Clinical Cancer Research

Dedifferentiated liposarcoma is a rare mesenchymal malignancy driven by amplification of chromosome 12q13-15, which includes the oncogenes CDK4 and MDM2. CDK4 amplification provides a rationale for targeted therapy with CDK4/6 inhibitors, and abemaciclib has shown the most durable activity reported to date in this disease. Clinical responses, however, are incomplete and often transient, and the cellular features that allow tumor cells to continue proliferating during treatment are not well understood. To address this gap, we performed multiplexed single-cell imaging to quantify 17 cell-cycle regulators in both dedifferentiated liposarcoma cell line Lipo246 and surgically resected primary human cells exposed to abemaciclib. Both models contained a subpopulation of cells that retained phosphorylated retinoblastoma protein, a marker of cell proliferation, at the highest abemaciclib doses. These fractionally resistant cells were defined by selective enrichment of cyclin-dependent kinase 2 (CDK2), cyclin B1, and phosphorylated ribosomal protein S6 (pS6), and showed enhanced sensitivity to the CDK2 inhibitor, tagtociclib. Together, these findings reveal nongenetic cell cycle plasticity as a mechanism of escape from CDK4/6 inhibition in dedifferentiated liposarcoma and nominate CDK2 and the PI3K-mTOR pathway as candidate targets for combination therapy.

Background and ObjectivesIntravesical gemcitabine/docetaxel (Gem/Doce) is an effective therapy for Bacillus Calmette- Guerin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC), achieving 50% complete responses at 2 years. However, the genomic determinants underlying response and resistance to Gem/Doce remain poorly defined. Our objective was to define the mutational landscape of BCG-unresponsive NMIBC and nominate genomic features associated with response or resistance Gem/Doce. MethodsPatients with BCG-unresponsive NMIBC treated with Gem/Doce were classified as responders (recurrence-free survival [RFS] >12 months) or non-responders (RFS <12 months). Whole-exome sequencing was performed on tumors prior to Gem/Doce treatment (n=23). Single nucleotide variants were identified and annotated using a Cancer Genome Analysis pipeline. Copy number alterations were inferred with ABSOLUTE, and clonal architecture was reconstructed using PhylogicNDT. Key Findings and LimitationsResponders demonstrated significantly prolonged time to high-grade recurrence (3.5 vs 42 months, p<0.001) and cystectomy compared with non-responders (9.5 months vs not reached; p<0.001). Non-responders exhibited higher tumor mutational burden (13.66 vs 8.71; p=0.02) and more frequent whole-genome doubling (2/2 non-responders vs 0/1 responders; p=0.33). Phylogenetic analyses revealed clonal BAP1 and subclonal BRCA2 mutations in responders, whereas non-responders harbored clonal FGFR3 mutations. Limitations include small sample size and retrospective design. Conclusions and Clinical ImplicationsDistinct genomic features underlie differential response to Gem/Doce in BCG-unresponsive NMIBC. In responders, alterations in DNA repair pathways (e.g., BRCA2) may sensitize tumors to chemotherapy, while non-responders with FGFR3 mutations may benefit from alternative targeted strategies. These findings warrant validation in larger cohorts and support the development of biomarker-driven clinical trials. Patient summaryIn this report we analyzed bladder tumors and found that some tumors respond well to treatment because they have defects in repairing DNA, making them more vulnerable to chemotherapy. In contrast, tumors that do not respond to chemotherapy harbor different genetic changes that help them survive and grow. These findings may help physicians choose more effective and personalized treatments in the future.

Background: CD8+ tumor-infiltrating lymphocytes (TILs) are established prognostic markers in colorectal cancer, yet the clinical significance of CD103+CD8+ tissue-resident memory-like (TRM-like) T cells in locally advanced rectal cancer (LARC) after neoadjuvant chemoradiotherapy (NACRT) remains unknown. Methods: We quantified CD8+ and CD103+CD8+ T-cell densities in stromal and intratumoral compartments of post-NACRT resection specimens from 40 LARC patients using Cu-Cyto, a deep learning-based imaging cytometry platform. Associations with survival, pathological response, and adjuvant chemotherapy (AC) were examined. Treatment-induced T-cell dynamics were assessed in paired pretreatment biopsies and post-NACRT resections (n = 9). Results: High stromal CD103+CD8+ density independently predicted better 5-year RFS (67.4% vs. 12.1%, p < 0.001) and OS (80.0% vs. 26.6%, p = 0.016); intratumoral density showed no prognostic significance. Pathological response correlated with stromal CD8+ but not CD103+CD8+ density. Paired analysis revealed a selective non-expansion of the CD103+ subset: stromal CD8+ T cells increased significantly after NACRT while CD103+CD8+ density remained unchanged. AC may preferentially benefit patients with low stromal CD103+CD8+ density. Conclusions: Stromal CD103+CD8+ T-cell density is a robust independent prognostic biomarker in rectal cancer after NACRT that appears to reflect pre-existing rather than treatment-induced immunity. Given its stability across NACRT, pretreatment biopsy assessment may provide equivalent prognostic information, with potential implications for patient stratification before treatment initiation.

