JNCI: Journal of the National Cancer Institute

PurposeGlioblastoma (GBM) is characterized by poor clinical outcomes and marked resistance to radiotherapy. Because effective radiosensitizing strategies for GBM remain limited, we investigated whether inhibition of KRAS/RAS signaling could enhance radiation response in GBM. In particular, we evaluated the radiosensitizing potential of RMC-6236, an RAS(ON) multiselective inhibitor that suppresses active RAS signaling across multiple RAS-dependent states. Experimental DesignHuman GBM cell lines (U251, LN-18, ACPK1, and OSU61) were treated with radiation, with or without genetic or pharmacological KRAS inhibition. KRAS signaling was suppressed by siRNA-mediated knockdown or RMC-6236 treatment. Radiation-induced KRAS activation and downstream MAPK signaling were assessed by Raf-RBD pull-down assays and immunoblotting. Radiosensitivity was evaluated using clonogenic survival assay. DNA damage persistence, cell cycle distribution, and mitotic catastrophe were analyzed by {gamma}H2AX immunofluorescence, flow cytometry, and nuclear morphology assessment, respectively. In vivo therapeutic efficacy was examined in an orthotopic U251 xenograft model. ResultsRadiation-induced transient activation and increased KRAS protein expression of KRAS, accompanied by activation of ERK, JNK, and p38 signaling in GBM cells. siKRAS suppressed radiation-induced KRAS and MAPK activation, and significantly enhanced radiosensitivity in all four GBM cell lines. Similarly, RMC-6236 inhibited radiation-induced KRAS activation and attenuated downstream MAPK signaling without reducing the total KRAS protein expression. RMC-6236 significantly increased the radiosensitivity across all GBM cell lines, with dose enhancement factors ranging from 1.33 1.46. Mechanistically, combined treatment with RMC-6236 and radiation increased persistent {gamma}H2AX foci and enhanced mitotic catastrophe without producing consistent redistribution of cells into radiosensitive cell cycle phases. In an orthotopic GBM model, the combination of RMC-6236 and radiation significantly prolonged survival compared to that of the control and radiation alone. ConclusionsThese findings indicate that radiation-induced KRAS signaling is a functionally important mediator of radioresistance in GBM and demonstrate that inhibition of KRAS/RAS signaling enhances the radiation response in vitro and in vivo. RMC-6236 may represent a promising radiosensitizing strategy for GBM by suppressing adaptive RAS/MAPK signaling and promoting persistent DNA damage and mitotic catastrophe following irradiation. However, clinical trials of this combination are warranted.

Background: Genome-wide association studies (GWAS) have identified numerous lung cancer susceptibility loci based on single nucleotide polymorphisms (SNPs), yet a substantial proportion of heritability remains unexplained. We therefore evaluated germline copy number variants (CNVs) as an underexplored source of genetic susceptibility and potential contributors to genomic instability in lung cancer. Methods: We conducted a genome-wide analysis of germline CNVs using 19,342 cases and 15,917 controls from the Transdisciplinary Research in Cancer of the Lung (TRICL) consortium, with replication in two independent cohorts. High-confidence CNVs were identified by integrating two CNV callers including PennCNV and modSaRa2. Association analyses were performed using both gene-based and CNV region-based approaches. Polygenic risk scores (PRS) were constructed from top loci, and functional validation was conducted using siRNA-mediated knockdown in lung fibroblast cells. Results: We identified CNVs in four genomic regions (1p36.22, 2q31.2, 6p21.32, and 19q13.32) significantly associated with lung cancer risk. Two loci (1p36.22 and 2q31.2) were consistently supported across both analytical strategies. A CNV-based PRS constructed from key genes (CLCN6, NFE2L2, OPA3, and PSMB8) was significantly associated with lung cancer risk and replicated across independent datasets. Functional assays demonstrated that knockdown of NFE2L2 and OPA3 increased endogenous DNA damage, supporting a role in genomic stability. Conclusions: Germline CNVs contribute to lung cancer susceptibility and may influence carcinogenesis through mechanisms related to genomic instability. Impact: These findings expand the genetic architecture of lung cancer and highlight CNVs as potential biomarkers for improving risk stratification and informing precision prevention strategies.

