Abstract Background: Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with a median survival of less than two years. Poor outcomes are driven in part by glioma stem cells (GSCs) that resist radiation and repopulate the tumor. The voltage gated potassium channel hERG1 (KCNH2) is upregulated in GBM and linked to therapy resistance, suggesting that its inhibition might improve radiotherapy efficacy. Methods: Human GBM cell line LN-229 and patient derived GSC line GNS144 were cultured as spheres. IC values for the selective hERG1 inhibitor E-4031 were determined (192.9 µM for LN-229, 94.23 µM for GNS144). Cells were treated with the respective IC concentration of E-4031 (or vehicle) and irradiated with a single fraction of 0 Gy or 10 Gy. Sphere formation assays were performed and quantified 7-14 days later. Cell-cycle distribution was assessed by flow cytometry. All experiments were performed in triplicate; statistical significance was evaluated with Tukey’s or Fisher’s LSD tests (p < 0.05). Results: In LN-229 spheres, combination therapy with E-4031 and radiation reduced sphere formation more than radiation alone (+14.4 %, p = 0.0041) or E-4031 alone (+23.56 %, p = 0.0002). In GNS144, the combination also performed better than radiation alone (+25.80 %, p = 0.0303) and E-4031 alone (+26.15 %, p = 0.0283). Cell cycle analysis of LN-229 showed that E-4031 decreased the S-phase population by 5.95 % (p = 0.0054) compared with controls, and when combined with radiation it reduced the S-phase population by an additional 7.36 % (p = 0.0016). Conclusions: Selective blockade of hERG1 with E-4031 markedly radiosensitizes GBM cells, producing synergistic reductions in sphere forming capacity, a surrogate for stemness. Moreover, hERG1 inhibition decreases the radioresistant S-phase cell population. These findings suggest that hERG1 blockade may be beneficial for radiosensitization in GBM, and further exploration of hERG1 targeted strategies could improve outcomes for patients diagnosed with glioblastoma. Citation Format: Hannah E. Goen, Naya Ohuabunwa, Jonathan D. Rodgers Gochicoa, Tingting Huang, Kelli B. Pointer. Blocking hERG1 enhances radiation sensitization in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7195.