BackgroundConvection-enhanced delivery of topotecan enables sustained local chemotherapy for recurrent glioblastoma and was associated with reduced tumor proliferation in our previous phase 1B clinical trial. That trial incorporated a paired pre- and post-treatment biopsy design - rare in glioblastoma clinical research - enabling tissue-anchored assessment of drug effect without reliance on radiographic or survival endpoints, which are notoriously difficult to interpret in this disease. However, the cellular and molecular consequences of local chemotherapy within the treated tumor microenvironment remain incompletely defined. MethodsWe integrated paired, MRI-localized pre- and post-treatment biopsies from a first-in-human CED-topotecan trial (n=5), leveraging the paired biopsy architecture, in which each patient serves as their own control and post-treatment specimens are spatially annotated relative to the MRI-defined infusion zone, to generate tissue-based evidence of drug effect without requiring large patient numbers. These biopsies were integrated with complementary experimental models, including a time-resolved syngeneic murine glioma CED model, acute patient-derived glioblastoma slice cultures, and in vitro human microglial and glioma systems. Clinical biopsies were analyzed by bulk RNA-seq, cell-type deconvolution, and multiplex immunofluorescence. Murine tumors were analyzed by survival, immunofluorescence, and single-cell RNA-seq; patient-derived slice cultures were profiled by single-cell RNA-seq. ResultsIn paired human biopsies, CED-topotecan induced spatially restricted transcriptional remodeling within the infusion zone, characterized by suppression of proliferative tumor programs and enrichment of inflammatory, interferon, hypoxia, and mesenchymal signatures. Cell-type deconvolution and immunofluorescence linked this response to myeloid remodeling, including enrichment of monocyte-derived tumor-associated macrophage states, increased MARCO-positive myeloid populations, and pH2AX-positive genotoxic stress within Iba1-positive myeloid cells. In the murine CED model, topotecan prolonged survival and reduced tumor cellularity, while also inducing inflammatory and DNA-damage programs in tumor-associated macrophages that evolved by 7-days toward hypoxia, angiogenesis, TGF-{beta} signaling, and mesenchymal/tissue-remodeling programs. Human slice culture and in vitro microglial systems confirmed stress-coupled inflammatory and DNA-damage responses in human myeloid cells. ConclusionsLocal topotecan delivery produces spatially structured tumor cytotoxicity together with a genotoxic, stress-coupled inflammatory myeloid response that evolves toward mesenchymal macrophage remodeling. By integrating paired clinical biopsies with time-resolved and mechanistic experimental models, this study provides a framework for understanding how local chemotherapy reshapes the glioblastoma microenvironment and for future studies evaluating dose, schedule, treatment duration, and combination strategies. These findings demonstrate that paired, spatially annotated tissue sampling from small, precisely characterized clinical cohorts can yield mechanistic insight that conventional radiographic and survival endpoints cannot provide, and support tissue-based response assessment as the appropriate paradigm for evaluating novel locoregional therapies in glioblastoma.

Aims Active surveillance (AS) allows selected men with localized prostate cancer to defer curative therapy and reduce treatment morbidity. Conversion from AS to treatment is commonly triggered by Gleason grade group (GGG) upgrading on confirmatory biopsy. We developed and validated a digital pathology artificial intelligence (DPAI) biomarker to predict GGG upgrading in AS-eligible patients. Materials & Methods The DPAI model was trained using histopathology image features from diagnostic biopsies of 998 patients and validated in an independent cohort of 296 patients meeting criteria for AS. Logistic regression estimated the probability of confirmatory-biopsy GGG increase, and feature selection identified the most predictive variables. Results AI-GUR (Artificial Intelligence-Gleason Upgrade Risk) predicted GGG reclassification at confirmatory biopsy (OR 1.60; p=0.0003), and provided information beyond conventional stratification (risk group, CAPRA) and cribriform morphology (all p<0.01). Predicted risks were similar across time from diagnosis (~10-15% to ~85% at 1, 1.5, or 2 years; p for time=0.50), consistent with initial biopsy mischaracterization rather than time-dependent progression. Conclusions AI-GUR provides individualized estimates of confirmatory-biopsy GGG upgrading for AS candidates. Using DPAI may improve shared decision-making by complementing standard clinicopathologic tools and molecular testing using the same biopsy specimen, while informing the likelihood of grade upgrade at confirmation.

