Clinical Cancer Research

Outcomes for high-grade pediatric brain tumor patients remain poor, but there is optimism that chimeric antigen receptor (CAR) T cell therapy can improve prognosis. We present the results from a phase I clinical trial of IL13BBz-CAR T cells infused weekly into the cerebral ventricles in pediatric and young adult patients with recurrent or refractory brain tumors. The trial met its primary objectives of feasibility, safety, and tolerability, with one dose-limiting toxicity. 8 of 16 patients evaluable for response experienced radiographic size decreases consistent with biologic activity and with an anti-tumor response. Two patients met protocol criteria for response. Median survival for patients receiving lymphodepletion was 20.5 months from diagnosis and 6.9 months from treatment for patients with midline glioma, and 187 months from diagnosis and 7.5 months from treatment for patients with ependymoma. Importantly, patients who did not receive lymphodepletion developed anti-CAR humoral and cellular immune responses detectable in the CSF and peripheral blood, whereas patients receiving lymphodepletion had no evidence of CSF anti-CAR immunity. Taken together, these findings demonstrate the safety, tolerability, and biological activity of locoregionally-delivered IL13BBz-CAR T cells for children and young adults with CNS tumors. Moreover, we show that anti-CAR immune responses arise in patients not receiving lymphodepletion, but not in the CSF of patients receiving systemic lymphodepletion. Further investigation of adoptive cellular therapies combined with immunosuppression is warranted in this patient population. ClinicalTrials.gov registration: NCT04510051.

Purpose: Response-adapted bladder preservation has emerged as a potential alternative to immediate radical cystectomy for selected patients with muscle-invasive bladder cancer (MIBC), but biomarkers to guide treatment de-escalation are lacking. We report the clinical outcomes of the phase II RETAIN2 trial together with a retrospective circulating tumor DNA (ctDNA) analysis of the RETAIN1 and RETAIN2 studies. Patients and Methods: RETAIN2 prospectively evaluated neoadjuvant accelerated methotrexate, vinblastine, doxorubicin, and cisplatin (AMVAC) plus nivolumab followed by response-adapted management based on clinical restaging. A retrospective tumor-informed ctDNA analysis evaluated longitudinal ctDNA dynamics and associations with clinical outcomes. Results: Seventy one evaluable patients were enrolled in RETAIN2. The trial met its primary endpoint, with a 2 year metastasis free rate of 77.5% after a median follow-up of 34.7 months. Among 22 patients managed with active surveillance, 15 (68.2%) remained metastasis free with an intact, non irradiated bladder and 3 (13.6%) developed metastatic disease. In a sensitivity analysis using time to metastasis, the Kaplan Meier estimated 2 year metastasis free probability was 83.7% overall and 85.5% with active surveillance. Retrospective ctDNA analyses were performed in 111 patients from RETAIN1 and RETAIN2. Baseline and post-treatment ctDNA positivity were associated with metastatic progression and inferior overall survival. Among patients managed with active surveillance who were ctDNA-negative after treatment, the 2 year Kaplan Meier estimated metastasis free probability and overall survival were 91% and 97%, respectively. Plasma ctDNA predicted metastatic progression but not intravesical recurrence. Conclusion: Response adapted bladder preservation after neoadjuvant AMVAC plus nivolumab achieved encouraging long term outcomes in selected patients with MIBC. Retrospective ctDNA analyses suggest that plasma ctDNA reflects occult systemic disease rather than bladder confined recurrence and may refine patient selection for bladder preservation. These findings support prospective evaluation of ctDNA guided strategies while emphasizing the continued need for bladder directed surveillance and complementary urinary biomarkers.