Background: Lung cancer in never-smokers is rising, with a substantial proportion harboring the EGFR mutation. While fine particulate matter (PM2.5) is a recognized risk factor, other intervenable pollutants and built environmental factors remain unknown. Objectives: To identify urban characteristics associated with EGFR-mutant (vs. wild-type) lung cancer using high-resolution spatiotemporal data. Methods: We analyzed 2,699 lung cancer patients with documented EGFR status treated at a high-volume academic medical center in New York City. Patient residential addresses were linked to high-resolution (300m x 300m) 5-year cumulative exposures to 3 air pollutants and 26 urban features. We developed Light Gradient Boosting Machine (LightGBM) models to classify EGFR status, comparing a basic clinical model with established predictors (Asian, female, never-smoking status, and adenocarcinoma histology) to an extended model with additional urban factors. Predictive performance was assessed based on discrimination (AUC). Results: We included 2,699 patients, of whom 54.1% were female and 25.8% self-identified as Asian, 11.2% as Black, and 7.4% as Hispanic; and 29% had EGFR-mutated cancer. The extended model showed modest improvements in discrimination (AUC: 0.775 [95% CI, 0.739-0.809] vs. 0.768 [0.723-0.811]), compared to the clinical model. Newly identified factors for EGFR-mutant status included black carbon (BC), nitrogen dioxide (NO2), proximity to airports, reduced access to public transportation, elevated noise levels, and lead exposure. Conclusions: Traffic-related pollutants (BC, NO2) from diesel engines and motor vehicles, and proximity to airports, were among the novel spatiotemporal features associated with EGFR-mutant lung cancer. These results may inform policy interventions.

Background: Prostate cancer survival varies by stage at diagnosis, and Black men experience a disproportionate burden of advanced disease. We examined whether neighborhood deprivation, measured by Area Deprivation Index (ADI), contributes to racial differences in metastatic presentation. Methods: We conducted a population-based study of men diagnosed with prostate cancer in the Ohio Cancer Incidence Surveillance System from 1996 to 2016. The primary endpoint was distant-stage disease at diagnosis. Generalized additive models assessed nonlinear associations of ADI and diagnosis year with metastatic risk. Inverse probability of treatment weighting (IPTW) models estimated odds ratios comparing Black with White men after sequential adjustment for diagnosis year, age, insurance, and ADI. Results: Among 135,095 men, 18,690 were Black and 116,405 were White. Distant-stage disease occurred in 7.0% of Black men and 5.0% of White men. Black men had higher median ADI (60.9 vs. 47.3). Medicaid-insured men had the highest unadjusted odds of metastatic presentation (OR, 4.68; 95% CI, 4.13-5.31), exceeding uninsured men (OR, 2.91; 95% CI, 2.54-3.34). In IPTW models without age adjustment, the odds ratio decreased from 1.54 to 1.24 after adding insurance and ADI. In age-adjusted IPTW models, the odds ratio decreased from 1.79 to 1.41 after adding insurance and ADI. Generalized additive models showed increasing metastatic risk at higher ADI values and after 2008. Conclusions: Neighborhood deprivation and insurance-related access explained part, but not all, of the excess odds of metastatic diagnosis among Black men. Impact: Integrating ADI into cancer surveillance may improve identification of populations at risk for late-stage diagnosis.

Background. Tobacco smoke exposure, quantified by serum cotinine, is associated with cardiovascular, metabolic, and sleep-related health risks. The relationship between biomarker-verified tobacco smoke exposure and objectively measured, free-living wrist-worn ambient light patterns has not been examined in a nationally representative U.S. adult sample. Methods. We analyzed NHANES 2011-2014 cross-sectional data from 6,937 adults aged >20 years with valid serum cotinine and wrist-worn Physical Activity Monitor (PAM) ambient light data. Seven light outcomes were modeled using survey-weighted linear regression with log2(cotinine+1) as the continuous exposure across four covariate adjustment levels. Benjamini-Hochberg false discovery rate (FDR) correction was applied across the 7 outcomes within each model. Results. In Model 2 (adjusted for age, sex, race/ethnicity, education, poverty-income ratio, BMI, and survey cycle; N = 6,350), higher serum cotinine was associated with significantly higher nighttime light (beta = +0.024, 95% CI: 0.010, 0.038; p-FDR = 0.014) and lower evening light (beta = -0.031, 95% CI: -0.055, -0.008; p-FDR = 0.042). In exploratory behavioral models without alcohol (Model 3a; N = 5,766), both nighttime and evening associations remained FDR-significant. After additional adjustment for alcohol, which substantially reduced the sample due to 37.6% missingness (Model 3b; N = 3,866), the nighttime association attenuated below the FDR threshold, while the evening association remained FDR-significant. Categorical analyses showed progressively higher nighttime light across cotinine groups, and a hypothesis-generating sex interaction was identified (p-interaction = 0.001). Conclusions. Higher serum cotinine concentrations were associated with higher nighttime and lower evening ambient light after sociodemographic adjustment. Attenuation after behavioral adjustment and the cross-sectional design preclude causal inference. Longitudinal studies with formal mediation analyses are needed to clarify the temporal ordering and mechanisms linking tobacco smoke exposure, smoking-related behaviors, and personal light-dark cycle patterns.