BackgroundRadiotherapy efficacy is constrained by an immunosuppressive tumor microenvironment (TME) enriched in extracellular adenosine and suppressive myeloid populations that attenuate cytotoxic T-cell responses. The CD73-adenosine-A2a/A2b receptor axis represents a key metabolic immune checkpoint; however, the relative contributions of tumor cell-intrinsic versus host-derived adenosine signaling to radiotherapy response remain incompletely defined. MethodsUsing orthotopic murine breast carcinoma models, we interrogated radiation-induced adenosine dynamics and downstream immune remodeling through quantitative adenosine measurements, bulk RNA sequencing, and multiparameter flow cytometry. Genetically engineered models were employed to dissect the roles of tumor-derived CD73 and host A2a/A2b receptors in regulating radiosensitivity. Therapeutic studies evaluated combinatorial targeting of CD73 and A2a/A2b receptors with radiotherapy and anti-PD-1, followed by comprehensive immune profiling in breast carcinomas. ResultsTumor cell-intrinsic CD73 and host A2A receptor signaling cooperatively drive radioresistance and tumor progression. Radiotherapy induces a rapid surge in intratumoral adenosine, triggering transcriptional and cellular programs consistent with myeloid-mediated immunosuppression and lymphocyte dysfunction. Although T-cell infiltration increases at later time point post-irradiation, effector function remains constrained. Pharmacologic inhibition of CD73 and A2a/A2b receptors partially restores T-cell functionality but is insufficient for durable tumor control as monotherapy. In contrast, concurrent blockade of adenosine signaling during radiotherapy, followed by adjuvant PD-1 inhibition, amplifies adaptive antitumor immunity and significantly enhances tumor control. ConclusionsThese findings define a mechanistic link between radiation-induced adenosine signaling and immune dysfunction in the TME. Targeting the CD73-A2a/A2b axis in combination with radiotherapy and checkpoint blockade represents a rational strategy to overcome radioresistance and improve antitumor immunity. STATEMENT OF SIGNIFICANCEThe tumor and immune cell contributions to adenosine signaling play a central role in shaping the therapeutic outcomes of tumor irradiation. Therapeutic targeting of the adenosine signaling axis improves radiosensitivity and efficacy of checkpoint blockade.

BackgroundTumour-associated macrophages (TAMs) play critical roles within the tumour microenvironment regulating immune evasion and therapeutic response. Previously, we have shown that the combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea (LDHU) reprograms the tumour immune microenvironment to a pro-inflammatory status. MethodsWe investigated a tumour-restricted Fcgr4 (Cd16.2) expressing macrophage population in multiple murine tumour models and the impact of CHK1i+LDHU on this population, using conventional and imaging flow cytometry as well as single-cell sequencing. ResultsTranscriptional profiling using CITE-seq and single-cell RNA sequencing reveals that Fcgr4 TAMs closely resemble Fcgr4- TAMs but display modest enrichment of interferon-associated and inflammatory gene programs, consistent with a functionally biased state rather than a distinct lineage. Importantly, we show that a highly tumour selective CHK1i+LDHU therapy shifts TAMs toward a more inflammatory phenotype while preserving dominant immunosuppressive features. Depletion of CSF1R macrophages enhanced CD8 T cell activation without influencing tumour growth but significantly augmented therapeutic efficacy of CHK1i+LDHU. ConclusionTogether, these findings define a novel TAM population and establish how targeted therapy reshapes, but does not fully overcome, TAM-mediated immune regulation.