Purpose: Radiation-associated lower cranial neuropathy (LCNP) is a debilitating late complication among head and neck cancer (HNC) survivors, leading to progressive dysphagia, aspiration, and loss of nutritional independence. No proven therapies exist to reverse LCNP. This Phase I dose-finding trial (XXXX, XXXX) and parallel registry study prospectively evaluated the safety, feasibility, tolerability, and symptomatic response of high-dose corticosteroid therapy for radiation-associated LCNP. Methods and Materials: Eligible participants were disease-free oropharyngeal cancer survivors [≥]2 years post-radiotherapy with LCNP involving CN XII +/- X and no structural or malignant etiology. Phase I trial participants received oral prednisone 1 mg/kg (Dose 1, n = 3) or 3 mg/kg (Dose 2, n = 5) daily for 5 days followed by a 2-week taper. A parallel registry (n = 6) enrolled broader HNC survivors treated with 1 mg/kg. Phase I dose escalation decisions were based on the balance of tolerability and symptom response. The primary endpoint was change in MDASI-HN Top 5 mean symptom score from baseline to 1-2 weeks post-taper; a [≥] 1.315 unit decrease indicated clinically meaningful improvement. Secondary endpoints included clinician-graded measures of bulbar function, electromyography (EMG) and patient-report outcome measures. Results: All regimens were feasible and well tolerated. Insomnia (n = 4, Grade 1) was the most frequent adverse event and there were no treatment discontinuations. At 1-2 weeks post-taper, the median MDASI-HN Top 5 change was -0.2 in Dose 1 and -1.6 in Dose 2; three of five Dose 2 patients achieved clinically meaningful improvement versus one in Dose 1. By 6-10 weeks, symptom improvement persisted in a subset but diminished overall. Dose 2 met pre-specified criteria for Phase II progression. No consistent improvements were observed in objective functional or electrophysiologic measures, though exploratory trends favored higher dosing. Conclusions: High-dose corticosteroid therapy at 3 mg/kg/day was feasible and tolerable in long-term HNC survivors with LCNP and showed preliminary evidence of short-term symptomatic benefit. Phase II evaluation is warranted.

Background: Trastuzumab deruxtecan (T-DXd) is active in HER2-expressing solid tumours, but trials excluded HER2 immunohistochemistry (IHC) 1+ disease, and data in pretreated ovarian cancer are lacking. We evaluated real-world T-DXd activity and genomic correlates in pretreated ovarian cancer, predominantly high-grade serous (HGSOC). Methods: HER2 expression was assessed in an unselected ovarian cancer cohort (N=74). Fifteen patients receiving off-label T-DXd (14 HGSOC, 1 clear cell; IHC 1+ to 3+) had HER2 status centrally confirmed using gastric-type criteria. Activity was assessed by intra-patient growth modulation index (GMI; progression-free survival [PFS] on T-DXd divided by PFS on the prior line; [≥] 1.33 considered meaningful). Patients on treatment at data cut-off were censored. Objective response (RECIST 1.1) was assessed centrally where imaging was available (n=8). Results: Of the 40 HER2-expressing tumours, 15 received T-DXd, limited mainly by reimbursement. Among 14 evaluable patients (median 5 prior lines), 9 reached a GMI [≥] 1.33 (median 1.69); 8 remained on treatment at cut-off, making durability preliminary. Confirmed partial responses occurred across the HER2 spectrum. Benefit was independent of homologous-recombination (HR) status: one HR-proficient, CCNE1-wild-type patient achieved prolonged control and was rendered disease-free after radiotherapy to an oligoprogressive lesion. Exploratory analysis showed all four evaluable CCNE1-amplified tumours had reduced or non-durable benefit. Conclusions: T-DXd shows preliminary, clinically meaningful activity in HER2 IHC 1+ ovarian cancer independent of HR status. CCNE1 amplification may attenuate benefit, a candidate biomarker for WEE1-inhibitor combinations. Approval restricted to IHC 3+ disease would exclude most responders in this cohort. Prospective validation is required.