Abstract Background: Glioblastoma (GBM) is the most common primary brain tumor in adults and has a median survival of less than two years despite surgery, chemotherapy, and radiotherapy. A subpopulation of glioma stem-like cells (GSCs) survives radiation and repopulates the tumor. Recent studies have shown that GSCs rely heavily on mitochondrial fatty-acid oxidation (FAO) for ATP production, NAD+ regeneration, and redox balance. Carnitine-palmitoyl-transferase-1 (CPT-1) catalyzes the rate limiting step of FAO by shuttling long-chain fatty acids into the mitochondrial matrix. We therefore hypothesized that pharmacologic inhibition of CPT-1 will limit GSC metabolism and sensitize GSCs to radiation. Methods: Human GBM cell line U-118 and a patient-derived GSC line (GNS144) were treated for 72 h with perhexiline (5 µM), etomoxir (10 µM), or vehicle control.Cells then received a single fraction of 0, 2, 4, 6, or 8 Gy. Cell viability was measured 48 h later. Sphere formation assays were performed 7-14 days after radiation; sphere number and mean diameter were recorded. Immunoblotting was performed to evaluate for stem cell markers CD44, Nestin, and Vimentin. All experiments were performed in triplicate; statistical significance was assessed by two-way ANOVA with Tukey post-hoc test (p < 0.05). Results: Perhexiline + radiation and etomoxir + radiation produced a dose dependent decline in cell viability compared with radiation alone (p < 0.001). Combination treatment reduced sphere number by 55-70 % across the 4-8 Gy range (p ≤ 0.005) and lowered mean sphere diameter by 30 % (p ≤ 0.005). Immunoblotting analysis showed a 2-fold reductions in CD44, Nestin, and Vimentin expression in the drug + radiation arms versus radiation alone (p < 0.01). Conclusions: These results suggest that inhibition of CPT-1-mediated FAO with perhexiline or etomoxir enhances radiation induced cell death, suppresses sphere forming capacity, and downregulates key GSC markers in both standard and patient-derived GBM models. These findings should be investigated further and suggest that CPT-1 blockade could be integrated with current standard of care regimens to overcome stem cell driven radiation resistance. Citation Format: Kenneth M. Austin, Tingting Huang, Todd Miller, Kelli B. Pointer. Targeting CPT-1-mediated fatty acid oxidation causes radiation sensitization in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6612.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with a median survival of less than two years despite multimodal therapy, including surgery, radiation, chemotherapy, and tumor treating fields. GBM's dismal prognosis is largely due to its high recurrence rates. Emerging evidence implicates ion channels in promoting tumor progression and treatment resistance across various malignancies. Specifically, the potassium voltage-gated channel hERG1, encoded by the KCNH2 gene, has been associated with worse patient outcomes in GBM. However, the mechanisms underlying hERG1’s contribution to treatment resistance, particularly radiation resistance, remain poorly understood. Here, we investigated the role of hERG1 in driving radiation resistance in GBM. RNA-sequencing data from 350 GBM patient tumors in The Cancer Genome Atlas (TCGA) were analyzed to assess KCNH2 expression and its correlation with overall survival. Patients were stratified into quartiles by KCNH2 expression, and Kaplan-Meier survival analyses were performed using R Studio (v4.3.1). The human GBM cell line LN-229 was used for in vitro studies. A drug screen determined the IC25 for an FDA-approved, non-cardiotoxic hERG1 inhibitor ketoconazole. Cell viability was assessed after 72 hours of drug treatment. Cells were treated with IC25 concentrations of inhibitors for three days, with radiation (0 Gy or 8 Gy) administered on day two, followed by two additional days of drug treatment. All experiments were performed in triplicate for statistical rigor. KCNH2 expression in the upper quartile was 9.7 times higher than in the lower quartile. Patients in the upper quartile exhibited worse median overall survival compared to those in the lower quartile (11.8 months vs. 12.9 months, p = 0.00053). The IC25 for ketoconazole was 28μM. Ketoconazole combined with radiation decreased cell viability by 54.4% compared to radiation alone (p =0.01) and by 78.8% compared to vehicle control (p < 0.0001). High KCNH2 expression is associated with worse survival outcomes in GBM patients. FDA-approved, non-cardiotoxic hERG1 inhibitor ketoconazole decreased cell viability and enhanced radiation sensitivity in vitro. These findings suggest that combining a non-cardiotoxic hERG1 inhibitor with radiation may represent a promising therapeutic strategy to improve GBM patient outcomes. Johnathan Rodgers Gochicoa, Tingting Huang, Kelli B. Pointer. FDA approved non-cardiotoxic hERG1 inhibitor ketoconazole enhances radiation sensitivity in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4697.