Radiotherapy is delivered to more than half of all patients with cancer yet is prescribed using uniform physical doses despite well-established interpatient variability in biological response. The genomic-adjusted radiation dose (GARD), derived from the radiosensitivity index (RSI), integrates tumor transcriptomics with radiation dose to estimate patient-specific treatment effect, and has been clinically validated as a predictor of radiotherapy benefit across diverse disease sites, including breast, lung, head and neck, glioma, sarcoma, rectal, and endometrial cancers. However, further clinical validation and deployment has been limited by reliance on microarray-based expression. Here we develop an RNA sequencing-based formulation of RSI (RSI-seq) and show that it preserves the functional properties of the original model across measurement platforms. RSI-seq maintains concordance with microarray RSI, including preservation of patient rank ordering (pooled Spearman{rho} = 0.86), and, when integrated into GARD, reproduces predicted changes in biological effect under clinically relevant dose perturbations (R2 [≥] 0.78 for {Delta}GARD in both directions). This preservation of interventional prediction is robust to expression noise and invariant to normalization strategy, enabling consistent application across RNA-seq pipelines. Application across the TCGA pan-cancer transcriptomic atlas demonstrates scalability across tumor types, with cohort medians agreeing closely with previously published microarray RSI medians (Spearman{rho} = 0.68, Pearson r = 0.85 across 20 matched cohorts). By bringing a clinically validated radiogenomic dose model into the RNA-sequencing era, RSI-seq makes biologically personalized radiotherapy directly accessible, retrospectively in existing RNA-seq cohorts and prospectively in modern clinical sequencing workflows, across the full range of tumor types treated with radiation.

Importance: While gut dysbiosis is known to impair response to immune checkpoint inhibitors (ICIs), the relative clinical impact of antibiotic timing (pre- vs. post-ICI initiation) remains unclear. Objective: To evaluate whether antibiotic timing differentially influences overall survival (OS) in a large, multi-institutional pan-cancer cohort. Design, Setting, and Participants: This retrospective cohort study utilized deidentified electronic health record data from six academic medical centers within the University of California Health system. We included 21,108 adults with any malignancy who received PD-1, PD-L1, or CTLA-4 inhibitors between January 2014 and December 2024. Exposures: Antibiotic exposure windows were categorized as pre-only (-60 to -1 days), post-only (+1 to +60 days), both windows, or none. Main Outcomes and Measures: The primary outcome was overall survival (OS) calculated from the first ICI dose. Multivariable Cox proportional hazards models adjusted for demographics, tumor type, line of therapy, and baseline health indicators (albumin, NLR, and recent hospitalization). Results: Among 21,108 patients, 17.3% had pre-only exposure, 13.3% had post-only exposure, and 60.6% had no exposure. In the multivariable model, post-only exposure (HR, 1.27; 95% CI, 1.20-1.35) and combined pre- and post- exposure (HR, 1.31; 95% CI, 1.23-1.40) were significantly associated with higher mortality. Pre-only exposure was not significantly associated with OS (HR, 1.04; 95% CI, 0.99-1.10). Subgroup analyses by tumor type showed consistent trends across major malignancies, including head and neck (Post HR, 1.46) and renal cell carcinoma (Post HR, 1.26). Conclusions and Relevance: In contrast to some smaller studies, this large-scale analysis indicates that antibiotic exposure after ICI initiation carries a greater risk than exposure prior to treatment. These findings highlight the need for rigorous antibiotic stewardship strategies specifically during the early phases of immunotherapy treatment.