Purpose: Exceptional responses are frequent in patients with HER2-positive (HER2+) metastatic breast cancer (MBC), but predictive biomarkers are lacking. We aimed to investigate the association between detection of minimal residual disease (MRD) via circulating tumor DNA (ctDNA) and exceptional response to first-line HER2 targeted therapy for MBC. Patients and Methods: We identified exceptional (real-world progression-free survival [rwPFS] [&ge;]3 years) and conventional (rwPFS <3 years) responders treated with first-line HER2 targeted therapy for HER2+ MBC and plasma collected at landmark timepoints (e.g., baseline, year [Y] 1, Y2, Y3, at progression). We generated personalized, tissue-informed MRD assays using MAESTRO mutation enrichment sequencing in a pooled format. The primary endpoint was the association between MRD status at Y1 and rwPFS. Results: Of 70 patients, 63 (90%) (40 exceptional and 23 conventional responders) had sufficient samples and successful assay design; MAESTRO was run on 149 samples. A median of 1,823 (range 387-5,000) tumor-specific mutations were tracked per patient. MRD was detected in 49 (32%) samples (median tumor fraction [TFx] 936 ppm; range 3.8-164,068 ppm); 15 (31%) samples had TFx <100 ppm. MRD was associated with outcomes: 0/27 [0%] exceptional versus 9/12 [75%] conventional responders (p<0.001) had detectable MRD at Y1. Exceptional responders who remained progression-free were always MRD-negative (n=30) or cleared MRD by Y1 (n=3). Six exceptional responders experienced late progression, and four of them had a Y3 sample: MRD was detected in three patients (lead time range 2.77-13.47 years), one patient had breast-only progression and was MRD-negative. Conclusions: MRD status at key timepoints is associated with exceptional response and late distant progression, supporting prospective clinical trials implementing MRD testing with highly sensitive tumor-informed assays to guide treatment de-escalation.

Background and purpose. Patients with oropharyngeal cancer (OPC) treated with radiotherapy (RT) exhibit heterogeneous courses of radiation-associated dysphagia (RAD) during recovery, yet most survivorship models typically treat RAD uniformly. This study aimed to identify distinct, data-driven RAD longitudinal Phenotypes based on imaging-graded swallow function from pre-treatment to 30 months post-RT and to characterize their baseline predictors. Materials and methods. Heterogeneous linear mixed-effects latent class trajectory modeling was applied to longitudinal DIGEST scores from the Stiefel MDA-OPC prospective registry. Eligible patients had [&ge;]3 Modified Barium Swallow (MBS) assessments between baseline and 30 months post-RT. Models were evaluated across functional forms and 1-5 latent classes; final selection used the Bayesian Information Criterion. Baseline predictors of class membership were identified via binary logistic regression. Results. The cohort comprised 650 OPC patients (2,116 MBS assessments; mean age 61 years, 89% male, 93% HPV-positive). Four RAD Phenotypes were identified: No/Minimal RAD (n=385/650, 59%), Mild/Moderate RAD (n=104/650, 16%), Moderate/Severe Transient RAD (n=94/650, 15%), and Moderate/Severe Progressing RAD (n=67/650, 10%). Classification quality was acceptable (mean posterior probabilities 0.78-0.89; entropy 0.69). Baseline DIGEST impairment, base-of-tongue primary, advanced T stage, and age [&ge;]60 independently predicted membership in higher-burden Phenotypes (AUC=0.845; 10-fold CV-AUC=0.835). Conclusion. RAD following RT for OPC comprises four biologically and clinically distinct longitudinal Phenotypes, predictable from pre-treatment characteristics. These findings support trajectory phenotyping as outcome framework for RAD research and risk-adaptive survivorship care.

Epithelioid sarcoma (EpS) is an ultra-rare, aggressive soft tissue sarcoma (STS) driven by INI1 loss and consequent hyperactivation of the chromatin-modifying enzyme EZH2. Although the EZH2 inhibitor tazemetostat has shown clinical activity, responses remain limited, highlighting the need for improved treatment strategies. Here, using two in-house generated patient-derived xenograft models and matched cell lines derived from INI-1 deficient EpS, we investigated EZH2 inhibition in combination with doxorubicin, the first-line standard for advanced STSs, identifying distinct patterns of response and resistance. Integrated transcriptomic and functional analyses revealed that response to EZH2 inhibition-based therapy was associated with chromatin remodeling, characterized by increased H3K27 acetylation and downregulation of histone deacetylase (HDAC)-related transcriptional programs. Conversely, the intrinsically resistant model failed to undergo this epigenetic transition despite EZH2 inhibition. Pharmacological HDAC inhibition restored H3K27 acetylation, promoted apoptosis, and enhanced the activity of EZH2 inhibition-based therapy. These findings identify failure to accumulate H3K27 acetylation as a hallmark of resistance to EZH2 inhibition-based treatment, and show that pharmacological HDAC inhibition can restore this chromatin transition and re-sensitize resistant tumors, providing a rationale for combined epigenetic targeting strategies in INI1-deficient malignancies. Translational relevanceProspective trials are challenging in rare tumors such as epithelioid sarcoma (EpS), limiting the level of evidence for existing therapies and the development of new agents. This is particularly relevant for EpS, where drug regimens are those used for all soft tissue sarcomas (STSs), and the specific mechanisms of drug response remain poorly understood. This preclinical study of tazemetostat in combination with doxorubicin shows differential outcomes in two INI1-deficient proximal-type EpS models, providing evidence of the heterogeneity that exists even within the same tumor subtype and fostering the need to better understand the molecular mechanisms driving drug sensitivity/resistance in this disease. In addition, we demonstrated the potential to treat EpS models through modulation of epigenetic mechanisms, showing that HDAC inhibition may restore sensitivity to EZH2-targeted therapy. These findings support the rationale for developing combination strategies incorporating epigenetic modulators and provide a preclinical framework for overcoming resistance to current therapies in EpS.

