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.

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.

PurposeLimited 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. PatientsLP-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. ResultsPatients 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. ConclusionsLP-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. Statement of translational relevanceTreatment failure in glioblastoma reflects inadequate drug brain exposure and DNA repair- mediated resistance. LP-184, a novel acylfulvene alkylator, generates MGMT-independent DNA lesions predominantly repaired by transcription-coupled NER. In a Phase 1a dose finding trial, LP-184 was well-tolerated at the recommended dose for expansion (RDE) in participants with advanced cancers, including recurrent glioblastoma. Plasma drug levels achieved predicted effective systemic exposures but not brain concentrations based on projected 20% brain penetrance. Pharmacokinetic modeling indicates that NER inhibition could increase tumor chemosensitivity with the addition of spironolactone. The optimal dosing regimen for spironolactone combined with LP-184 was identified in orthotopic PDX models, facilitating advancement to Phase 1b/2a testing of LP-184 plus spironolactone.

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.

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.

BackgroundMen with aggressive, localized prostate cancer (PC) undergo definitive radiotherapy (RT) with androgen deprivation therapy (ADT). The prospective, phase II ARIEL trial evaluates a quantitative MRI biomarker, Restriction Spectrum Imaging restriction score (RSIrs), at three time points (before treatment, after ADT and after RT) for treatment response assessment. RSIrs highlights intracellular restricted diffusion and is correlated with high-grade PC. DesignParticipants are men with unfavorable-intermediate-risk or high-risk localized PC undergoing definitive RT with neoadjuvant and concurrent ADT, and MRI-RSI acquisitions at three time points: before therapy, after neoadjuvant ADT but before RT, and after RT. The primary aim is to evaluate performance of RSIrs for identifying patients who will experience early biochemical recurrence. Change in RSIrs within visible tumors after ADT and RT is the primary independent variable. Results97 patients met inclusion criteria and received [&ge;]1 MRI. On central review, visible PI-RADS lesions were identified in 88 patients: 80 patients had one lesion, and 8 patients had two lesions. After neoadjuvant ADT, 40% of lesions were not clearly visible. Those still visible had shrank by median 55.8% (IQR: 42.8-69.0%), much more than the prostate volume decrease of 21.5% (11.9-31.6%). RSIrs maximum within visible lesions decreased from mean 329 (SD:185) pre-ADT to 209 (SD:125) pre-RT (p<0.01), and to 107 (SD:61) post-RT (p<0.01). Conventional apparent diffusion coefficient (ADC) changes were less consistent. Follow-up is ongoing to assess whether imaging response is related to future recurrence risk. ConclusionARIEL has completed accrual and preliminary results demonstrate changes in RSIrs after treatment, which may indicate tumor response. Primary results will be presented when the primary endpoint is reached. With neoadjuvant ADT, both pre- and post-ADT MRI are likely necessary for accurate focal RT boost targeting. Concurrent commencement of ADT and RT simplifies workflows and facilitates accurate gross tumor volume delineation.

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.

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.

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.

PurposeHPV-associated carcinomas (HPV+ cancers) account for 5% of all cancers. Circulating tumor HPV DNA (ctHPVDNA) assays for HPV+ cancer surveillance have limited prognostic utility at the time of cancer diagnosis. While HPV integration into the host genome is a proven tissue-based biomarker predicting poor clinical outcomes, existing clinically utilized ctHPVDNA assays cannot classify the viral physical state. MethodsWe previously developed HPV-DeepSeek, a multi-feature HPV whole-genome sequencing liquid biopsy with 99% diagnostic accuracy at the time of HPV+ oropharynx cancer diagnosis. We test the diagnostic accuracy of HPV-DeepSeek in a cohort of 235 HPV+ cancers across nine anatomic sites and employ a novel blood-based computational classifier to infer HPV genome physical state from plasma, termed HPV-SIGNAL, to assess its prognostic potential. ResultsHPV-DeepSeek demonstrated a sensitivity and specificity of 99%. In 181 eligible samples, HPV-SIGNAL identified four viral physical states: episomal-only (N = 69), episomal-rearranged (N = 48), integrated-mixed (N = 55), and integrated-clonal (N = 9), which were confirmed and further elucidated via three orthogonal tissue and blood approaches. Patients harboring integrated viral states in the blood exhibited significantly worse progression-free survival (HR 3.28, 95% CI 1.63-6.58, p = 0.00084) and overall survival (HR 2.98, 95% CI 1.16-7.64, p = 0.023) compared to patients with episomal states. ConclusionHPV whole-genome sequencing liquid biopsy has high diagnostic accuracy across HPV+ cancer types and can be used to identify and classify HPV physical state from blood. Patients with integrated viral states detected in the blood demonstrated worse progression-free and overall survival, suggesting blood-based HPV physical state classification could be used as a prognostic tool at the time of cancer diagnosis. Translational RelevanceCurrent circulating tumor HPV DNA assays for HPV-associated cancer surveillance have limited prognostic utility at the time of cancer diagnosis. While HPV integration into the host genome is a proven tissue-based biomarker predicting poor clinical outcomes, existing circulating tumor HPV DNA assays cannot classify the viral physical state. Here, we show that HPV-SIGNAL, a novel blood-based computational classifier to infer HPV genome physical state from plasma using output from HPV-DeepSeek, an HPV whole genome sequencing liquid biopsy, accurately identifies and classifies HPV physical state from blood and is prognostic of progression-free and overall survival across HPV-associated cancer types.

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.