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a median survival of less than two years. Despite standard therapy of surgery, radiation, and chemotherapy, GBMs recur. These recurrences may partially be due to glioblastoma stem cells (GSCs). Various subpopulations of GSCs exist, likely attributing to different resistant niches. The mesenchymal (MES) subpopulation may drive radiation (RT) resistance. The transcriptomic shifts that define GSC adaptation to RT remain unknown. This study explores the transcriptomic reprogramming of MES GSCs following RT to identify resistance mechanisms. Using dataset GSE162931 from the GEO database and MES gene signatures from PMID:31327527, we analyzed two patient-derived GBM cell lines pre- and post-10 Gy RT. Data for the two cell lines were preprocessed and integrated with Seurat, followed by clustering and UMAP visualization with 2D kernel module-score weighted density plots. Cluster proportion changes between conditions were statistically examined using Chi-squared or Fisher’s tests with adjusted p-values. Differential expression analysis revealed conserved and unique gene markers distinguishing treated from naïve conditions, visualized in volcano plots and heatmaps. A final score integrating module scores and cluster proportions quantified MES traits at the single-cell level. All analyses were conducted in R studio version 4.2.3. UMAP, density plots, and cluster proportion changes indicate significant shifts in MES GSC distributions post-RT, suggesting RT selection for specific transcriptomic profiles. Highly MES subclusters exhibit distinctive RT-responsive gene expression patterns. The final scoring revealed enhanced MES traits in post-RT clusters. Distinct clustering patterns in MES GSCs highlight diverse adaptive strategies. Differential expression analysis reveals upregulation of DNA repair genes (SAT1, NDRG1, GADD45A), enhancing genomic stability post-RT, and stemness markers (NES, GDF15), emphasizing self-renewal capacity. Genes supporting oxidative stress management (SQSTM1, DDIT4) were upregulated, while downregulated metabolic genes (PGK1, LDHA) indicate a shift towards oxidative phosphorylation. Gene Set Enrichment Analysis identified enriched pathways related to DNA repair, cell migration, and differentiation regulation. These findings underline the transcriptomic plasticity in MES GSCs that drive RT resistance. Potential targets like AKAP12 and S100A4, essential for maintaining stem-like traits and MES phenotypes, present new therapeutic targets that could be used to combat resistant GSC populations, aiming to reduce tumor recurrence and enhance GBM patient outcomes. Grace Rosner, Abigail Goen, Godwin Peasah-Darkwah, Jonathan Rodgers Gochicoa, Alos Diallo, Gokul Srinivasan, Elisa Bu Sha, Joshua Levy, Kelli B. Pointer. Radiation-induced transcriptomic reprogramming of mesenchymal glioblastoma stem cells reveals key pathways and potential targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7502.

Purpose/Objective(s) Diffuse midline glioma (DMG) is an aggressive brain tumor. DMGs represent 15-20% of all pediatric central nervous system tumors, with 200-300 new cases diagnosed per year in the United States. Roughly 70% of DMG samples contain a H3K27M mutation. There has been no significant progress in treatment for DMG made in the last 50 years with the standard of care mainly being radiation therapy. Despite treatment, H3K27M DMG is universally fatal. Due to the rare nature of H3K27M DMGs, further understanding of what drives prognosis and outcomes is needed. The purpose of this study was to determine demographic, clinical, and treatment variables that impact overall survival (OS) in H3K27M DMG. Materials/Methods Data from the Surveillance, Epidemiology, and End Results (SEER)-Medicare Database from 2018-2020 was used to investigate prognostic factors that impact OS in patients with H3K27M DMG. 206 patients under the SEER category “1.1.3 Diffuse midline Glioma H327KM” were included in the study. Age, race, region in the United States, rural-urban location, household income, year of diagnosis, months from diagnosis to treatment, grade, laterality, extent of disease, tumor size, chromosome 19q and 1p loss of heterozygosity, chemotherapy, radiation, surgery, and surgery/radiation sequence were analyzed as factors that may impact OS. Univariate analysis was performed. Variables with a p value < 0.05 were analyzed using the multivariate Cox proportional hazards model. Statistical software was used for statistical analysis. A p value < 0.05 was considered statistically significant. Results The median age at diagnosis was 12 years old, 44.2% of the patients were male, and 55.8% of the patients were female. The median OS for patients with H327KM DMG was 14 months (95% CI = 11.574-16.426) with a 1-year survival rate of 54.1% and a 2-year survival rate of 26.8%. On multivariate analysis radiation treatment, sex, and age were statistically significant factors that influenced OS. Individuals who received radiation treatment survived significantly longer than individuals who did not receive radiation (p = 0.042); males survived significantly longer than females (p = 0.006); and younger individuals (£12) had a statistically significant better OS than older individuals (>12) (p = 0.008). There were no differences in OS based on other variables. Conclusion Radiation, sex, and age were identified as variables that were statistically significant for OS in individuals with H3K27M DMG. These data provide useful insight about factors that influence H3K27M DMG.