Background The value and optimal timing of circulating tumour DNA (ctDNA) analysis in locally advanced oesophageal adenocarcinoma (OAC) is uncertain. We hypothesised that perioperative detection would predict event-free (EFS) and overall (OS) survival, and that 3-6 months post-operative detection would predict recurrence. Methods In this prospective multi-centre cohort study, tumour-informed ctDNA assays were designed for 49 patients using whole-exome sequencing. Bloods were collected for ctDNA detection up to 8 days prior to surgery, and at 3-6 weeks and 3-6 months post-surgery, then correlated with clinicopathological characteristics, EFS and OS. Results Pre- and early post- surgery ctDNA positivity were associated with worse EFS (HRs 7.97 (95% confidence interval, CI 2.64-24.04), p<0.0001; 8.18 (95%CI 3.23-20.69), p<0.0001) and OS (HRs 7.82 (95%CI 2.22-27.54), p=0.00018; 13.69 (95%CI 4.52-41.49), p<0.0001). In pre-surgery positive patients, post-surgery ctDNA clearance associated with improved EFS and OS, and predicted better OS in those with a poor histopathological response to neoadjuvant treatment. 3-6 month ctDNA-positivity preceded standard-of-care recurrence detection by median 53.5 (interquartile range 39.3-200.0) days. Conclusions Perioperative ctDNA positivity associates with worse EFS and OS in OAC, identifying a subgroup with improved outcomes despite adverse pathological features. ctDNA testing at 3-6 months predicts recurrence earlier than standard-of-care surveillance.

Background: Circulating tumor DNA (ctDNA) is an emerging biomarker of treatment response and recurrence risk, while residual cancer burden (RCB) after neoadjuvant treatment (NAT) is a well-established risk factor for distant recurrence. Here, we examined the association between high ctDNA concentration at diagnosis and risk of distant recurrence after neoadjuvant treatment (NAT), in the context of RCB. Methods: The study included 712 patients with high-risk breast cancer in the neoadjuvant I-SPY2 trial. Tumor-informed ctDNA test results at diagnosis were used to stratify patients into ctDNA-negative and ctDNA-positive groups. For this analysis, the ctDNA-positive group was divided into tertiles (low, intermediate, high) based on ctDNA concentration reported as mean tumor molecules per mL [MTM/mL] of plasma. Correlations between MTM/mL at diagnosis and ctDNA dynamics during NAT, residual cancer burden (RCB), and distant recurrence-free survival (DRFS) were examined across all subtypes. Results: In all subtypes, high ctDNA concentration at diagnosis was associated with worse DRFS, whereas low ctDNA concentration or ctDNA-negative status was associated with improved DRFS, even with high tumor burden after NAT (RCB-II/RCB-III). We also found that patients with high ctDNA concentration, regardless of subtype, were less likely to experience early ctDNA clearance; however, those who did had a significantly higher likelihood of achieving a favorable response (RCB-0/RCB-I) than those with late or no ctDNA clearance. Furthermore, across all subtypes, patients with early ctDNA clearance, including those with substantial residual cancer (RCB-II/RCB-III) after NAT, had improved DRFS, irrespective of the ctDNA concentration at diagnosis. Conclusions: Across all subtypes, pathologic response and ctDNA clearance reduce the risk of distant recurrence associated with high ctDNA concentration at diagnosis. ctDNA concentration at diagnosis and ctDNA clearance dynamics during NAT may facilitate the prediction of treatment response and further stratify the risk of metastatic recurrence in non-responders.

