Clinical Cancer Research

In a phase 2 trial, local-regionally advanced HPV-positive oropharyngeal carcinoma (OPC) patients received ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) as induction immunotherapy and concurrently with radiotherapy (NCT03799445). Co-primary endpoints achieved included 6-month complete metabolic response rate (94%) and 2-year progression-free survival (84%). Induction yielded a 46% major histological response rate. Single-cell profiling revealed responders had higher baseline intratumoral tissue-resident memory (TRM) CD8+ T cells and NK cells expressing Fc Gamma Receptor IIIa (FCGR3A). Decreases in effector regulatory T (eTreg) cells, which highly expressed CTLA4, occurred only in responders, suggesting ipilimumab-dependent depletion by FCGR3A+ NK cells. eTreg depletion correlated with increased Interferon Gamma (IFNG)+ effector CD8+ T cells. CD8+ T-cell clonotypes transitioned from TRM to effector memory and IFNG+ effector cells in responders, whereas clonotypes transitioned to exhausted TRM and proliferating cells in nonresponders. We conclude that eTreg depletion is critical for major response to induction dual immune checkpoint blockade.

We investigated the effectiveness of navtemadlin (KRT-232) in treating recurrent glioblastoma. A surgical window-of-opportunity trial (NCT03107780) was conducted on 21 patients to determine achievable drug concentrations within tumor tissue and examine mechanisms of response and resistance. Both 120 mg and 240 mg daily dosing achieved a pharmacodynamic impact. Sequencing of three recurrent tumors revealed an absence of TP53-inactivating mutations, indicating alternative mechanisms of resistance. In patient-derived GBM models, the lower range of clinically achieved navtemadlin concentrations induced partial tumor cell death as monotherapy. However, combining navtemadlin with temozolomide increased apoptotic rates while sparing normal bone marrow cells in vitro, which in return underwent reversible growth arrest. These results indicate that clinically achievable doses of navtemadlin generate significant pharmacodynamic effects and suggest that combined treatment with standard-of-care DNA damaging chemotherapy is a route to durable survival benefits. Statement of significanceTissue sampling during this clinical trial allowed us to assess mechanisms of response and resistance associated with navtemadlin treatment in recurrent GBM. We report that clinically achievable doses of navtemadlin induce pharmacodynamic effects in tumor tissue, and suggest combinations with standard-of-care chemotherapy for durable clinical benefit.

PurposeRelapse is a major cause of failure in human papillomavirus (HPV)-independent head and neck squamous cell carcinoma (HNSCC). Clinicopathologic criteria for adjuvant treatment remain imprecise and have not changed for decades. We investigated whether circulating tumor DNA (ctDNA) in lymphatic exudate collected via surgical drains ("lymph") 24-hours after surgery identified molecular residual disease (MRD) and compared its performance to time-matched plasma. Experimental DesignUsing an ultra-sensitive tumor-informed sequencing approach, tumor variants were called in lymph and plasma to classify patients as ctDNA-positive or ctDNA-negative, trained in an initial cohort of 36 patients and replicated in an independent cohort of 37 patients. Progression-free survival (PFS) was compared in ctDNA+ vs. ctDNA-patients. ResultsLymph identified MRD in two independent multi-site cohorts (initial cohort sensitivity = 76%, specificity = 63%, P = 0.01; replication cohort sensitivity = 65%, specificity = 70%, P = 0.04). Lymph performance was enhanced in locoregional relapse (sensitivity = 78%, specificity = 67%, P = 0.0004) and generalized to early-stage patients. Analysis of matched plasma collected at this early timepoint was not predictive of recurrence (sensitivity = 35%, specificity = 72%, P = 0.7). In patients with intermediate-risk pathology, lymph ctDNA was associated with recurrence (sensitivity = 88%, specificity = 67%, P = 0.0008), suggesting an opportunity for improved stratification of patients who may benefit from additional adjuvant treatment. ConclusionPostoperative lymph is a novel, proximal, and early source of MRD with the potential to introduce more precision into adjuvant therapy decision-making and improve outcomes, especially for intermediate-risk HPV-independent HNSCC patients. Translational RelevancePostoperative lymphatic exudate represents a proximal analyte for MRD detection in HPV-independent HNSCC designed specifically for use in the immediate post-surgical window when adjuvant therapy decisions must be made. Accurate MRD identification at this early timepoint has the potential to augment traditional pathology and personalize adjuvant treatment paradigms in HPV-independent HNSCC.