Background: Nitrogen dioxide (NO2) is a surrogate for traffic and industrial air pollution associated with adverse respiratory outcomes. Whether elevated NO2 and temperature jointly influence adult-onset asthma (AOA) risk is unclear, especially among subgroups with varying lifestyle and exposure profiles. We investigated further in the prospective All of Us research program. Methods: Among 596,926 U.S. participants who consented to electronic health record release, annual average NO2 concentrations from satellite data were linked to residential locations for 376,535 individuals. We used multivariable Cox regression to estimate associations between NO2, temperature, and incident AOA, adjusting for co-pollutants and potential confounders. We analyzed 4-category cross-classification variables between NO2 (high>75th percentile vs. low<=75th percentile) and maximum or average temperature (high>median vs. low<=median). We also stratified by sex, age, income, and smoking status. Additive interactions were estimated using Relative Excess Risk due to Interaction, Attributable Proportion, and Synergy Index. Results: We identified 10,413 incident AOA cases over an average 4-year follow-up. Participants with the highest categories of NO2 and temperature exposure had significantly higher risk compared to those with the lowest (HRHigh NO2 x High Max. Temp.=1.37, 95%CI:1.26-1.49; HRHigh NO2 x High Average Temp.=1.49, 95%CI:1.38-1.61). The joint association of high NO2 and high maximum temperature was more pronounced among ever-smokers (HR=1.59, 95%CI:1.40-1.81) than never-smokers (HR=1.26, 95%CI:1.13-1.41). Interaction analyses supported super-additive interactions of high NO2 and high average temperature on AOA risk, particularly among ever smokers, lower-income participants, and younger adults. Conclusion: Our findings highlight the respiratory health threat of long-term joint exposure to elevated NO2 and average temperature, particularly among vulnerable subgroups.

Purpose: Elective nodal (EN) irradiation (ENI) during radiotherapy for locally advanced head-and-neck squamous cell carcinoma (LA-HNSCC) influences hematotoxicity, anti-tumor immunity, and synergy with immunotherapy. We evaluated whether EN-sparing upfront boosts affect DNA damage, systemic immune signaling in peripheral blood lymphocytes (PBLs), and radiation-induced lymphopenia (RIL). Methods and Materials: Twenty-eight patients with LA-HNSCC were randomized to either adjuvant or definitive chemoradiotherapy with standard ENI or EN-sparing upfront boost (adjuvant: 2x2 Gy; definitive: 5x2 Gy). Blood was collected pre-radiotherapy, 15 min, and 24 h after the first fraction, and before the sixth fraction. DNA damage in PBLs was assessed via {gamma}H2AX and 53BP1 foci and dicentric chromosome (DIC) assay. RNA sequencing was performed in two patients per group (definitive setting) at pre-CRT, before the sixth fraction, and at therapy end. Absolute lymphocyte counts (ALCs) were monitored weekly to assess RIL. Results: DNA damage in PBLs correlated with planning target volume and whole-body dose, both of which were reduced by EN-sparing by 9.9-fold and 4.4-fold, respectively (p < 0.001 each). Correspondingly, EN-sparing significantly reduced radiation-induced foci and DIC levels in PBLs (3-4-fold, p < 0.001) and lowered the fraction of radiation-damaged PBLs per fraction (11% vs. 23% with ENI, p < 0.001). EN-sparing preserved baseline ALCs during week 1 of chemoradiotherapy and delayed RIL, whereas ENI caused an immediate ALC decline and RIL. Lymphocyte counts after week 1 negatively correlated with planning target volume, whole-body dose, and DNA damage in PBLs (p < 0.01). Transcriptomics showed metabolic and interferon signaling associated with EN-sparing, versus sterile inflammatory and damage-associated patterns with ENI. Conclusions: EN-sparing by an upfront boost significantly reduced PBL damage and early RIL with distinct immune responses associated with lymphocyte viability and immune maturation. These findings support upfront EN-sparing strategies to mitigate RIL and improve radiotherapy-immunotherapy synergy in HNSCC.