Background: Liquid biopsy-based monitoring of circulating tumor DNA (ctDNA) holds promises for real-time assessment of tumor burden and treatment response in precision oncology. However, mutation-based approaches alone show limited sensitivity, particularly in low-shedding tumors. We evaluated whether integrating epigenomic biomarkers (specifically LINE-1 retrotransposon (L1PA) hypomethylation and copy number variation (CNV)) with standard mutation-based ctDNA analysis could improve cancer detection and longitudinal monitoring in patients enrolled in the SHIVA02 precision oncology trial. Methods: We performed a retrospective analysis of 32 patients with advanced or metastatic solid tumors who received molecularly matched targeted therapies within the SHIVA02 trial (NCT03084757). Plasma samples were collected at baseline and longitudinally every two months and at progression. Multimodal ctDNA profiling was performed using the DRAGON targeted NGS panel covering SNVs, indels and focal CNVs in 571 genes and the DIAMOND assay profiling L1PA methylation and genome-wide CNV. A three-step classification algorithm integrating maximum variant allele frequency (MaxVAF), L1PA methylation-based cancer probability (MethPCancer), and genome-wide CNV scores was developed to maximize ctDNA detectability. Results: At baseline, mutation-based profiling detected ctDNA in 62.5% of patients. L1PA hypomethylation alone identified ctDNA in 78.1% of patients, including cases with undetectable mutations. The three-step integrative model increased overall detectability to 93.8%. Concordance analyses between tumor tissue and plasma revealed that 80.6% of mutations and 60% of CNVs identified in tumor biopsies were detectable in matched ctDNA. The three modalities showed limited pairwise correlation at baseline, supporting their complementarity. Longitudinal analysis demonstrated that changes in MaxVAF, MethPCancer, and L1PA CNV scores over time were informative on treatment response across tumor types, with ctDNA detected in 94.5% of samples collected at disease progression. In selected patients, ctDNA alterations preceded radiological progression by four months. Conclusions: Multimodal ctDNA profiling integrating mutation analysis, CNV profiling, and LINE-1 hypomethylation substantially improves ctDNA detection at baseline and during treatment in a pan-cancer precision oncology setting. These complementary genomic and epigenomic biomarkers provide a more comprehensive and dynamic assessment of tumor burden than single-modality approaches, supporting prospective validation in larger cohorts for integration into precision oncology workflows.

Circulating prostate-specific antigen (PSA) discriminates poorly in the diagnostic gray zone (3.0-9.99 ng/mL), where ~75% of biopsies yield no clinically significant prostate cancer (PCa). We evaluated whether urinary creatine riboside (CR), a tumor-derived metabolite excreted through the prostatic urethra, complements PSA for gray-zone detection and independently predicts prostate-cancer-specific mortality (PCSM). In the NCI-Maryland PCa Case-Control Study (951 cases, 962 controls; 47.6% African American men; median follow-up 11.5 years), urinary CR was quantified by UPLC-MS/MS. Within the PSA gray zone (n = 668), urinary CR was complementary to PSA, with markedly higher single-marker discrimination than PSA (AUC 0.93, 95% CI 0.88-0.98 vs 0.77, 0.66-0.89) and additive when combined ({Delta}AUC +0.17, p < 0.001; 91.4% sensitivity at 80% specificity). After adjustment for 11 clinical and sociodemographic covariates, urinary CR independently predicted PCSM complementary to PSA (Fine-Gray SHR 1.72, 1.35-2.19 for CR; 1.35, 1.08-1.68 for PSA; Harrell's C 0.85 for CR + PSA vs 0.77 for PSA alone), with strongest signal in African American men (SHR 2.43, 1.57-3.75 for CR). We conclude that urinary CR is a candidate non-invasive biomarker complementary to PSA - improving gray-zone triage and predicting PCSM; prospective validation in biopsy-referred cohorts is warranted.