BackgroundOnce the treatment starts, early prediction of treatment benefit and its correlation with overall survival (OS) remains challenging in metastatic castration-resistant prostate cancer (mCRPC). Existing prognostic models require long-term follow-up, limiting their ability to inform timely treatment decisions. To address this gap, we evaluated tumor growth rate (g-rate)-based survival models across multiple treatment lines to assess their ability to predict OS and support early clinical decision-making. MethodsWe developed GxSurv, a Random Survival Forest (RSF)-based framework that incorporates baseline clinical variables and g-rate calculated from serial on-treatment PSA, to construct line-specific prediction models of OS, a direct measure of treatment outcome. Three variants were developed: G3Surv, using the 3-month g-rate; G6Surv, using the 6-month g-rate; and GfSurv, using the final observed g-rate. Model performance was evaluated using Harrells C-index, Unos C-index, Integrated Brier Score (IBS), time-dependent area under the curve (tAUC). Model interpretability was assessed using permutation importance to quantify predictor contributions within the GxSurv framework. FindingsThe study included 15912 treatment records from 11014 patients with mCPRC across four lines of therapy. We found that incorporation of g-rate consistently improved model performance across all treatment lines, with all GxSurv models outperforming Cox proportional hazards (CoxPH). As the earliest prognostic model, our G3Surv demonstrated strong early predictive performance, with Harrells C-index values ranging from 0{middle dot}700 to 0{middle dot}746 and tAUC values of 0{middle dot}766 to 0{middle dot}822 across all lines, representing 5-8% and 4-5% improvements over CoxPH, respectively. These results indicate that G3Surv accurately predicts individual treatment outcomes at 3 months after treatment initiation. Feature importance analyses consistently identified g-rate as a top predictor, followed by baseline PSA and hemoglobin, with relative variation across treatment lines. InterpretationIntegrating g-rate calculated from on-treatment PSA values enables accurate, line-specific prediction of treatment outcomes in mCRPC, with the 3-month g-rate providing robust early prognostic information to support timely, personalized clinical decision-making. FundingU.S. National Science Foundation, National Institutes of Health, American Cancer Society.

The addition of pembrolizumab to preoperative radiotherapy (RT) improved disease-free survival (DFS) for patients with stage III undifferentiated pleomorphic sarcoma (UPS) and dedifferentiated/pleomorphic liposarcoma (LPS) in the randomized SU2C-SARC032 trial. To precisely identify patients who benefit from pembrolizumab and RT, we performed comprehensive multi-omics profiling of pre- and post-treatment tumor and blood samples, including bulk RNA-seq, flow cytometry, and cytometry by time of flight. Additionally, we built a single-cell RNA-seq atlas spanning 65,786 cells from UPS and LPS to recover single-cell states in bulk tumor samples using digital cytometry. Two opposing tumor microenvironments (TMEs), immune-cold sarcoma ecotype 1 (SE1) and immune-hot sarcoma immune class E (SIC E), benefited from pembrolizumab. Pembrolizumab combined with RT caused an overall increase in activated CD8+ T cells, CD56low NK cells, and T cell receptor diversity, while diminishing matrix-remodeling stromal cells and sarcoma cells. Our findings identify different mechanisms of response to pembrolizumab in localized, high-risk UPS/LPS and suggest that sarcoma TME signatures may identify patients most likely to benefit from adding pembrolizumab to preoperative RT.

Purpose: Limited CNS bioavailability and pharmacodynamics are obstacles to effective systemic therapies for glioblastoma. One strategy to overcome these challenges is drug combinations enhancing CNS penetration and/or tumor chemosensitivity. LP-184, a synthetic acylfulvene class alkylator, induces DNA damage and inhibits glioblastoma cell viability in pre-clinical models. LP-184 is a prodrug converted to active metabolites by intracellular prostaglandin reductase 1 (PTGR1) that is over-expressed in >70% of glioblastoma. DNA damage induced by LP-184 is MGMT agnostic and reversed by transcription-dependent NER. Patients: LP-184 was evaluated in a Phase 1a study (NCT05933265) in 63 adult patients with advanced malignancies including 16 patients with recurrent glioblastoma. All patients with glioblastoma received prior standard-of-care therapy and most had received 1 or more additional therapies before enrollment. Results: Patients with glioblastoma experienced more frequent transaminitis, Grade 1-2 nausea and a trend towards more frequent and severe thrombocytopenia compared to the non-glioblastoma cohort. Otherwise, overall toxicity profiles were similar. Clinical pharmacokinetic analysis combined with published pre-clinical intra-tumoral bioavailability data (~20% penetration) predicted that LP-184 at the recommended dose for expansion (RDE) would achieve cytotoxic levels if combined with spironolactone, a BBB permeable ERCC3 degrader and TC-NER inhibitor that sensitizes glioblastoma cells to LP-184 3-6-fold. We show that three daily doses of spironolactone deplete orthotopic glioblastoma PDX ERCC3 protein by ~ 80% and increases tumor LP-184 cytotoxicity 2-fold. Conclusions: LP-184 is well tolerated at the RDE, and we establish a clinically translatable scheme for dosing spironolactone in combination with LP-184 for a future Phase 1b clinical trial.