Therapeutic resistance remains a major challenge in advanced and recurrent endometrial cancer (EC). Aberrant NOTCH signaling has been associated with aggressive tumor behavior and therapeutic resistance across multiple malignancies, yet its therapeutic significance in EC remains incompletely defined. We investigated whether auranofin (AuR), a noncanonical modulator of NOTCH signaling through the transcriptional effector RBPJ, alters platinum responsiveness in EC models. Elevated NOTCH3 copy-number was associated with poorer overall survival in the TCGA-UCEC cohort. AuR treatment reduced RBPJ occupancy at canonical NOTCH target loci, including HES1 and HES4, across multiple EC models. Stable NOTCH3 depletion altered AuR responsiveness in a context-dependent manner while significantly enhancing cisplatin (CDDP) sensitivity in AN3CA cells. Pharmacologic AuR treatment similarly potentiated CDDP response in AN3CA cells and AN3CA xenografts, resulting in reduced tumor burden and prolonged endpoint-free survival following combination treatment. In contrast, ARK-1 xenografts demonstrated limited additional benefit from combined AuR plus CDDP therapy despite detectable suppression of RBPJ occupancy. Together, these findings identify context-dependent NOTCH-associated therapeutic vulnerabilities in EC and support further development of biomarker-guided AuR-based platinum-sensitization strategies. Statement of significanceAuranofin suppresses RBPJ-associated transcriptional activity and enhances cisplatin response in biologically distinct subsets of endometrial cancer.

Background Endometrial cancer exhibits marked molecular and immune heterogeneity that is only partially explained by established genomic biomarkers. We investigated whether T cell receptor (TCR) repertoire architecture captures complementary dimensions of antitumor immunity beyond conventional molecular classification. Methods Paired tumor and peripheral blood samples from eight patients with molecularly characterized endometrial cancer underwent TCR repertoire profiling. Diversity, clonality, and tumor blood overlap metrics were integrated with genomic variables, including tumor mutational burden (TMB), genomic instability metric (GIM), and POLE status. Principal component analysis and correlation analyses were used to identify major dimensions of repertoire organization. Composite Immune Focusing and Immune Sharing Scores were derived to summarize dominant repertoire patterns. Results The first two principal components explained 70.1% of total repertoire variance and revealed substantial heterogeneity independent of histological subtype. TMB was strongly associated with reduced repertoire diversity and increased clonal dominance, resulting in a robust association with the Immune Focusing Score ({rho} = 0.88, p = 0.004). POLE mutated tumors occupied the extreme end of this focusing continuum. In contrast, genomic instability was associated with increased tumor blood repertoire overlap and preserved diversity, reflected by a strong correlation between GIM and the Immune Sharing Score ({rho} = 0.76, p = 0.027). The two immune scores showed minimal correlation with each other ({rho} = -0.24, p = 0.57), indicating that they capture largely independent aspects of immune organization. Conclusion Integrative analysis of TCR repertoire architecture and tumor genomics identifies distinct immunogenomic states in endometrial cancer that are not fully captured by conventional molecular classification. If validated in larger cohorts, immune focusing and immune sharing metrics may provide complementary biomarkers for patient stratification and immunotherapy-oriented precision oncology

While prostate cancer (PC) is defined as immunologically cold, limiting the efficacy of immune checkpoint inhibitors, therapeutic vaccination targeting tumor-associated antigens represents an attractive strategy to promote disease control in low volume metastatic patients. The UV1 cancer vaccine is based on immunization with tripeptide fragments from human telomerase reverse transcriptase (hTERT) and a phase II clinical trial demonstrated induction of robust T cell response in men with de novo metastatic castration-sensitive prostate cancer (mCSPC). Comparison with long-term survival data of non-metastatic CSPC patients as reference showed that despite metastatic disease at diagnosis, UV1-treated patients who mounted an early vaccine-induced immune response achieved progression-free and overall survival comparable to non-metastatic patients. We examined biological determinants of clinical benefit following UV1 vaccination including tumor transcriptome and T cell receptor (TCR) profiling from circulating and tissue resident T-cells of the 22 men enrolled. Analysis of diagnostic and post-UV1 treatment biopsies revealed that low baseline exhaustion of T cells and higher CD8+ T cell abundance are associated with early immune response to the vaccine and longer survival. Moreover, we identified specific TCR motifs relative to early responders, that can indicate potential benefit from UV1 vaccination. These findings indicate that baseline intratumoral T cell exhaustion state and repertoire shape responsiveness to hTERT vaccination and long-term outcome. Overall, our study underlines how baseline immune profiling may be used as a companion biomarker to predict mCSPC patients most likely to benefit from therapeutic vaccination.