Background: Reirradiation (reRT) is increasingly offered following progression in diffuse intrinsic pontine glioma (DIPG) and diffuse midline glioma (DMG), though optimal patient selection remains a challenge. This study evaluated clinical outcomes after reRT in a contemporary cohort of patients with DIPG/DMG. Methods: Patients <26 years old with DMG/DIPG treated with radiation therapy between 2011-2025 were retrospectively reviewed. Primary endpoints included overall survival (OS2) and progression-free survival (PFS2), measured from first progression, and change in neurologic symptoms after reRT. Survival was estimated using Kaplan Meier methods, with Cox proportional hazards modeling for prognostic factors. Results: Fifty eight patients were included; 37 (63.8%) underwent reRT. Tumors were predominantly pontine (74.1%). ReRT was associated with improvement in motor function (51.4% vs. 9.5%, p=0.002), cranial nerve function (29.7% vs. 4.8%, p=0.044), and gait ataxia (35.1% vs. 9.5%, p=0.059). Median OS2 and PFS2 were improved with reRT (OS2: 9.67 vs. 2.57 months, p<0.001; PFS2: 5.63 vs. 1.57 months, p<0.001). OS2 was independently associated with reRT (HR 0.27, p<0.0001), pontine location (HR 2.94, p=0.004), and steroid use at progression (HR 4.12, p=0.001). PFS2 was independently associated with reRT (HR 0.23, p < .0001) and distant pattern of failure (HR 2.83, p=.037). Among reRT patients, non-pontine location was associated with improved OS2 (p=0.02), and local failure was associated with improved PFS2 (p=0.003). Conclusion: ReRT was associated with neurologic improvement and prolonged survival. Patients with non-pontine tumors or local-only failure might derive the greatest benefit. Prospective studies are warranted to define optimal dose/fractionation and refine patient selection.

Importance: Enfortumab vedotin (EV) is an antibody-drug conjugate approved for the treatment of locally advanced or metastatic urothelial cancer (la/mUC). Cutaneous adverse events (cAEs) are common during EV therapy, with prior studies suggesting an association between EV-related cAEs and improved survival; however, there is insufficient data to delineate the survival benefit of EV-induced cAEs from those associated with concurrent immune checkpoint inhibitors (ICIs). Objective: This study aims to evaluate the association of EV-induced cAEs and survival, and to characterize the timing and morphology of EV-induced cAEs. Design: We conducted a multi-institutional retrospective study of patients with la/mUC treated with EV between 2020 and 2025. Setting: Multicenter academic referral center. Participants: A total of 449 EV-treated patients were included. Patient characteristics were extracted manually, and likelihood scoring was used to attribute cAEs to either EV or other etiologies. Exposure: EV treatment. Main Outcomes and Measures: We estimated progression-free (PFS) and overall (OS) survival using Kaplan-Meier method. Multivariable time-varying and landmark Cox regression models were used to evaluate associations between EV-induced cAE and survival. Sensitivity analyses were performed at landmarks from 15 to 105 days. Results: Of 449 patients, 206 (45.9%) developed a cAE; 39 (18.9%) were high-grade and 127 (61.7%) were attributed to EV. The most common cAEs were pruritus (41.3%), unspecified and desquamating dermatitis (37.3%), and morbilliform dermatitis (27.7%). Across all treatment groups, survival was longer in patients with EV-induced cAEs. Developing an EV-induced cAE was protective across all examined landmark times, with hazard ratio (HR) 0.60 (95% CI: 0.43-0.82, p<0.001) for PFS and HR 0.46 (95% CI: 0.31-0.67, p<0.001) for OS at primary landmark time of 30 days. Early-onset EV-induced cAEs were protective at all landmark times and high-grade EV-induced cAEs were not associated with worse survival. Conclusions and Relevance: EV-induced cAEs were independently associated with improved PFS and OS in patients with la/mUC, even after accounting for immortal time bias and ICI exposure. Distinguishing EV-induced cAEs from other etiologies in timeline and morphology may help guide oncology and dermatology management.