1.The ABACUS study was a single arm, phase II trial evaluating neoadjuvant atezolizumab in operable urothelial carcinoma (UC). Initial bulk transcriptomic and immunohistochemistry analyses suggested links between immune activation, tissue remodeling, and resistance pathways such as transforming growth factor {beta} (TGF{beta}) that were associated with clinical outcome. To further characterize spatial and phenotypic changes at high resolution, artificial intelligence-assisted digital image analysis of hematoxylin and eosin sections and spatial transcriptomics (10x Genomics Visium) were performed on paired tissue samples. In baseline samples, cells residing in lymphoid aggregates and tertiary lymphoid structures (LAs/TLSs) were more abundant in stable disease than in relapse and exhibited gene expression programs associated with improved survival in UC. Most spatial features reflected shared pharmacodynamic changes between stable disease and relapse; however, carcinoma-endothelial adjacency was reduced significantly following treatment and differed between groups, accompanied by distinct transcriptional programs. Together, these findings indicate that atezolizumab induces localized immune and stromal remodeling within the tumor microenvironment, while non-response despite immune expansion is associated with persistent spatial immune exclusion and carcinoma-endothelial adjacency. Spatial and phenotypic biomarkers identified here may inform rational combination strategies for immune checkpoint inhibitor-refractory urothelial carcinoma.

PURPOSE: Newly-diagnosed glioblastoma (nGBM) is a devastating tumor with median survival of only 14-18 months despite aggressive standard of care (SOC). Dendritic cell (DC) homologous antigenic double-loading provides a powerful pattern-based signal that initiates cDC1-like skewing of monocytic precursors, inducing downstream development of CD8+ memory effectors. Here we report phase I results for DOC1021 (dubodencel), a novel DC vaccine regimen integrated with SOC. METHODS: In this dose-escalating study, DC prepared from mobilized peripheral blood were doubly loaded with autologous tumor lysate and amplified tumor mRNA and administered bilaterally near the deep cervical node chains in three biweekly courses given with weekly peg-IFN after conclusion of chemoradiation. Four dose levels from 3.5x106 to 3.6x107 total cells were tested. Patients with subtotal resection or tumor progression prior to vaccination were not excluded. RESULTS: Eighteen patients (median age 61 years (range 47-73), 94% MGMT unmethylated, 25% subtotal/partial resected) completed vaccination (16 nGBM, 2 recurrent) with no dose-limiting toxicities. Attributable AE were mostly mild and flu-like or injection-site reactions. Twelve-month OS among the newly-diagnosed cohort was 88% compared to an expected ~60% for SOC alone. Patients who received observation rather than reoperation in response to worsening MRI contrast-enhancement demonstrated gradual lesional resolution and improved OS. Immunophenotyping revealed post-vaccination elevations in CD4 and CD8 memory T-cells in peripheral blood, and spatial transcriptomic analysis revealed foci of activated inflammatory complexes at the primary tumor site. CONCLUSIONS: DOC1021 was safe, feasibly integrated within SOC, and associated with more favorable outcomes in this challenging patient population. Patients who received observation rather than reoperation for worsening MRI contrast-enhancement exhibited superior survival, suggesting an immune-reactive tumor microenvironment manifesting as pseudo-progression. These data supported initiation of a randomized Phase II trial (NCT06805305) for nGBM.