Background The relationship between total cell-free DNA (cfDNA) concentration and multi-cancer early detection (MCED) sensitivity is non-obvious on account of competing considerations. On the one hand, this concentration is elevated in cancer and increases in advanced disease, suggesting higher concentrations may be associated with more biologically active tumors that are easier to detect. On the other hand, this elevation is known to be largely leukocyte-derived, which may dilute tumor-derived DNA (ctDNA) and make detection harder. The net direction of these competing effects on detection sensitivity has not been systematically examined. Methods EMERGE is an observational case-control study conducted at 43 Indian sites from June 2022-February 2025. It prospectively enrolled and analyzed 1,030 treatment-naive participants with malignant or benign conditions, most presenting symptomatically, along with 450 controls aged [&ge;]50 years without prior malignancy. Plasma cfDNA underwent targeted hybrid-capture enzymatic methylation sequencing. Classifiers were trained for cancer detection and tissue-of-origin prediction, and tested on the independent validation set. Primary outcomes were the associations between total cfDNA concentration and (i) detection sensitivity and (ii) tissue-of-origin accuracy, evaluated in an independent validation cohort. Results After adjustment for cancer type, stage, demographic and technical covariates, cfDNA concentration was significantly associated with detection sensitivity (p=6x10-4) but not with tissue-of-origin accuracy (p=0.67). At 0.986 specificity (95% CI: 0.968-1.000), stage I sensitivity rose monotonically from 0.52 (95% CI: 0.34-0.69) in the lowest cfDNA concentration tertile to 0.85 (95% CI: 0.73-0.97) in the highest. This association was mechanistically supported by a region-specific increase in hypermethylation scores within regions identified as differentially hypermethylated in TCGA tumor tissue, while panel-wide scores declined. The dissociation between the concentration-sensitivity and concentration-tissue-of-origin associations, together with inverse or insignificant correlations between ctDNA fraction and cfDNA concentration at early stages in published datasets, suggests that the concentration-sensitivity association is partly independent of ctDNA fraction. Conclusions Total cfDNA concentration is a routinely measured determinant of MCED assay sensitivity, reflecting enrichment of tumor-associated aberrant methylation partly independent of ctDNA fraction, an association likely most pronounced in symptomatic cohorts. Standardized reporting of cfDNA concentration could improve cross-study benchmarking. Study Registration Clinical Trials Registry, India: CTRI2022/05/042936 Keywords Cell-free DNA (cfDNA), Tumor Fraction, Circulating Tumor DNA (ctDNA), Circulating Mutant Allele Frequency (cMAF), Multi-cancer early detection (MCED), cfDNA Concentration, Tissue-of-Origin (TOO), Methylation, Epigenomics

PurposeSWI/SNF (BAF) chromatin remodeling complex alterations are common in urothelial carcinoma, yet no biomarker-directed therapeutic strategies have been established for this population. We investigated whether BAF alterations delineate a biologically distinct, therapeutically actionable urothelial carcinoma subtype. Experimental DesignWe performed integrative genomic and transcriptomic analyses of 792 urothelial carcinoma tumors from the Oncology Research Information Exchange Network (ORIEN) and validated findings in the TCGA-BLCA cohort. Mechanistic studies incorporated RNA sequencing and ATAC-seq following histone deacetylase (HDAC) inhibition. Functional dependencies were assessed using patient-derived xenograft organoids and cell line models. Clinical relevance was explored in a biomarker-enriched investigator-initiated trial. ResultsApproximately half of urothelial carcinoma tumors exhibited BAF alterations, defining a previously unrecognized chromatin-altered molecular subtype characterized by activation of proliferative programs, loss of lineage identity, and altered metabolic signaling. This subtype was enriched for transcriptomic programs associated with HDAC inhibitor sensitivity and depleted of HDAC inhibitor resistance signatures. Mechanistically, HDAC inhibition induced widespread chromatin remodeling with reduced accessibility at AP-1 and TEAD-associated regions, and downregulation of E2F- and MYC-driven transcriptional networks. Functional studies confirmed enhanced HDAC inhibition sensitivity in ARID1A-mutated cell lines and a patient-derived organoid model. Early clinical observations demonstrated a durable responder treated with HDAC inhibitors and immunotherapy. ConclusionsBAF alterations define a chromatin-dependent tumor state in urothelial carcinoma that is selectively vulnerable to HDAC inhibition. Integrating genomic, epigenomic, functional, and early clinical evidence, these findings provide a rationale for biomarker-enriched clinical trials and HDAC inhibitor-based combination strategies in urothelial carcinoma.