Glypican-3 (GPC3) is an oncofetal protein widely being explored as a diagnostic and therapeutic target in hepatocellular carcinoma (HCC). Given that radiotherapy in the form of external beam and radioembolization are standard-of-care treatments for HCC, we aimed to determine whether there was any relationship between GPC3 and response to radiotherapy. Here, we demonstrate that GPC3 expression confers radioresistance in liver cancer through integrated in vitro, in vivo, and patient-level clinical analyses. Stable GPC3-knockout in liver cancer cell lines (HepG2, Hep3B, Huh7) and ectopic GPC3 expression in GPC3-negative liver cancer cells (SNU449), as well as in non-hepatic A431 cells, demonstrated that GPC3-mediated radioresistance is not restricted to hepatic lineage. Following irradiation, GPC3-deficient cells exhibited reduced proliferation, impaired clonogenic survival, persistent DNA damage, prolonged G2/M arrest, and increased apoptosis. Transcriptomic profiling demonstrated enrichment of cell-cycle and DNA damage response pathways in irradiated GPC3-deficient cells compared with GPC3-positive cells, and protein analyses confirmed sustained activation of the ATM/CHK2 axis. In vivo, GPC3 deletion markedly enhanced radiation-induced tumor growth delay in both HepG2 and A431 xenograft models. Consistent with these findings, high GPC3 expression was associated with inferior clinical outcomes in patients with HCC undergoing external-beam radiotherapy or radioembolization. Together, these findings identify GPC3 as a determinant of radioresistance in liver cancer and suggest its potential utility as a biomarker to guide radiotherapeutic strategies. Significance statementRadiotherapy is an important treatment option for HCC, but biomarkers that predict tumor response remain limited. GPC3 is highly expressed in most HCCs and is being investigated as an important biomarker for diagnosis and treatment of this disease, yet its relationship, if any, on radiosensitivity has not been previously reported. Here, we identify GPC3 as a modulator of radioresistance. GPC3 loss enhances radiosensitivity and is associated with persistent unresolved DNA damage, prolonged G2/M arrest, and sustained activation of the ATM/CHK2 pathway, resulting in delayed tumor growth after irradiation. In a clinical cohort of patients treated with radiotherapy, high GPC3 expression was associated with poorer overall survival. These findings suggest that GPC3 expressing tumors may necessitate either more dose-intense radiotherapy, radiobioligically ablative and/or combined with other modalities, or alternative therapeutic modalities to adequately treat HCC.

Background. Meningiomas exhibit well-established hormonal biology, yet no study has examined whether myeloid immune infiltration interacts with estrogen-responsive transcription in this tumor type. Methods. We applied three-method consensus immune deconvolution (EPIC, MCPcounter, CIBERSORTx) to 968 harmonized meningioma RNA-seq transcriptomes from five public datasets, stratified by Thirimanne et al. (2024) transcriptomic subtypes. Competitive gene set enrichment compared macrophage-high versus macrophage-low tertiles with sex-adjusted, purity-adjusted, and method-independent sensitivity analyses. Survival modeling tested both total macrophage burden and a decomposed microglia-to-macrophage ratio validated against single-cell ground truth (pseudo-bulk r = 0.77). Results. Macrophage-high tumors showed significant suppression of estrogen response gene sets (FDR = 4.9 x 10-5) despite paradoxical ESR1 upregulation (log2FC = +0.40, FDR = 2.5 x 10-26) and PGR downregulation (log2FC = -0.34, FDR = 2.7 x 10-3), indicating post-receptor transcriptional disruption. This signal strengthened after sex adjustment (FDR = 1.9 x 10-6) and was confirmed across a multi-layer sensitivity battery (eleven analyses including reference-matrix-independent, purity-adjusted, rotation-based self-contained, and empirical-null tests; all FDR < 3 x 10-4 in the relevant convergent tests). Myeloid infiltration was strongly subtype-dependent (Kruskal-Wallis p = 7.4 x 10-16) but grade-independent (p = 0.399), with CSF1R enriched in the macrophage-dominant Cluster B. Neither total macrophage score (HR = 0.90, p = 0.53; N = 102) nor a decomposed microglia/macrophage ratio (HR = 0.92, p = 0.46; N = 101) predicted recurrence-free survival. Conclusions. The pre-registered primary endpoint - macrophage infiltration score predicting recurrence-free survival - was not supported; the estrogen-immune dissociation emerged from secondary exploratory gene-set analysis and requires independent validation. Macrophage-infiltrated meningiomas exhibit a previously unreported dissociation between maintained ESR1 expression and suppressed estrogen-responsive transcription, with implications for hormonal therapy stratification.