PurposeTarlatamab is a DLL3-directed bispecific T-cell engager demonstrating clinically meaningful activity in relapsed small cell lung cancer (SCLC) in the phase II DeLLphi-301 trial. Determinants of tarlatamab sensitivity and resistance are incompletely understood, and thus we sought to identify genomic and transcriptional correlates of tarlatamab sensitivity using a clinical sequencing pipeline at a single comprehensive cancer center. Experimental DesignWe performed a retrospective, single-institution analysis of 12 patients with SCLC treated with tarlatamab. Whole-exome sequencing (WES) and exome-capture whole-transcriptome sequencing (WTS) were performed on 12 samples, and two matched samples after treatment with tarlatamab. Integrative analysis examined correlation between molecular features and clinical outcomes. ResultsThe overall response rate was 50%, which was consistent with outcomes reported in the DeLLphi-301 trial. Differences between SCLC driver alterations and tumor mutational burden were not significant between responders and non-responders, but homologous recombination deficiency scores were higher in responsive tumors. DLL3 expression was significantly greater in responders and demonstrated predictive discrimination for clinical response (AUC 0.83). Tumors responsive to tarlatamab were predominantly ASCL1-driven (SCLC-A) and demonstrated increased immune activation, such as enrichment of cytotoxic T-cell, NK-cell, and T cell transcriptional programs. Transcriptional subtype and a composite metric consisting of DLL3 expression and immune activity (DLI score) further discriminated between responders and non-responders (sensitivity 0.83, specificity 1). Paired post-treatment sample analysis identified loss of ASCL1 lineage and emergence of YAP1 expression and downregulation of DLL3, consistent with lineage plasticity as a mechanism of acquired resistance. ConclusionsSensitivity to tarlatamab is correlated with a combination of increased DLL3 expression, ASCL1-driven lineage, and an increased immune activation. Lineage state reprogramming and decrease in DLL3 expression accompany acquired resistance to tarlatamab. These findings highlight the utility of RNA based biomarkers which integrate target expression, lineage state, and immune context to guide tarlatamab therapy in SCLC. Prospective validation of the whole-transcriptome DLI score and transcriptional subtype will inform tarlatamab response prediction.

Circulating tumor cells (CTCs) captured from the bloodstream of patients with solid tumors have the potential to accelerate precision oncology by providing insight into tumor biology, disease progression and response to treatment. However, their potential is hampered by the lack of standardized CTC enrichment platforms across tumor types. EpCAM-based CTC enrichment, the most commonly used platform, is limited by EpCAM downregulation during metastasis and the low EpCAM expression in certain tumor types, including the highly prevalent and lethal NSCLC. In this study we demonstrate that Transferrin Receptor (TfR) is a selective, efficient biomarker for CTC identification and capture in patients with prostate, pancreatic and NSCLC. TfR identifies significantly higher CTC counts than EpCAM, and TfR+-CTC enumeration correlates with disease progression in metastatic prostate and pancreatic cancers, and overall survival and osimetrinib-resistance in non-small cell lung cancer (NSCLC). Profiling of TfR+-CTCs provides a snapshot of the molecular landscape of each respective tumor type and identifies potential mechanisms underlying treatment response to EGFR TKi and immune checkpoint inhibitors in NSCLC. One sentence summaryTransferrin Receptor identifies circulating tumor cells in solid tumors

Despite high rates of post-surgical recurrence in men with high-risk localized prostate cancer (PCa), there is currently no role for neoadjuvant therapy. Tumor infiltrating regulatory T cells (TI-Tregs) limit the antitumor effects of presurgical androgen deprivation therapy (ADT). Therefore, we designed a neoadjuvant clinical trial to test whether Treg depletion via a non-fucosylated anti-CTLA-4 antibody (BMS-986218) is feasible and augments response to ADT. In this single-center, two-arm, open-label study, 24 men with high-risk localized PCa were randomized to ADT with or without BMS-986218 prior to radical prostatectomy. Treatment was well tolerated and feasible. Mechanistic studies indicated BMS-986218 depleted TI-Tregs by engaging CD16a/FCGR3A on tumor macrophages, modulated dendritic cells (DCs), and augmented T cell priming. Depth of Treg depletion and increased DC frequencies were quantitatively associated with improved clinical outcome. Overall, this study supports the feasibility and biological activity of neoadjuvant immunotherapy with ADT + Fc- enhanced anti-CTLA-4 in high-risk localized PCa. Statement of SignificanceNext-generation antibodies targeting CTLA-4 have been engineered for enhanced tumor Treg depletion in patients, yet their mechanisms of action remain incompletely defined. We performed the first single cell multi-omic correlative analyses of response to a glycoengineered anti-CTLA-4 antibody and defined mechanisms associated with clinical outcome in patients with high-risk localized prostate cancer.

IntroductionCerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring neoplastic processes of the central nervous system. We hypothesize that analysis of CSF-tDNA in patients with advanced lung cancer improves the sensitivity of leptomeningeal disease (LMD) diagnosis and enables central nervous system response monitoring. MethodsWe applied CAPP-Seq using a lung cancer-specific sequencing panel to 81 CSF, blood, and tissue samples from 24 patients with advanced lung cancer who underwent lumbar puncture (LP) for suspected LMD. A subset of the cohort (N = 12) participated in a prospective clinical trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment with. ResultsCSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearmans {rho}, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). ConclusionsDetection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA may improve the sensitivity of LMD diagnosis, enable improved prognostication, and drive therapeutic strategies that account for spatial heterogeneity in resistance mechanisms.