Importance: Non-medullary thyroid cancer (NMTC) and melanoma are associated with inherited long telomeres due to germline pathogenic/likely pathogenic variants (PV/LPV) in POT1, TINF2, and ACD resulting in long-telomere syndrome (LTS) and they commonly have somatic TERT promoter mutations. The genetic relationship between these variants and their clinical associations are defined incompletely and may inform clinical practice. Objective: To test the hypothesis that germline LTS-associated PV/LPV are exclusive from functional somatic TERT variants and assess clinical/genetic associations. Design: Retrospective observational cohort study with/without germline LTS variants, that have somatic sequencing and pathology data. Setting: Participants were enrolled through 18 cancer centers participating in the Oncology Research Information Exchange Network (ORIEN). Participants: 995 adults with NMTC and 993 with melanoma between 2013 and 2025. All adult patients at an ORIEN center were offered enrollment Exposures: All patients with NMTC or melanoma are included. There are no required exposures. Main Outcomes and Measures: The presence/absence of a germline or somatic long-telomere variant; secondary outcomes are associations with tumor stage, telomerase expression, and oncogenes. Results: Germline and somatic variants in POT1/TINF2/ACD, somatic TERT promoter variants, TERT fusions, oncogenes, and telomerase mRNA expression were evaluated in 995 NMTC and 993 melanoma patients. In NMTC, 13 (1.5%) had a germline LTS variant while 0/12 with tumor sequencing had somatic TERT promoter variants/fusions. In melanoma, 7 (0.7%) had a LTS variant; 0/2 with tumor sequencing had a TERT promoter variant/ fusion. Meta-analysis including NMTC and melanoma in the current study, a recent thyroid cancer study, and thyroid TCGA, germline LTS-associated PV/LPV and somatic TERT variants/fusions were mutually exclusive (p=0.036). High telomerase mRNA levels were associated with TERT promoter variants/fusions (p<4e-11) and larger NMTC/distant metastases (p=0.016), but not germline LTS variants. NMTCs with somatic TERT promoter variants/fusions had higher tumor mutation burden (p<0.02) versus tumors from patients with a germline LTS variant. TERT promoter mutant variant allele frequency was lower in smaller and non-metastatic vs larger/metastatic NMTC. Conclusion and Relevance: Germline LTS-associated variants appear to be exclusive from somatic TERT promoter variants/fusions but are not associated with aggressive NMTC, suggesting common roles in tumorigenesis but different biological impacts.

Background Cancer treatment response is highly variable, even among patients with the same tumor type and treatment. Exceptional responders (ERs), who are individuals who experience unusually favorable outcomes, provide critical insights into the biological factors driving treatment success. While prior studies have highlighted the role of somatic changes, the contribution of germline rare variants remains underexplored. This study aimed to uncover the genetic underpinnings of exceptional responses by identifying rare, non-silent and predicted deleterious germline mutations enriched among ERs compared to typical cancer patients. Methods The Network of Enigmatic Exceptional Responders (NEER) project collected clinical and germline whole-genome sequencing (WGS) data from 53 ERs. After quality control procedures and ancestry background checks, 51 ERs were left for final analysis. While non-silent mutations were identified based on allele frequencies and mutation types, multiple pathogenicity predictors were applied for predicted deleterious variants. These were compared to a harmonized and comparable subset from the Pan-Cancer Analysis of Whole Genomes (PCAWG) cohort (n=414) using Fisher's exact tests. Kaplan-Meier survival analysis applied to evaluate prognostic associations in PCAWG patients. Additionally, Fisher's exact tests were conducted stratified by cancer type and treatment regimen to identify potential associations between rare germline variants and therapeutic responses. Results Variants in immune-related genes such as CCL26 and GPRC5D were prevalent, suggesting enhanced immune regulation among ERs. Fourteen genes with non-silent and eight with predicted deleterious mutations showed significantly different frequencies between NEER and PCAWG cohorts (FDR < 0.05). IRX3 emerged as a protective gene enriched in ERs, whereas OR6B2 was associated with poor survival in PCAWG lung cancer patients. Moreover, rare non-silent germline variants in drug target genes were enriched among ERs treated with cisplatin and doxorubicin, implicating altered DNA repair and drug-binding mechanisms in their remarkable outcomes. Conclusions This study reveals a distinctive germline mutation landscape in exceptional cancer responders, marked by immune-related and drug-target-associated variants that may enhance therapy response and prolong survival. The findings highlight potential novel prognostic biomarkers, such as IRX3 and OR6B2, providing a foundation for developing personalized cancer treatments informed by rare genetic variation.

ContextPancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy often diagnosed at advanced stages due to the lack of early clinical symptoms. DNA methylation alterations arise early in PDAC tumorigenesis and may serve as promising biomarkers for blood-based cancer detection. ObjectiveTo evaluate the performance of EPISEEK, a laboratory-developed blood-based multi-cancer early detection (MCED) assay, for detecting PDAC across disease stages. DesignA retrospective cohort study included 97 patients with stage I-IV PDAC and 201 asymptomatic healthy controls. Sensitivity, specificity, area under the curve (AUC), and stage-specific performance were assessed. EPISEEK-MCED performance was also compared with CA 19-9 alone and in combination with CA 19-9. ResultsEPISEEK-MCED classified 65 of 97 PDAC cases as positive, corresponding to an observed sensitivity of 70.1% (95% CI, 60.3% - 78.3%) at 99.5% specificity. The assay demonstrated strong discrimination between PDAC cases and healthy controls, with an AUC of 0.916 (95% CI, 0.88 - 0.952). Sensitivity increased with advancing stage while remaining substantial in early-stage disease, measuring 53.6% for stage I and 65.1% for stage II PDAC, 100% for stage III and 94.7% for stage IV. Across stages, EPISEEK-MCED outperformed CA 19-9 alone, particularly in early-stage disease. Combined analysis of EPISEEK-MCED and CA 19-9 further improved detection performance, achieving sensitivity of 57.1% and 81.4% for stage I and II, respectively. ConclusionsEPISEEK-MCED demonstrated high specificity and sensitivity for PDAC detection across disease stages, including early-stage disease. Combining EPISEEK-MCED with CA19-9 further improved performance, supporting its clinical utility for PDAC detection.

PurposeLow-dose radiation-induced adaptive response (LDRIAR) is well documented, but its role in early DNA damage signalling remains unclear. This study aimed to investigate whether adaptive response influences initial DNA double-strand break (DSB) recognition, as reflected by {gamma}H2AX foci formation, and to evaluate its time-dependent expression in human lymphocytes. Materials and MethodsPeripheral blood lymphocytes from three healthy donors were exposed to a priming dose followed by a challenging dose at defined time intervals. DNA damage was assessed using {gamma}H2AX foci analysis, comparing acute and split-dose exposures in both PHA-stimulated (large) and non-stimulated (small) lymphocytes. ResultsA clear time-dependent adaptive response was observed. No significant reduction in {gamma}H2AX foci was detected at 1 h (p > 0.05). At 2 h, a significant decrease was observed ([~]7-8% in large and [~]13% in small lymphocytes; p < 0.01), which increased at 4 h ([~]12% and [~]22%, respectively; p < 0.001). The maximal response occurred at 15 h, with reductions of [~]40- 43% in large and [~]27% in small lymphocytes (p < 0.001). Small lymphocytes exhibited an earlier response, while large lymphocytes showed a greater magnitude at later time points. The temporal trend was consistent across donors, with minor variability at later intervals. ConclusionsThe findings demonstrate that LDRIAR is reflected at the level of DNA damage signalling and follows a defined temporal pattern with cell-type specificity. This suggests that adaptive response may influence early DSB-associated processes, contributing to a better understanding of radiation response mechanisms in radiobiology.

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

Tobacco smoking induces DNA methylation (DNAm) changes in blood and other tissues, which may influence chronic health outcomes. However, the breadth of smoking-related DNAm changes remains unmapped, offering a space for employing novel technologies. To expand our understanding of smoking impacts on DNAm, we conducted an epigenome-wide association study (EWAS) comparing ever smokers to never smokers, using blood from a multiethnic U.S. study population (n=887). We employed the newly developed Illumina Methylation Screening Array (MSA) covering 269,094 unique sites, including 123,776 CpGs not assayed in previous EWAS. Trans-ethnic meta-analysis identified 152 differentially methylated positions (DMPs) associated with ever-smoking status (n=764); European-specific analysis yielded 129 DMPs (n=674), including 106 overlapping with trans-ethnic analysis. A separate, large-scale replication EWAS (n=2,190) confirmed 91 trans-ethnic and 77 European-specific DMPs. Among our findings, we identified 61 DMPs at CpGs novel to the MSA platform, including near both new and known smoking-associated genes. Most notably, we uncovered a dense cluster of 12 DMPs within a 1117 bp region of ECEL1P1, forming the most long-lasting, persistent smoking-associated DMR ever detected, even among former smokers who quit decades prior. We also detected new signals at AHRR, a well-known locus for smoking-related DNAm changes. eFORGE analysis revealed that detected smoking-associated DNAm changes are predominantly located in hematopoietic stem and progenitor cell (HSPC) DNase I hotspots, aligning with gene set enrichment analyses that highlighted pathways related to hematopoietic stem cell differentiation. Our findings suggest that HSPCs serve as a reservoir for an epigenetic memory of smoking. Additionally, we observed short-term cell-specific smoking-associated DNAm changes in myeloid cells. Our results demonstrate the utility of the MSA in expanding our knowledge of both transient and persistent environmental exposure-associated DNAm changes.