The PRESERVE trial (NCT04972097) is a prospective, single-arm pivotal IDE study evaluating focal irreversible electroporation (IRE) using the NanoKnife System for intermediate-risk prostate cancer. Men with Gleason Grade Group 2-3 disease underwent focal IRE and were followed for durability of oncologic control and safety. At 24 months, 68 patients completed follow-up with no new treatment failures identified. PSA levels were below baseline in 97% of patients, and one clinically triggered biopsy was negative for cancer. No new device- or procedure-related adverse events occurred beyond 12 months. These findings demonstrate durable efficacy and sustained safety of focal IRE.

PurposeTo develop and validate a multimodal recurrence-risk model integrating histology, genomic testing, and clinical variables. MethodsWe developed AI-Path, a whole-slide image biomarker for recurrence prediction trained in CALGB 9344, and validated it in three independent cohorts: TAILORx, a multi-site Chicago cohort, and the MDX-BRCA cohort. We then integrated AI-Path with Oncotype DX Recurrence Score (RS), tumor size, and nodal status into a Cox model, PathClinRS, fit using 60% of cases from TAILORx, with the remaining 40% held out for validation. The primary end point was distant recurrence-free interval. Performance was assessed using Harrells concordance index (C-index) and Kaplan-Meier analyses. ResultsA total of 12,418 patients were included. In TAILORx, AI-Path outperformed RS for distant recurrence (C-index, 0.682 vs 0.647; P = .038), driven by superior prediction of late recurrence (0.656 vs 0.567; P < .001). In node-negative disease, PathClinRS outperformed RSClin in the TAILORx fitting (0.72 vs 0.70; P = .016) and validation sets (0.74 vs 0.70; P = .004). In node-positive disease, PathClinRS outperformed RSClinN+ in Chicago (0.94 vs 0.74; P < .001) and MDX-BRCA (0.71 vs 0.66; P = .004) cohorts. Compared with NATALEE eligibility, PathClinRS identified nearly twice as many high-risk node-negative patients while maintaining a comparable 10-year distant recurrence risk (16.7% vs 16.6% per NATALEE eligibility in TAILORx fitting; 21.0% vs 19.4% in TAILORx validation). PathClinRS identified 68% of intermediate risk premenopausal patients as low-risk with no evidence of chemotherapy benefit, compared to only 36% identified as low risk by standard clinicopathologic criteria. ConclusionDigital histopathology provides prognostic information complementary to genomic assays and has the potential to personalize therapy beyond existing clinicogenomic tools.

MotivationThe tumor microenvironment (TME) dictates cancer progression and therapeutic response, yet translating TME subtypes into robust clinical biomarkers remains a significant challenge. Existing classification models typically rely on static gene signatures and cohort-dependent normalization, making them ill-suited for application to the small, unbalanced datasets common in early-phase clinical trials. To better guide drug development, methods are required that offer the flexibility to target specific biological contexts and bridge the gap between the discovery of tumor archetypes and their robust translation to individual patient samples. ResultsWe developed TumorArchetypeR, a modular R package that unifies unsupervised subtype discovery with the generation of rank-based, single-sample classifiers. By leveraging a systematic parameter grid search, the framework identifies stable, data-driven subtypes rather than relying on arbitrary defaults. Crucially, to ensure clinical translatability, the package includes a module to train a robust classifier using binary gene-pair rules, enabling prediction without cohort-level preprocessing. Applying TumorArchetypeR to colorectal cancer, we resolved the heterogeneity of fibrotic tumors, distinguishing an immunosuppressive "Immune-enriched/Fibrotic" state from an immune-excluded "Fibrotic/Myeloid" phenotype. Furthermore, we identified a distinct "Th/B-cell enriched" archetype associated with superior survival, a group largely obscured by existing pan-cancer models. With our rank-based classifier demonstrating robust performance on previously unseen samples, these findings highlight TumorArchetypeR as a scalable, end-to-end solution for refining patient stratification and optimizing precision oncology strategies. The TumorArchetypeR package and documentation are openly available on GitHub at https://github.com/lutgem/TumorArchetypeR.