Outcomes for adult patients with a high-grade glioma continue to be dismal and new treatment paradigms are urgently needed. To optimize the opportunity for discovery, we performed a phase 0/1 dose-escalation clinical trial that investigated tumor pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics following combined ribociclib (CDK4/6 inhibitor) and everolimus (mTOR inhibitor) treatment in recurrent high-grade glioma. Patients with a recurrent high-grade glioma (n = 24) harboring 1) CDKN2A/B deletion or CDK4/6 amplification, 2) PTEN loss or PIK3CA mutations, and 3) wild-type retinoblastoma protein (Rb) were enrolled. Patients received neoadjuvant ribociclib and everolimus treatment and no dose-limiting toxicities were observed. The median unbound ribociclib concentrations in Gadolinium non-enhancing tumor regions were 170 nM (range, 65 - 1770 nM) and 634 nM (range, 68 - 2345 nM) in patients receiving 5 days treatment at the daily dose of 400 and 600 mg, respectively. Unbound everolimus concentrations were below the limit of detection (< 0.1 nM) in both enhancing and non-enhancing tumor regions at all dose levels. We identified a significant decrease in MIB1 positive cells suggesting ribociclib-associated cell cycle inhibition. Single nuclei RNAseq (snRNA) based comparisons of 17 IDH-wild-type on-trial recurrences to 31 IDH-wild-type standard of care treated recurrences data demonstrated a significantly lower fraction of cycling and neural progenitor-like (NPC-like) malignant cell populations. We validated the CDK4/6 inhibitor-directed malignant cell state shifts using three patient-derived cell lines. The presented clinical trial highlights the value of integrating pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics to assess treatment effects in phase 0/1 surgical tissues, including malignant cell state shifts. ClinicalTrials.gov identifier: NCT03834740.

BackgroundImmune checkpoint inhibitors (ICIs) improve survival in advanced non-small cell lung cancer (NSCLC), yet current biomarkers such as PD-L1 expression and response criteria (RECIST v1.1) align poorly with long-term survival. Radiomics has been proposed as a source of novel biomarkers, but standard radiomic approaches suffer from limited biological interpretability and poor generalizability across treatment settings. We address these gaps by developing the Quantitative Vessel Tortuosity (QVT) Score, a biologically interpretable imaging biomarker that quantifies tumor vascular complexity -a known mediator of immune evasion - from routine imaging. We hypothesized that QVT Score would improve prognostication and enable treatment response monitoring in ICI-treated NSCLC, independent of current biomarkers. MethodsThis retrospective, multicenter study analyzed 1,301 CT scans from 682 ICI-treated NSCLC patients. An automated pipeline segmented lesions and tumor-associated vasculature within each scan, extracting 910 QVT features measuring vascular shape and complexity. Unsupervised clustering of these features in a discovery cohort (N=375) was performed to identify fundamental vascular phenotypes. A continuous QVT score was then derived using regularized logistic regression to map patients along this phenotypic spectrum. QVT Score was externally validated in ICI monotherapy (N=172) and chemoimmunotherapy (N=135) cohorts. In a longitudinal cohort (n=143), early on-treatment QVT Score changes were evaluated for overall survival (OS) association. ResultsTwo robust vascular phenotypes emerged in the discovery cohort: a highly vascularized, chaotic "QVT High" phenotype with poor post-ICI OS and a "QVT Low" phenotype with normalized vasculature and improved ICI outcomes. The continuous QVT Score was prognostic for ICI monotherapy (HR = 1.17 per 0.1 increase, p = 0.0028) and chemoimmunotherapy (HR = 1.23 per 0.1 increase, p = 4.9x10-). High QVT status remained prognostic for both treatments after adjustment for PD-L1 and clinical variables (adjusted HR range: 2.13-2.38, p [&le;] 0.002). Early decreases in QVT Score during therapy, indicating vascular normalization, were associated with improved OS (HR = 1.93, p = 0.0022) independent of RECIST best overall response and tumor volume change. ConclusionsQVT Score is a novel, biologically interpretable imaging biomarker that quantifies vascular complexity. It enables automated, non-invasive prediction and monitoring of ICI outcomes by capturing treatment-induced vascular remodeling. Integrating QVT Score into clinical decision-making and drug development can address critical gaps in precision oncology.