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Checkmate 227 established ipilimumab and nivolumab (ipi/nivo) as a first line treatment option for stage IV non-small cell lung cancer (NSCLC), providing a median overall survival of 17.1 months. Stereotactic body radiotherapy (SBRT) provides high rates of metastasis control and may be additive to immunotherapy by providing direct cytoreduction and preventing early progression at existing sites of disease, leading to durable improved outcomes. We designed a phase I study to examine the safety and efficacy of SBRT and ipi/nivo in first line treatment of metastatic NSCLC.

Our findings distinguish the genomic and transcriptomic effects of RT versus RT+ICB and challenge the prevailing paradigm that local ablative RT positively stimulates the immune response. We propose the use of tumor aneuploidy as a biomarker in personalizing treatment approaches for patients with various cancers.

Purpose: There are limited opportunities for mentorship for underrepresented in medicine (URM) trainees and physicians in radiation oncology (RO). The purpose of this study was to create and evaluate a formal mentorship program open to URMs and allies with interests in diversity, equity, and inclusion. Methods and Materials: A mentorship program incorporating a virtual platform was designed by the Association of Residents in Radiation Oncology Equity and Inclusion Subcommittee. It was structured to include 6 sessions over 6 months with matched mentor-mentee pairs based on responses to a publicized online interest form. A compilation of evidence-based guidelines was provided to optimize the mentorship relationship. Linked pre- and postprogram surveys were administered to collect demographic data, define baseline goals and level of support, and evaluate program satisfaction. Results: Thirty-five mentor-mentee pairs were matched; 31 mentees completed the preprogram survey and 17 completed the postprogram survey. Preprogram, only 3 mentees (9.7%) reported satisfaction with current mentorship and 5 (16%) reported mechanisms or mentorship in place at their program to support URMs. On the postprogram survey, mentees reported high satisfaction with areas of mentorship, mentor attributes, and the program overall. Opportunities for improvement include implementation of mechanisms to enhance communication with mentor-mentee pairs and maintain longitudinal engagement. Conclusions: In the first tailored mentorship program in RO for URMs and those with diversity, equity, and inclusion interests, our results demonstrate that there is self-reported interest for better mentorship for URMs in RO, and that a nationwide structured mentorship program can address participants' goals with high satisfaction. Program expansion could provide URMs and allies in RO more opportunities for career development and promote a greater sense of community and inclusion within the field. (c) 2023 Published by Elsevier Inc.

The number of patients who received each combination of hERG blockers is shown.

Abstract Identifying predictive and prognostic markers for glioblastoma (GBM) is an area of intense investigation. The objective of this study was to evaluate OLIG2 and Ki-67 as prognostic biomarkers. A tissue microarray with clinical data was created for 70 patients diagnosed with GBM between 2002-2007 at a single institution. Immunohistochemistry was performed to evaluate expression of OLIG2 and Ki-67. Overall survival and progression free survival (PFS) were calculated using the Kaplan-Meier method. Univariable (UVA) and multivariable (MVA) analysis was performed using Cox proportional hazards modeling. The median age of diagnosis was 63, 66% of patients were male, 80% had a KPS ≥70, 64% had a subtotal resection or biopsy, and 36% had a gross total resection. Median OS and PFS were 13.3 months and 9.5 months respectively. For patients who received resection plus chemoradiation, median OS and PFS were 16.7 months and 9.5 months respectively. 36.4% of patient samples had Ki-67 expression fraction > 20%. 18.0% had heavy OLIG2 staining. On MVA, lower KPS was associated with shorter OS and radiation therapy was associated with longer OS. OLIG2 was not associated with OS or PFS. Patients who had a Ki-67 < 20% had a shorter PFS, but not OS on MVA. Our data demonstrate higher Ki-67 levels correlate with improved PFS without an OS benefit. This association persisted for patients receiving standard of care treatment. OLIG2 levels did not correlate with PFS or OS in either cohort. Our study indicates that further investigation is needed to determine whether Ki-67 is useful in predicting recurrence risk and potentially changing treatment strategies for these patients, as well as the mechanism behind the associated improvement seen with higher Ki-67 levels. These data are currently limited by lack of IDH and MGMT status but suggest Ki-67 should be further investigated as a biomarker for progression.