SUMMARYChordoma, a rare malignant notochordal tumor of the skull base and spine, is typically resistant to chemotherapy and radiotherapy and exhibits aggressive local recurrence. Here we show that chordoma recurrence correlates with a coordinated upregulation of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs), a low PFA/MUFA ratio and an adaptive, lipid peroxidation-resistant state that protects against DNA damage and cell death. Single-cell metabolic profiling identified a tumor subpopulation marked by a fatty acid biosynthesis-high state coupled to stemness. RT-tolerance was directly linked to elevated FASN and lipid droplet (LD) expansion, and MUFA-loading phenocopied RT-tolerance in chordoma cells. Mechanistically, LDs accumulated in response to RT via generation of ROS, and subsequent activation of ER-stress, SREBP1 and Fatty Acid Synthetase (FASN). DESI-MS showed that low-dose irradiation was sufficient to increase MUFAs early and build peroxidation resistant MUFA-LDs, whereas PUFA induction required a higher radiation dose. In a spatially defined manner in a patient-derived xenograft. Finally, in silico knockout and pharmacologic FASN blockade restored radiosensitivity and apoptosis in vitro and in vivo. Collectively, our result support a unifying model in which RT resistance in chordoma is shaped by an adaptive fatty acid metabolic program that buffers oxidative injury and increases survival of RT-resistant, stem-like tumor subpopulations. These findings further support FASN inhibition as a practical radiosensitization strategy for chordoma particulary where RT dose escalation is constrained by anatomy. KEYPOINTSO_LIRecurrent chordoma exhibits fatty acid-associated metabolic reprogramming. C_LIO_LIMUFA-associated lipid droplet accumulation is linked to radioresistance in chordoma cells. C_LIO_LITargeting FASN restores radiotherapy sensitivity of chordoma in vitro and in vivo. C_LI IMPORTANCE OF STUDYThis study underscores the clinical importance of targeting metabolic vulnerabilities to restore radiosensitivity in chordoma. By integrating transcriptomics, metabolomics, and in vitro and in vivo models, we identified adaptive fatty acid metabolic reprogramming as a central mechanism of RT resistance in chordoma. Recurrent tumors were characterized by coordinated enrichment of unsaturated fatty acids, especially monounsaturated fatty acids (MUFAs), together with a low PUFA/MUFA ratio and a lipid peroxidation-resistant state. Mechanistically, RT-tolerance chordoma cells exhibited a high-FASN state driven by activation of the ROS-ER stress-PERK/SREBP1/FASN axis, leading to intracellular lipid droplet expansion. Importantly, genetic and pharmacologic inhibition of FASN restored radiosensitivity and enhanced apoptosis in both in vitro and in vivo models, suggesting a translatable therapeutic strategy. Together, these findings link adaptive metabolic reprogramming to RT resistance and support new therapeutic approaches for chordoma management.

Liquid biopsies targeting circulating tumor DNA enable noninvasive cancer detection but lack sensitivity in pre- and early-cancer stages, where clinical benefits would be greatest. Human papillomavirus (HPV) causes six cancer types, accounting for 5% of all cancers worldwide. Targeting HPV cell-free (cf)DNA offers a compelling opportunity to overcome current liquid biopsy constraints due to its unique tumor-specific origin, lack of sequence homology to the human genome, and the high viral-to-human copy ratio per cell. Utilizing HPV-associated anal cancer and precancer as a model, here we applied a custom, multi-feature HPV whole-genome liquid biopsy to biobanked and prospective screening cohorts spanning the HPV infection-precancer-cancer continuum. HPV cfDNA was detected years before cancer diagnosis and as early as the infection stage, with increasing detection as stages advanced. Genomic hallmarks of HPV malignancy, including HPV integration, PIK3CA mutations, and 3q amplification, were detected exclusively in cancer, while precancers exhibited distinct HPV genotypes. Fragmentomics analysis of HPV cfDNA revealed stage-informative signatures reflecting viral epigenetic changes during carcinogenesis. A unified classifier incorporating genomic and fragmentomics features achieved a mean AUC of 0.77 for identifying cancer and high-grade precancer, stages requiring clinical intervention. Together, these findings demonstrate the feasibility of blood-based screening and molecular risk stratification for HPV-associated cancer and precancer. TeaserProfiling blood HPV cell-free DNA detects cancer years early and distinguishes precancers needing intervention from surveillance