In approximately half of endometrial carcinoma (EC), PTEN loss-of-function and activating PI3K mutants coexist. Unlike cells with either single mutation, PTEN/PIK3CA coexistent alterations result in elevated membrane phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels and mTORC1 hyperactivation, rendering PI3K or AKT inhibition ineffective in blocking mTORC1 activity and tumor growth. The bi-steric mTORC1 kinase inhibitor, RMC-6272, suppresses mTORC1 activity and cell growth by reducing protein translation and cell cycle progression. In vivo, RMC-6272, but not PI3K inhibitors, effectively suppressed mTORC1 and growth of EC PDXs with coexistent PTEN/PIK3CA lesions. These findings are consistent with a phase I trial of bi-steric mTORC1 inhibitor RMC-5552, showing anti-tumor activity in patients with EC. PDXs with KRAS co-mutations regrew after RMC-6272 treatment, which was prevented by the addition of the RAS(ON) multi-selective inhibitor RMC-7977. Overall, these data suggest that mTORC1 hyperactivation drives ECs with coexistent PTEN/PIK3CA mutations, explain the limited antitumor activity of PI3K and AKT inhibitors, and support clinical evaluation of mTORC1 inhibitors as potential therapy for EC. SignificanceWe have found the mechanistic consequences of PTEN/PIK3CA co-alterations in endometrial tumors and that these mutations result in a profound hyperactivation of mTORC1 signaling. Single mutant tumors are sensitive to PI3K inhibition but those with both mutations are insensitive to PI3K or AKT inhibition but are exquisitely dependent on mTORC1 kinase. This provides strong preclinical rationale for targeting mTORC1, alone or combined with RAS inhibition (in RAS co-mutant tumors), as an effective therapeutic strategy.

Even though multiple resistance mechanisms and pathways for cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) have been discovered, the complete landscape of resistance is still being elucidated. Moreover, the optimal subsequent therapy to overcome resistance remains uncertain. To address this, we carried out a phase I/II clinical trial of exemestane plus everolimus and palbociclib, triplet therapy for CDK4/6i-resistant hormone receptor-positive (HR+), HER2-metastatic breast cancer, one of the first trials evaluating CDK4/6i after CDK4/6i progression. With an observed clinical benefit rate of 18.8% (n = 6/32), the trial did not meet its primary efficacy endpoint. However, we leveraged the multi-omics tumor data from these patients to study the landscape of CDK4/6i resistance and to identify correlates of response to triplet therapy. We generated whole exome sequencing from 24 tumor and 17 ctDNA samples and transcriptome sequencing from 27 tumor samples obtained from 26 patients in the trial. Genomic and evolutionary analysis recapitulated the spectrum of known resistance genes (ERBB2, NF1, AKT1, RB1, ESR1) and pathways (RTK/MAPK, PI3K/AKT/mTOR, cell cycle, estrogen receptor), discovered potential new mechanisms of resistance in these pathways (ERBB2 amplification, BRAFV600E, MTORT1977R), and identified a patient with co-existing tumor lineages with distinct activating ERBB2 mutations, potentially the first case of convergent evolution of HER2 activation following CDK4/6i therapy. Joint genomic and transcriptomic analysis revealed that genomic resistance mechanisms were associated with transcriptomic features in their respective pathways, suggesting that transcriptomic features could be used to identify the pathways driving resistance. In particular, the mutually exclusive ESR1 and ERBB2/BRAF mutations, were each linked with high activity in distinct pathway signatures (estrogen receptor pathway vs RTK/MAPK pathway, respectively) and were exclusive to distinct molecular subtypes (Luminal A or Luminal B vs HER2-E, respectively). Overall, incorporating clinical and multi-omics features in CDK4/6i-resistant tumors enabled identification of known or putative drivers of resistance to the prior CDK4/6i and anti-estrogen therapies in nearly every patient (n = 22/23), including several patients in which transcriptomic features were the sole drivers. Genomic and transcriptomic features - particularly PI3K/AKT/mTOR mutations and/or high mTORC1 pathway activity - suggested that clinical benefit to combined estrogen receptor, CDK4/6, and mTOR inhibition was correlated with activation of the mTOR pathway. Our results illustrate how transcriptome sequencing provides complementary and additional information to genome sequencing, and how integrating both may help better identify patients likely to respond to CDK4/6i therapies. SignificanceCombined endocrine, CDK4/6 inhibitor, and mTOR inhibitor therapy showed limited benefit in patients with HR+ metastatic breast cancer who had progressed on a prior CDK4/6 inhibitor. Multi-omics analysis of tumors from this trial identified novel genomic and transcriptomic drivers of CDK4/6i resistance, known or putative drivers of resistance in 22/23 patients, and correlates of response to the trial therapy. Integrated genome and transcriptome sequencing may better identify factors that determine response to CDK4/6i therapy and help select optimal therapy.