Tumor-infiltrating lymphocytes (TILs) are widely used to assess antitumor immunity in breast cancer but may not reflect the functional competence of adaptive immune responses. We show that immune organization, reflected by tertiary lymphoid structures (TLS) and coordinated humoral and cellular immune programs, represents a distinct dimension of tumor immunity beyond lymphocyte abundance. By integrating histologic, transcriptomic, spatial, and immune receptor profiling analyses across multiple breast cancer cohorts, we show that immune organization is associated with greater immune repertoire diversity, evidence of therapy-induced clonal selection, and improved clinical outcomes, independent of immune infiltration. Transcriptomic measures of immune organization retained independent prognostic value across external cohorts, whereas measures of immune infiltration did not. Furthermore, treatment-induced increases in immune organization, but not immune infiltration, were associated with therapeutic response. These findings identify immune organization as a dynamic and clinically measurable state of adaptive antitumor immunity with implications for prognosis, treatment monitoring, and therapeutic development in breast cancer.

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.

The limited efficacy of immune checkpoint inhibitor (ICI) therapy in triple-negative breast cancer (TNBC) highlights the need for combination strategies that enhance antitumor responses. Sorafenib, a multikinase inhibitor with anti-angiogenic and immunomodulatory activity, represents a rational partner for ICI-based combination therapy. However, therapeutic responses to such combinations are biologically complex and cannot be fully characterized by any single biomarker or imaging modality. Here, we evaluated ICI therapy combined with sorafenib in the aggressive and ICI-refractory orthotopic 4T1 TNBC model. Therapeutic responses were assessed using a unique longitudinal multimodal imaging framework integrating [Zr]Zr-DFO-anti-CD8 minibody and [{superscript 1}F]FDG PET, as well as perfluorocarbon (PFC)-based {superscript 1}F MRI and hyperpolarized {superscript 1}3C MRS, together with ex vivo analyses. Only the ICI-sorafenib combination suppressed tumor growth, whereas both monotherapies showed limited antitumor activity. Multimodal imaging, together with complementary ex vivo analyses, uncovered coordinated tumor microenvironment (TME) remodeling, including vascular normalization, elevated CD8 cell presence with modest enrichment in the tumor center, delayed increase in phagocyte-associated {superscript 1}F MRI signal coupled with reduced CD206 cell infiltration, and sustained metabolic activity. These findings support ICI-sorafenib combination therapy as a promising therapeutic strategy for TNBC. Therapeutic efficacy reflected coordinated vascular, immune, and metabolic remodeling. This multimodal imaging framework enables non-invasive longitudinal monitoring of these complementary TME changes, providing a comprehensive strategy for treatment assessment in immunotherapy-based combination therapies. One Sentence SummaryLongitudinal multimodal imaging identified a multidimensional TME response signature of effective ICI-sorafenib therapy in TNBC.

Introduction: Tumor mutational burden (TMB) is a biomarker for immune checkpoint inhibitor therapy, traditionally measured in tissue (tTMB). Blood-based TMB (bTMB), derived from circulating tumor DNA, is minimally invasive but shows modest concordance with tTMB. The significance of blood-tissue TMB discordance remains unclear. Methods: We retrospectively analyzed 105 patients with advanced NSCLC who underwent pretreatment blood and tissue next-generation sequencing between October 2020 and September 2024. The blood-to-tissue TMB ratio was defined as ln[(1 + bTMB)/(1 + tTMB)]. Outcomes were overall survival (OS) and progression-free survival (PFS). Survival was assessed using Kaplan-Meier methods and multivariable Cox models. Results: Median follow-up was 10 months. Patients in the lowest ratio tertile had longer OS than those in the upper two tertiles (median, 33 vs 11 months; hazard ratio [HR], 0.55; 95% confidence interval [CI], 0.32-0.97; p = 0.04), whereas PFS did not differ (HR, 0.89; p = 0.62). A higher ratio, analyzed continuously, was independently associated with shorter OS (HR per 1-unit increase, 1.60; 95% CI, 1.10-2.31; p = 0.01), but not PFS. The association persisted after adjustment for metastatic organ count and radiographic tumor burden. The high-bTMB/low-tTMB subgroup had the poorest OS (HR, 3.17 vs low-bTMB/high-tTMB; p = 0.01). Conclusions: A higher blood-to-tissue TMB ratio was independently associated with worse OS in advanced NSCLC. Directional discordance between bTMB and tTMB may reflect tumor heterogeneity and provide prognostic information beyond either measure alone.

