiotherapy . We report the outcomes from a tertiary centre alongside spatial patterns of recurrences in the above group Material/Methods For this retrospective cross-sectional study, consecutive patients contoured as per the ESTRO 2013 guidelines, treated between 2016-2022 were eligible if their diagnostic imaging demonstrated involvement of the internal mammary nodes. Radiotherapy (40 Gy/15#/3 weeks) was delivered to the residual breast / thoracic wall, SCF region corresponding to the ESTRO lymph node level 4 and internal mammary chain nodes. Residual IMN/ level 4 nodes received a boost of 10Gy/5#. Spatial mapping of sites of recurrence at the local site and three nodal sites (axilla, SCF and IMN) was performed using deformable image registration. Sites of recurrence at the local site and three nodal levels were contoured separately. Volumetric intersection of the recurrent gross tumour volume (GTV_recurrence) with treated clinical target volume (CTV) was calculated. Actuarial overall (OS), disease free survival (DFS) & cumulative incidence of local (LR), regional (RR) and loco-regional recurrence(LRR) were calculated using Kaplan Meier method. Univariate comparison of outcomes with or without residual disease was performed using the log rank test. Results The median age of the 61 eligible women was 49 years. 77% received neoadjuvant chemotherapy and the rest adjuvant chemotherapy. 82% patients had a mastectomy. Axillary lymph node dissection was done in 96.7%. Boosts to residual IMN and SCF nodes were delivered to 21(34.4%) and 2 (3.3%) respectively. Median follow up was 3.6 years. Out of the 61 patients, 42 patients were disease free with an estimated 3 year disease free survival of 75% (95% CI 64, 88%). Spatial mapping of locoregional recurrence was possible in all but 1 patient with local (only) recurrence who was lost to follow-up after mammogram only. Among the patients with loco regional recurrence 1 had recurrence in local site + SCF +axilla, 3 had recurrence in the SCF+axilla, 2 in the SCF+IMN and 1 in the axilla+SCF+IMN. Only one patient had isolated axillary recurrence or isolated SCF recurrence. There were no IMN only recurrences. Among the 8 patients with nodal recurrence, a total of 27 individual GTV_recurrence were identified in the axilla(n=11), SCF(n=11) and IMN (n=5). IMN recurrences showed complete or partial overlap with CTV. SCF recurrences were a mix with predominantly in-field recurrences while axillary recurrences occurred outside the treated volume.Four (6.6%) patients had Grade 2 lymphoedema as documented late side effect. Conclusion Aggressive treatment of IMN disease with adjuvant radiation is effective with good locoregional control. Systemic recurrences are common and may benefit from intensification strategies.

Background & Aims: Fatigue is a central symptom of chronic liver disease (CLD), substantially impacting health-related quality of life (HRQoL). This study aimed to further understand CLD symptomatology, including fatigue, and its impact on HRQoL from a patient perspective. Methods: Abbott Global Assessment of Patients unmet needs (aGAP) was a multinational, cross-sectional survey in adults with compensated CLD in China, India and Mexico, conducted between July and November 2024. Adult participants who self-reported that they had physician-diagnosed CLD and were experiencing fatigue completed a quantitative survey to assess symptom burden and included three HRQoL patient-reported outcome (PRO) questionnaires (Patient-Reported Outcomes Measurement Information System [PROMIS]-29+2, Work Productivity and Activity Impairment - Specific Health Problem version 2.0 [WPAI: SHP], Multidimensional Fatigue Inventory [MFI]). Results: Overall, 505 participants (China: 200; Mexico: 105; India: 200) completed the study. Participants reported that their CLD-related fatigue sometimes, often or always affected their self-esteem/confidence (45.1%) and ability to maintain or acquire new employment (38.6%). Most participants reported moderate (51.3%) or serious (26.9%) fatigue, with 33.5% experiencing fatigue every day or almost every day. Many participants felt their social life was negatively impacted by their fatigue (47.3%) and that there were related financial difficulties (53.9%). Use of validated PRO tools demonstrated severe fatigue (MFI: overall mean [SD] 13.9 [3.4] general fatigue and 13.4 [3.6] physical fatigue) as well as substantial levels of work and activity impairment (WPAI: SHP overall mean [SD] 53.0 [26.4]) and high levels of anxiety, pain interference, depression and sleep interference (PROMIS T-scores [&ge;]54). Conclusions: Fatigue has a substantial impact on HRQoL among adults with CLD across several countries, highlighting a global unmet need for targeted interventions to effectively identify and manage the condition.