Translational RelevanceDespite definitive local therapy, some men with prostate cancer develop biochemical recurrence (BCR-PCa) and progress to metastatic disease. Current standard-of-care, androgen receptor pathway inhibitors (ARPIs) with or without androgen deprivation therapy (ADT) carry substantial long-term morbidity. This trial tested a novel, non-hormonal approach of 5-azacitidine (AZA) plus all-trans retinoic acid (ATRA) to induce tumor cell dormancy via epigenetic reprogramming. The regimen was well tolerated, with manageable toxicities, and showed preliminary signals of delayed PSA progression and prolonged PSA doubling time in some patients, suggesting dormancy induction. One patient achieved durable disease stabilization with ATRA maintenance. These first-in-human findings indicate that epigenetic reprogramming may modulate dormancy in BCR-PCa, offering a potential strategy to delay or minimize ADT use and its toxicities. This approach highlights the translational potential of preventing or delaying overt metastases by activating dormancy pathways in early recurrent prostate cancer. PurposeBiochemical recurrence (BCR) after definitive local therapy remains a major clinical challenge in prostate cancer (PCa), with heterogeneous disease trajectories and few established strategies to delay further progression without prolonged androgen deprivation. This pilot study evaluated the combination of 5-azacitidine (AZA) and all-trans retinoic acid (ATRA) to induce tumor dormancy and delay clinical progression in patients with BCR. Experimental DesignIn a prospective, open-label, randomized, single-institution pilot trial, patients with BCR of PCa and no recent hormonal or definitive therapy received low-dose AZA and sequential ATRA. The co-primary endpoints were changes in prostate-specific antigen doubling time (PSADT) and time to next treatment (TTNT). Safety and biomarker analyses, including bone morphogenetic protein (BMP) signaling and dormancy marker NR2F1 in circulating tumor cells (CTCs), were evaluated to investigate treatment effects on minimal residual disease dormancy. ResultsFourteen patients were enrolled. Treatment resulted in an increase in median PSADT from 2.45 to 4.56 months. The median TTNT was 9.6 months, with 28.6% of patients experiencing TTNT over 12 months. No new safety signals were identified; adverse events were consistent with those expected for AZA and ATRA. Analysis of circulating BMP4 and BMP7 suggested that higher BMP4 levels may correlate with treatment response. Notably, all patients achieved testosterone recovery post-treatment, likely reflecting the avoidance of ongoing androgen deprivation. Across the cohort, treatment with AZA+ATRA led to a reduction in total CTC numbers and an apparent increase in the fraction of NR2F1-positive CTCs in responders, although the small cohort size limited statistical testing. ConclusionsThe combination of AZA and ATRA was feasible and prolonged PSA kinetics in a subset of patients with BCR of PCa, with a favorable safety profile. This epigenetic approach promoting tumor dormancy presents a potential strategy to defer progression and delay the need for continuous hormonal suppression. Larger studies are warranted to validate these findings and further explore biomarkers predictive of clinical benefit.

H3K27M-mutant diffuse midline gliomas (DMGs) express high levels of the GD2 disialoganglioside and chimeric antigen receptor modified T-cells targeting GD2 (GD2-CART) eradicate DMGs in preclinical models. Arm A of the Phase I trial NCT04196413 administered one IV dose of autologous GD2-CART to patients with H3K27M-mutant pontine (DIPG) or spinal (sDMG) diffuse midline glioma at two dose levels (DL1=1e6/kg; DL2=3e6/kg) following lymphodepleting (LD) chemotherapy. Patients with clinical or imaging benefit were eligible for subsequent intracerebroventricular (ICV) GD2-CART infusions (10-30e6 GD2-CART). Primary objectives were manufacturing feasibility, tolerability, and identification of a maximally tolerated dose of IV GD2-CART. Secondary objectives included preliminary assessments of benefit. Thirteen patients enrolled and 11 received IV GD2-CART on study [n=3 DL1(3 DIPG); n=8 DL2(6 DIPG/2 sDMG). GD2-CART manufacturing was successful for all patients. No dose-limiting toxicities (DLTs) occurred on DL1, but three patients experienced DLT on DL2 due to grade 4 cytokine release syndrome (CRS). Nine patients received ICV infusions, which were not associated with DLTs. All patients exhibited tumor inflammation-associated neurotoxicity (TIAN). Four patients demonstrated major volumetric tumor reductions (52%, 54%, 91% and 100%). One patient exhibited a complete response ongoing for >30 months since enrollment. Eight patients demonstrated neurological benefit based upon a protocol-directed Clinical Improvement Score. Sequential IV followed by ICV GD2-CART induced tumor regressions and neurological improvements in patients with DIPG and sDMG. DL1 was established as the maximally tolerated IV GD2-CART dose. Neurotoxicity was safely managed with intensive monitoring and close adherence to a management algorithm.