Background Spatial transcriptomics involves studying the spatial organization of gene expression within tissues, offering insights into the molecular diversity of tumors. While spatial gene expression is commonly amalgamated from 1-10 cells across 50-micron spots, recent methods have demonstrated the capability to disaggregate this information at subspot resolution by leveraging both expression and histological patterns. However, elucidating such information from histology alone presents a significant challenge but if solved can better permit spatial molecular analysis at cellular resolution for instances where Visium data is not available, reducing study costs. This study explores integrating single-cell histological and transcriptomic data to infer spatial mRNA expression patterns in whole slide images collected from a cohort of stage pT3 colorectal cancer patients. A cell graph neural network algorithm was developed to align histological information extracted from detected cells with single cell RNA patterns through optimal transport methods, facilitating the analysis of cellular groupings and gene relationships. This approach leveraged spot-level expression as an intermediary to co-map histological and transcriptomic information at the single-cell level. Results Our study demonstrated that single-cell transcriptional heterogeneity within a spot could be predicted from histological markers extracted from cells detected within a spot. Furthermore, our model exhibited proficiency in delineating overarching gene expression patterns across whole-slide images. This approach compared favorably to traditional patch-based computer vision methods as well as other methods which did not incorporate single cell expression during the model fitting procedures. Topological nuances of single-cell expression within a Visium spot were preserved using the developed methodology. Conclusion This innovative approach augments the resolution of spatial molecular assays utilizing histology as a sole input through synergistic co-mapping of histological and transcriptomic datasets at the single-cell level, anchored by spatial transcriptomics. While initial results are promising, they warrant rigorous validation. This includes collaborating with pathologists for precise spatial identification of distinct cell types and utilizing sophisticated assays, such as Xenium, to attain deeper subcellular insights.

Over 150,000 Americans are diagnosed with colorectal cancer (CRC) every year, and annually over 50,000 individuals will die from CRC, necessitating improvements in screening, prognostication, disease management, and therapeutic options. CRC tumors are removed en bloc with surrounding vasculature and lymphatics. Examination of regional lymph nodes at the time of surgical resection is essential for prognostication. Developing alternative approaches to indirectly assess recurrence risk would have utility in cases where lymph node yield is incomplete or inadequate. Spatially dependent, immune cell-specific (e.g., Tumor Infiltrating Lymphocytes- TILs), proteomic, and transcriptomic expression patterns inside and around the tumor - the tumor immune microenvironment (TIME) - can predict nodal/distant metastasis and probe the coordinated immune response from the primary tumor site. The comprehensive characterization of TILs and other immune infiltrates is possible using highly multiplexed spatial omics technologies, such as the GeoMX Digital Spatial Profiler (DSP). In this study, machine learning and differential co-expression analyses helped identify biomarkers from DSP-assayed protein expression patterns inside, at the invasive margin, and away from the tumor, associated with extracellular matrix remodeling (e.g., GZMB, fibronectin), immune suppression (e.g., FOXP3), exhaustion and cytotoxicity (e.g., CD8), PD-L1 expressing dendritic cells, neutrophil proliferation, amongst other concomitant alterations. Further investigation of these biomarkers may reveal independent risk factors of CRC metastasis that can be formulated into low-cost, widely available assays.