Cholangiocarcinoma (CCA) is a highly aggressive malignancy characterized by poor prognosis, limited therapeutic options, and a predominantly immunosuppressive tumor microenvironment. Protein arginine methyltransferase 1 (PRMT1), the major mediator of asymmetric arginine dimethylation, has been implicated in multiple oncogenic processes, although its role in CCA remains unknown. Here, we demonstrate that PRMT1 is frequently overexpressed in human CCA and is associated with aggressive molecular subtypes and immune-desert tumors. Genetic dependency analyses and pharmacological inhibition using type I PRMT inhibitors markedly impaired CCA cell proliferation, clonogenicity, and tumoroid growth. Transcriptomic profiling revealed that PRMT1 inhibition induces broad alterations in gene expression and alternative splicing, affecting pathways involved in proliferation, apoptosis, DNA damage response, metabolism, and immune signaling. Mechanistically, PRMT1 targeting promoted genomic stress, accumulation of cytosolic double-stranded DNA, and activation of the cGAS-STING-TBK1-IRF3 signaling axis, resulting in enhanced interferon signaling and increased expression of T cell-recruiting chemokines, including CXCL9 and CXCL10. PRMT1 inhibition also synergized with cisplatin, poly-ADP-ribose polymerase (PARP) inhibition, and PRMT5 blockade in vitro and in patient-derived tumoroids. Importantly, in an aggressive orthotopic murine model of intrahepatic CCA, combined treatment with the PRMT1 inhibitor GSK3368715 and anti-PD-1 antibodies significantly reduced tumor burden and increased CD4+ and CD8+ T-cell infiltration compared with monotherapies. Collectively, these findings identify PRMT1 as a critical regulator of CCA growth and immune evasion and support the therapeutic potential of PRMT1 inhibition, particularly in combination with immunotherapy.

Background: The use of immune checkpoint inhibitors (ICIs) in the treatment of cancer has rapidly expanded over the last decade. However, there are several knowledge gaps in understanding how tumor cells evade the immune system. There is paucity of data in HPV negative oral cancer, particularly of the gingivobuccal region. Understanding the mechanism of immune system evasion in this cancer is vital for improving patient outcomes. Methods: We characterized the baseline immune milieu of oral cancer using immunohistochemistry (IHC) on whole tumor sections from 124 cases. Tumors were classified as hot or cold and further stratified into high-risk and low-risk groups. High-risk patients included those with lymph node metastasis at diagnosis/recurrence or distant metastasis within 2 years of treatment completion. Patients without these features were categorized as low risk. Validation by RNA-Seq and Joint Enrichment Analysis of Oncogenic and Immunologic Pathways was carried out in a subset of 46 cases. Results: Hot high-risk tumors (by IHC) were distinguished by elevated PD-L1 expression and reduced NK-cell, PD1, and CTLA-4 expression. There was no difference in the expression levels of CD3+, CD8+, granzyme, or perforin compared to hot low-risk tumors, findings that align with the definition of hot tumors. RNA-Seq revealed a gene signature associated with exhausted T-cells in hot high-risk tumors. Gene and pathway analyses identified differential upregulation of isoform-specific TOX, TCF, CXCR, RUNX, IRF, BRD and BCL6 genes, implicating immune cell exhaustion and tumor aggressiveness. Significantly downregulated genes included PDCD1, HAVCR2, ZAP70, and STAT, indicative of a disabled immune microenvironment. These findings support that a state of immune exhaustion in HHR tumors is driven by progenitor exhausted T-cells and terminally exhausted T-cells; independent of PD1-TIM3. Conclusion: These findings suggest that combining TOX/TCF/BCL6 inhibitors with immune checkpoint inhibitors in the adjuvant setting might benefit patients with hot high-risk tumors. Given the results, testing for a targeted exhaustion-related gene panel at diagnosis is recommended for oral cancers to stratify tumors as high-risk or low-risk. Larger validation studies and clinical trials are now warranted.

Functional ex vivo assays using live tumor tissues have demonstrated strong predictive accuracy for response to immune checkpoint inhibitors (ICIs) but are not scalable, requiring manual processing of large resections collected at academic centers. Here, an ex vivo live tumor fragment (LTF) platform was developed using standard-of-care biopsies from 228 patients with suspected malignancy collected across prospective, multicenter observational trials and biobanks. Hierarchical clustering of ICI-mediated changes in cytokine production identified two groups: responders and nonresponders. A binary classifier (elive index) using 8 cytokines achieved an AUC of 0.99 for cluster prediction. elive index correctly predicted clinical benefit in 93% (26/28) of patients (P = 3.2x10-5) and accurately identified 83% (10/12) of objective responders. Critically, elive responders were identified among biomarker-negative patients, highlighting the platform as a scalable approach that complements existing companion diagnostics and expands the population of patients identified to benefit from ICI therapy.