PURPOSEIdentification of discrete sub-groups associated with treatment response and resistance in localized Ewing sarcoma (EWS) remains a challenge. The primary objective of the Childrens Oncology Group biology study AEWS18B1-Q was to perform molecular characterization of a large cohort of patients with localized Ewing sarcoma treated on prospective trials with modern standard of care therapy. METHODSWe analyzed clinical and molecular features from patients with localized EWS enrolled on AEWS0031, AEWS1031, or INT-0154 frontline trials. All patients had available FFPE tissue, frozen tissue, or whole-genome amplified material. Sequencing was performed for identification of canonical fusions, recurrent copy number alterations (CNAs), and alterations in TP53 and STAG2. Where available, tissue was analyzed for loss of STAG2 protein expression. Molecular features were evaluated for their association with cumulative incidence of relapse in univariate and multivariable analyses. RESULTSThree hundred and fifty-one cases had sufficient tissue, which in most cases was extracted from two FFPE slides. EWS canonical fusions were identified in 282 cases (80.3%). Pathogenic mutations in TP53 and STAG2 were identified in 5.1% and 7.6% of cases, respectively and 63.1% of cases were found to have recurrent CNAs. In univariate analysis, there was an increased cumulative incidence of relapse in patients with TP53 mutation (5-year cumulative incidence of relapse 43%, CI [17%, 67%] vs. 22%, CI [17%, 27%]; Grays test P = 0.039), STAG2 mutation (53%, CI [29%, 73%] vs. 21%, CI [16%, 26%]; P < 0.001), and recurrent CNAs (30%, CI [22%, 37%] vs. 16%, CI [9%, 24%]; P = 0.005). In a multivariable analysis, STAG2 mutation was the only molecular biomarker that remained prognostic. CONCLUSIONThis is a prospective validation of the molecular prognostic features of localized EWS receiving standard of care therapy on therapeutic clinical trials. Building on prior work, patients with STAG2 mutations were at high risk of relapse. CONTEXTO_ST_ABSKey ObjectiveC_ST_ABSTo determine if molecular features can identify clinically relevant disease sub-groups applicable to frontline clinical trial design among patients with localized Ewing sarcoma. Knowledge GeneratedAmong 351 patients with localized Ewing sarcoma treated on prospective trials, canonical fusions were identified in 80% of cases. In a multivariable analysis of patients with genomically defined Ewing sarcoma, the presence of STAG2 mutations identified a high-risk population. Relevance (added by Associate Editor)

Many studies have reported biomarkers predictive of response to immune checkpoint inhibitors (ICI) based on retrospective analyses. However, few clinical trials have tested their value prospectively. The DUTRENEO trial (EudraCT: 2017-002246-6) investigated whether an 18-gene Tumour Inflammation Signature (TIS) that can robustly identify patients who respond to ICI in multiple tumour types could stratify patients with localized muscle-invasive bladder cancer (MIBC) to receive neoadjuvant ICI (durvalumab+tremelimumab) or standard cisplatin-based neoadjuvant chemotherapy (NAC). Patients with TIS-high tumors were randomized to ICI or NAC, while patients with TIS-low tumors received NAC. A total of 73 patients were treated. Pathological complete response (pCR) rates were 38.5% (TIS-High, ICI), 30% (TIS-High, NAC), and 55% (TIS-Low, NAC) (p = 0.349), indicating that - as applied - the TIS score did not significantly enrich in responders to ICI. Post-hoc analysis showed that higher TIS thresholds improved prediction of response to ICI but excluded many responders. Multi-omics analyses of pre-treatment samples, including whole-exome sequencing, bulk RNA sequencing, and spatial transcriptomics (Visium, Xenium), revealed that bulk RNA response signatures originated mainly from cancer cells. Spatial transcriptomics showed that ICI response was associated with proximity between cancer cells and adaptive immune cells, while resistance to NAC was linked to phenotypic plasticity of cancer cells despite low genetic diversity. The unique design of the DUTRENEO trial underscores the challenges of translating retrospective biomarkers into clinical practice and highlights the importance of spatial features in understanding tumour-immune interactions. These findings suggest that integrating spatial and multi-modal biomarkers in well-designed clinical trials could improve stratification and response prediction to select neoadjuvant therapy.