Introduction: Previous studies have evaluated stereotactic body radiotherapy (SBRT) in oligometastatic patients with NSCLC, including multimodality treatment with anti- programmed cell death protein-1 monotherapy. Questions remain regarding the timing of SBRT and immunotherapy, safety with dual checkpoint blockade, and the utility in widely metastatic patients. This randomized phase 1 trial combined nivolumab and ipilimumab with sequential or concurrent multisite SBRT in patients with stage IV NSCLC to evaluate safety and obtain preliminary activity data. Methods: Treatment-naive patients with metastatic NSCLC were randomized to concurrent (SBRT with immuno-therapy) or sequential (SBRT followed by immunotherapy) treatment. A maximum of four treatment fields received SBRT. Nivolumab and ipilimumab were continued until clinical progression, development of toxicity, or after 2 years. Dose-limiting toxicity was defined as greater than or equal to grade 3 toxicity to the relevant organ system attributed to SBRT and immunotherapy occuring within 3 months. Results: A total of 37 patients were assessable. No dose-limiting toxicity occurred in the concurrent cohort (n 1/4 18). The sequential cohort required a dose reduction in the central lung group owing to two grade 4 pneumonitis events (2 of 19). Overall best response was as follows: 5.4% (2 of 37) complete response, 40.5% (15 of 37) partial response, 16.2% (6 of 37) stable disease, and 37.8% (14 of 37) progressive disease. Median progression-free survival was 5.8 months (95% confidence interval: 3.6-11.4 mo), with median follow-up of 17.0 months. Median overall survival was not reached. Conclusions: Concurrent nivolumab, ipilimumab, and SBRT were not more toxic than sequential therapy, and multisite SBRT was well tolerated in widely metastatic patients. Multimodality therapy resulted in durable metastasis con-trol and encouraging early overall survival. (c) 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

PURPOSE:Thoracic stereotactic body radiation therapy (SBRT) is associated with high rates of local control but carries a risk of pneumonitis. Immunotherapy is a standard treatment for patients with metastatic disease but can also cause pneumonitis. To evaluate the feasibility and safety of thoracic SBRT with systemic immunotherapy, clinical outcomes of patients treated with immune checkpoint blockade (ICB) and SBRT on prospective trials were reviewed.METHODS AND MATERIALS:Three consecutive phase 1 trials of combination SBRT and ICB conducted between 2016 to 2020 for widely metastatic solid tumors were reviewed. The protocols mandated adherence to NRG BR001/BR002 organs at risk constraints, resulting in <100% coverage of some target volumes. ICB was administered either sequentially (within 7 days after completion of SBRT) or concurrently (before or at the start of SBRT), depending on protocol. End points included pneumonitis, dose-volume constraints, local failure, and overall survival. The cumulative incidence estimator and Kaplan-Meier method were used.RESULTS:In the study, 123 patients met eligibility with 311 metastases irradiated. The most common histologies included non-small cell lung cancer (33%) and colorectal cancer (12%). Median follow-up was 12 months. The overall rate of grade 3+ pneumonitis was 8.1%; 1-year local failure was 3.6%. Established dosimetric parameters were significantly associated with the development of pneumonitis (P < .05). In most patients, the lungs were not challenged with high doses of radiation, defined as receiving ≥75% of the maximum for a given lung dose-volume constraint. Patients who were challenged were not found to have a significantly higher risk of pneumonitis.CONCLUSIONS:In the largest series of thoracic SBRT and immunotherapy, local control was excellent with acceptable toxicity and support the conclusion that established dose-volume constraints for the lung are safe. However, these results highlight the potential value in reporting of organs at risk being challenged with doses approaching protocol specified limits.

Immune checkpoint blockade (ICB) improves outcomes for some patients with advanced or metastatic cancers. Despite demonstrable progress, many patients do not respond to ICB. Recently, clinical trials have focused on combinations of ICB with radiation therapy. Although two recent Phase III randomized trials demonstrated improved survival with adjuvant ICB following chemoradiation, other Phase I/II/III trials are either negative or inconclusive, but do yield suggestive results and promising insights into future therapeutic strategies. We provide a selective review of a subset of these trials and attempt to integrate with basic laboratory findings where relevant to define issues pertaining to the combination of radiotherapy and immunotherapy.