Group 3 medulloblastomas (G3MB) carry the worst prognosis among medulloblastoma subtypes, yet molecularly targeted therapies remain elusive. Standard treatments cause severe long-term morbidity in survivors. Here, we identify tumor-derived sphingosine kinase 2 (SPHK2) as an essential driver of G3MB initiation and progression. SPHK2 exacerbates local immunosuppression by suppressing cytotoxic T-cell and NK-cell activity while promoting regulatory T-cell infiltration. Genetic or pharmacologic SPHK2 inhibition using Opaganib attenuates pro-survival tumor signaling and restores anti-tumor immunity, significantly improving survival in syngeneic G3MB mouse models. Combining Opaganib with fractionated low-dose radiation (f-LDRT) further enhances antigen presentation and reprograms tumor-associated myeloid cells toward an anti-tumor phenotype. This combination therapy markedly prolongs survival without inducing significant toxicity. Overall, our study establishes SPHK2 as a previously unrecognized therapeutic target and presents a safe, effective, microenvironment-reprogramming regimen for G3MB. One Sentence SummaryDirect inhibition of tumor-derived SPHK2 overcomes local immunosuppression and downregulates pro-survival signaling in Group 3 medulloblastoma, while combination with fractionated low-dose radiation further enhances anti-tumor immunity and significantly improves survival.

Radiopharmaceutical therapies (RPT) deliver non-uniform radiation dose in tumors and the impact of this on response is poorly understood. Dose heterogeneity could engender treatment resistance in low dose regions, yet we hypothesize that a broader array of dose-dependent immuno-radiobiological mechanisms in tumor microenvironments (TME) and preservation of immune function in low-dose regions could promote adaptive anti-tumor immunity and response. In murine models, non-uniform lutetium-177 delivering <2.5 Gy to >20 Gy in a TME induced broader immunomodulatory effects and T cell-dependent survival improvement compared to more uniform distributions. Preserving low-dose regions promoted dendritic cell activation and TME infiltration of clonally expanded CD8+ T cells. In three independent cohorts of patients with prostate cancer, heterogeneous tumor dose distribution strongly correlated with improved clinical outcomes. These findings defy expected radiobiological dose-response and define a novel mechanism of action for RPT, supporting clinical investigation of dose distribution for optimizing patient selection and personalized dosing.

TFCP2-rearranged rhabdomyosarcoma is an exceptionally rare and highly aggressive malignancy driven by TFCP2 gene fusions and associated with a dismal clinical prognosis. Because standardized treatment regimens are lacking, developing representative preclinical models is critical for identifying effective therapies. Here, we present a case of a 29-year-old male with rapidly progressive, metastatic pelvic intraosseous rhabdomyosarcoma (iRMS) harboring a FUS::TFCP2 fusion and anaplastic lymphoma kinase (ALK) overexpression. To evaluate therapeutic vulnerabilities, we established a patient-derived xenograft (PDX) model that faithfully recapitulated the histologic, immunohistochemical, and molecular hallmarks of the primary tumor. High-throughput in vitro pharmacological screening of PDX-derived cells demonstrated notable resistance to standard cytotoxic chemotherapies and revealed a paradoxical and selective sensitivity profile across ALK inhibitors. The PDX-derived cells were susceptible to crizotinib, brigatinib, and ceritinib, yet resistant to the more selective second- and third-generation inhibitors alectinib and lorlatinib. Notably, next-generation ROS1/pan-TRK inhibitors (entrectinib, repotrectinib, and taletrectinib) demonstrated superior efficacy compared to the fourth-generation ALK inhibitor NVL-655. Our findings establish a validated preclinical PDX model for FUS::TFCP2 iRMS and suggest that multi-targeted tyrosine kinase inhibition may offer a more viable therapeutic strategy than narrow-spectrum ALK targeting or conventional chemotherapy.