Radiotherapy and immunology.

The majority of cancer patients receive radiotherapy during the course of treatment, delivered with curative intent for local tumor control or as part of a multimodality regimen aimed at eliminating distant metastasis. A major focus of research has been DNA damage; however, in the past two decades, emphasis has shifted to the important role the immune system plays in radiotherapy-induced anti-tumor effects. Radiotherapy reprograms the tumor microenvironment, triggering DNA and RNA sensing cascades that activate innate immunity and ultimately enhance adaptive immunity. In opposition, radiotherapy also induces suppression of anti-tumor immunity, including recruitment of regulatory T cells, myeloid-derived suppressor cells, and suppressive macrophages. The balance of pro- and anti-tumor immunity is regulated in part by radiotherapy-induced chemokines and cytokines. Microbiota can also influence radiotherapy outcomes and is under clinical investigation. Blockade of the PD-1/PD-L1 axis and CTLA-4 has been extensively investigated in combination with radiotherapy; we include a review of clinical trials involving inhibition of these immune checkpoints and radiotherapy.

Hormonal Therapy and Radiation Therapy in Prostate Cancer: 5-Year Outcomes From a Trial Evaluating Combined Androgen Blockade With 5-Alpha Reductase Inhibitors as an Alternative to Gonadotropin Releasing Hormone Agonists.

BACKGROUND: We previously reported that for men undergoing combined androgen deprivation therapy (ADT) and radiation therapy (RT) for prostate cancer, substitution of LHRH-agonists with 5-α- reductase inhibitors (5-ARIs) led to improved preservation of 6-month hormonal quality of life (hQOL). With longer term follow-up, we evaluated disease control. METHODS: In this non-randomized trial, men with unfavorable intermediate or high-risk prostate cancer, aged ≥70 years or with Charlson Comorbidity Index ≥2, were treated with RT (78-79.2 Gy in 39-44 fractions) and either oral ADT (oADT; 5-ARI with antiandrogen) or standard of care ADT (SOC; leuprolide with antiandrogen) for up to 28 months. The primary endpoint was EPIC hQOL; secondary endpoints included biochemical control and survival as well as changes in cholesterol and hemoglobin levels. RESULTS: Between 2011 and 2018, 70 men were enrolled (40 in oADT; 30 in SOC). Median follow-up was 65 months [IQR 36-94]. Five-year freedom from biochemical failure for oADT and SOC was 89% versus 86%, disease free survival was 62% versus 69%, cancer-specific survival was 100% versus 96%, and overall survival was 70% versus 81% (all P>.1). Testosterone (2 mo through 3 yr) and hemoglobin levels (2 mo through 2 yr) were higher, and cholesterol levels (1 yr) were lower in the oADT groups (all P < .05). CONCLUSIONS: In this non-randomized study, men treated with combined RT and oADT had better preservation of hQOL and comparable 5-year disease outcomes to men treated with SOC. Eugonadal testosterone with this approach may yield measurable benefits in cholesterol and hemoglobin levels.

Radiotherapy Enhances Metastasis Through Immune Suppression by Inducing PD-L1 and MDSC in Distal Sites.

PURPOSE: Radiotherapy (RT) is a widely employed anticancer treatment. Emerging evidence suggests that RT can elicit both tumor-inhibiting and tumor-promoting immune effects. The purpose of this study is to investigate immune suppressive factors of radiotherapy. EXPERIMENTAL DESIGN: We used a heterologous two-tumor model in which adaptive concomitant immunity was eliminated. RESULTS: Through analysis of PD-L1 expression and myeloid-derived suppressor cells (MDSC) frequencies using patient peripheral blood mononuclear cells and murine two-tumor and metastasis models, we report that local irradiation can induce a systemic increase in MDSC, as well as PD-L1 expression on dendritic cells and myeloid cells, and thereby increase the potential for metastatic dissemination in distal, nonirradiated tissue. In a mouse model using two distinct tumors, we found that PD-L1 induction by ionizing radiation was dependent on elevated chemokine CXCL10 signaling. Inhibiting PD-L1 or MDSC can potentially abrogate RT-induced metastasis and improve clinical outcomes for patients receiving RT. CONCLUSIONS: Blockade of PD-L1/CXCL10 axis or MDSC infiltration during irradiation can enhance abscopal tumor control and reduce metastasis.

Combined Stereotactic Body Radiation Therapy and Immune Checkpoint Inhibition for Liver Metastases: Safety and Outcomes in a Pooled Analysis of 3 Phase 1 Trials.