Over 500 clinical trials are investigating combination radiotherapy and immune checkpoint blockade (ICB) as cancer treatments; however, the majority of trials have found no positive interaction. Here we perform a comprehensive molecular analysis of a randomized phase I clinical trial of patients with non-small cell lung cancer (NSCLC) treated with concurrent or sequential ablative radiotherapy and ICB. We show that concurrent treatment is superior to sequential treatment in augmenting local and distant tumor responses and in improving overall survival in a subset of patients with immunologically cold, highly aneuploid tumors, but not in those with less aneuploid tumors. In addition, radiotherapy alone decreases intratumoral cytotoxic T cell and adaptive immune signatures, whereas radiotherapy and ICB upregulates key immune pathways. Our findings challenge the prevailing paradigm that local ablative radiotherapy beneficially stimulates the immune response. We propose the use of tumor aneuploidy as a biomarker and therapeutic target in personalizing treatment approaches for patients with NSCLC treated with radiotherapy and ICB.

Recent research has highlighted the importance of key tumor microenvironment features, notably the collagen-rich extracellular matrix (ECM) in characterizing tumor invasion and progression. This led to great interest from both basic researchers and clinicians, including pathologists, to include collagen fiber evaluation as part of the investigation of cancer development and progression. Fibrillar collagen is the most abundant in the normal extracellular matrix, and was revealed to be upregulated in many cancers. Recent studies suggested an emerging theme across multiple cancer types in which specific collagen fiber organization patterns differ between benign and malignant tissue and also appear to be associated with disease stage, prognosis, treatment response, and other clinical features. There is great potential for developing image-based collagen fiber biomarkers for clinical applications, but its adoption in standard clinical practice is dependent on further translational and clinical evaluations. Here, we offer a comprehensive review of the current literature of fibrillar collagen structure and organization as a candidate cancer biomarker, and new perspectives on the challenges and next steps for researchers and clinicians seeking to exploit this information in biomedical research and clinical workflows.

Abstract Purpose Immune checkpoint inhibitors improve survival in metastatic diseases for some cancers. Multisite SBRT with pembrolizumab (SBRT + Pembro) was shown to be safe with promising local control using biologically effective doses (BEDs) = 95–120 Gy. Increased BED may improve response rate; however, SBRT doses are limited by surrounding organs at risk (OARs). The purpose of this work was to develop and validate methods for safe delivery of ultra‐high doses of radiation (BED10 > 300) to be used in future clinical trials. Methods and Materials The radiation plans from 15 patients enrolled on a phase I trial of SBRT + pembro were reanalyzed. Metastatic disease sites included liver (8/15), inguinal region (1/15), pelvis (2/15), lung (1/15), abdomen (1/15), spleen (1/15), and groin (1/15). Gross tumor volumes (GTVs) ranged from 80 to 708 cc. Following the same methodology used in the Phase I trial on which these patients were treated, GTVs > 65 cc were contracted to a 65 cc subvolume (SubGTV) resulting in only a portion of the GTV receiving prescription dose. Volumetric modulated arc therapy (VMAT) was used to plan treatments BED10 = 360 Gy. Plans utilizing both 6FFF and 10FFF beams were compared to clinical plans delivering BED10 = 112.50 Gy. The target primary goal was V100% > 95% with a secondary goal of V70% > 99% and OAR objectives per the trial. To demonstrate feasibility, plans were delivered to a diode array phantom and evaluated for fidelity using gamma analysis. Results All 30 plans met the secondary coverage goal and satisfied all OAR constraints. The primary goal was achieved in 12/15 of the 6FFF plans and 13/15 of the 10FFF plans. Average gamma analysis passing rate using criteria of 3% dose difference and 3, 2, and 1 mm were 99.1 ± 1.0%, 98.5 ± 1.6%, and 95.1 ± 3.8%, respectively. Conclusion Novel VMAT planning approaches with clinical treatment planning software and linear accelerators prove capable of delivering radiation doses in excess of 360 Gy BED10 to tumor subvolumes, while maintaining safe OAR doses.