Background: Severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are major dose-limiting toxicities of chimeric antigen receptor (CAR) T-cell therapy. Existing pre-infusion biomarkers offer modest discrimination, motivating non-invasive alternatives. Methods: We prospectively enrolled 26 patients with relapsed/refractory large B-cell lymphoma receiving axicabtagene ciloleucel. Pre-infusion (day -1) exhaled breath samples were analyzed by gas chromatography-mass spectrometry for 40 volatile organic compounds (VOCs). Candidates with univariate AUC > 0.65 for severe (grade >=2) CRS or ICANS were carried forward to sensitivity-maximization-at-given-specificity with LASSO regularization (SMAGS-LASSO), which selected separate panels for each outcome. Model performance was assessed by leave-one-out cross-validation with permutation p-values and Harrell bootstrap optimism correction. Results: The 4-VOC CRS panel (heptanal, benzaldehyde, 2-butanone, ethylbenzene) achieved LOOCV AUC 82.5% (80% sensitivity at 88% specificity) and the 3-VOC ICANS panel (nonanal, allyl methyl sulfide, levomenthol) achieved AUC 86.3% (67% sensitivity at 86% specificity). By tertile, severe CRS occurred in 8/9 (89%) high-risk versus 2/9 (22%) low-risk patients (Cox HR 6.82, 95% CI 1.41-32.9, p=0.017) and severe ICANS occurred in 8/9 (89%) versus 2/9 (22%) (HR 8.28, 95% CI 1.73-39.6, p=0.008). Each 1-SD score increase corresponded to a 3.80-fold higher hazard of severe CRS (p<0.001) and 4.36-fold higher hazard of severe ICANS (p<0.001). In head-to-head comparison, the 3-VOC ICANS panel outperformed the modified Endothelial Activation and Stress Index (mEASIX) (delta-AUC +0.36, DeLong 1-sided p=0.008). The 4-VOC CRS panel had numerically higher AUC than mEASIX (delta-AUC +0.19, p=0.150). Conclusions: Pre-infusion exhaled breath VOC panels stratify CAR T-cell recipients by severity and timing of severe CRS and ICANS, providing a non-invasive complement to existing serum biomarkers. Multi-institutional validation is warranted.

PurposeComprehensive genomic profiling (CGP) has changed the treatment paradigm for non-small cell lung cancer (NSCLC) with the advent of molecularly targeted therapies for actionable genomic alterations (AGA). Despite this, the use of CGP is suboptimal, particularly in squamous cell lung cancer (sqNSCLC), which is more closely associated with smoking exposure and a lack of AGAs. We hypothesized that the prevalence of AGAs is inversely correlated with the chronicity and extent of smoking exposure in patients with sqNSCLC. Experimental DesignWe retrospectively evaluated all patients with liquid biopsy testing via Guardant 360CDX or Guardant360 in the context of any sqNSCLC diagnosis at the City of Hope Comprehensive Cancer Center between 10/2020 and 7/2023. The data was obtained on 2/23/24. Social and clinical histories were evaluated to assess the frequency of AGAs in patients with no or remote smoking history. ResultsOf the 56 patients in the initial evaluation, 24% (n=13) were non-smokers or remote smokers (greater than 20 years from cessation). Of these 13 patients, eight (61.5%) harbored AGA. Of these 8 patients, alterations observed included EGFR exon 19 deletion (50%, n=4), MET exon 14 skipping mutation (25%, n=2), EGFR G719S (13%, n=1), EGFR E114K (13%, n=1). Of those patients harboring AGAs that received NCCN-concordant matched targeted therapy, the objective response rate (ORR) with targeted agents was 50% and the clinical benefit rate (CBR) was 83.3%. ConclusionsThese data support the use of CGP in sqNSCLC particularly in patients with remote or no smoking exposure. Statement of translational relevanceThese data demonstrate high frequency of actionable genomic alterations (AGAs) in patients diagnosed with squamous cell lung cancer (sqNSCLC) with remote or no smoking history. Specifically, enrichment of EGFR and MET gene alterations were observed. These findings support the use of comprehensive molecular profiling in sqNSCLC. Furthermore, treatment outcomes demonstrate frequent objective responses and high clinical benefit rate supporting the use of targeted therapies in sqNSCLC harboring AGAs. This analysis provided rationale for further research of larger datasets investigating therapeutic approaches in sqNSCLC, which may have significant implications for consensus guideline recommendations and routine clinical practice.

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