PURPOSE: Stereotactic body radiation therapy (SBRT) safely and effectively controls liver metastases (LMs), but its safety and efficacy when combined with immune checkpoint inhibitors (ICIs) are not well characterized. This analysis of 3 phase 1 trials of combination SBRT and ICI evaluates whether LM-SBRT increases the risk for hepatotoxicity when combined with ICI and explores efficacy endpoints. METHODS AND MATERIALS: Data were analyzed from 3 phase 1 trials of combination SBRT and ICI for patients with metastatic solid tumors conducted between 2016 and 2020. ICI was administered per trial protocol with LM-SBRT delivered to 45 Gy in 3 fractions with mean liver dose <16 Gy and ≥700 cc of normal liver spared 17.1 Gy. Hepatic adverse events (HAEs) were defined as hepatic failure, autoimmune hepatitis, or elevation of aspartate transaminase, alanine transaminase, bilirubin, or alkaline phosphatase using Common Terminology Criteria for Adverse Events version 4.0. Cumulative incidence of HAEs and local failure were modeled with death as a competing risk. Competing risk regression was performed using Fine-Gray modeling. Survival was estimated via the Kaplan-Meier method. RESULTS: Two hundred patients were analyzed, including 81 patients with LM, 57 of whom received LM-SBRT. The 12-month rate of any grade ≥2 HAE was 11% and 10% in LM-SBRT and non-LM-SBRT patients, respectively non-significant (NS). Radiographic evidence for liver disease and dual-agent ICI was significantly associated with HAEs on univariable and multivariable analysis, but liver dose metrics were not. Patients with LM had significantly worse progression-free and overall survival compared with those without, and local failure of treated LM was significantly higher than for treated extrahepatic metastases (28% vs 4% at 12 months, P < .001). CONCLUSIONS: Combination LM-SBRT and ICI did not significantly increase the risk for HAEs compared with ICI without LM-SBRT, suggesting hepatotoxicity is largely driven by factors other than liver radiation therapy, such as choice of ICI. LM is associated with worse overall survival and local control outcomes.

Bladder-Preserving Trimodality Therapy With Capecitabine.

INTRODUCTION: Many patients with muscle-invasive bladder cancer are poor candidates for radical cystectomy or trimodality therapy with maximal transurethral resection of bladder tumor (TURBT) and chemoradiotherapy with cisplatin or mitomycin C. Given the benefit of chemotherapy in bladder-preserving therapy, less-intense concurrent chemotherapy regimens are needed. This study reports on efficacy and toxicity for patients treated with trimodality therapy using single-agent concurrent capecitabine. MATERIALS AND METHODS: Patients deemed ineligible for radical cystectomy or standard chemoradiotherapy by a multidisciplinary tumor board and patients who refused cystectomy were included. Following TURBT, patients received twice-daily capecitabine (goal dose 825 mg/m2) concurrent with radiotherapy to the bladder +/- pelvis depending on nodal staging and patient risk factors. Toxicity was evaluated prospectively in weekly on-treatment visits and follow-up visits by the treating physicians. Descriptive statistics are provided. Overall, progression-free, cancer-specific, distant metastasis-free, and bladder recurrence-free survival were estimated using the Kaplan-Meier method. RESULTS: Twenty-seven consecutive patients met criteria for inclusion from 2013 to 2023. The median age was 79 with 9 patients staged cT3-4a and 7 staged cN1-3. The rate of complete response in the bladder and pelvis was 93%. Overall, progression-free, cancer-specific, distant metastasis-free, and bladder recurrence-free survival at 2 years were estimated as 81%, 65%, 91%, 75%, and 92%, respectively. There were 2 bladder recurrences, both noninvasive. There were 7 grade 3 acute hematologic or metabolic events but no other grade 3+ toxicities. CONCLUSION: Maximal TURBT followed by radiotherapy with concurrent capecitabine offers a high rate of bladder control and low rates of acute and late toxicity.

RNA m6A methylation and MDSCs: Roles and therapeutic implications for radiotherapy.

Emerging evidence suggests that local tumor radiotherapy reshapes the repertoire of circulating myeloid-derived suppressor cells (MDSCs) and leads to their infiltration into the tumor microenvironment, which poses a major obstacle for radiotherapy efficacy. Recent findings have identified RNA m6A modification at the nexus of both anti-tumor immunity and radiation response. Here, we examine the mechanisms by which this RNA modification modulates the immune milieu of the radiation-remodeled tumor microenvironment. We discuss potential therapeutic interventions targeting m6A machinery to improve radiotherapy response.

Epitranscriptional regulation of TGF-ß pseudoreceptor BAMBI by m6A/YTHDF2 drives extrinsic radioresistance.

Activation of TGF-ß signaling serves as an extrinsic resistance mechanism that limits the potential for radiotherapy. Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) antagonizes TGF-ß signaling and is implicated in cancer progression. However, the molecular mechanisms of BAMBI regulation in immune cells and its impact on antitumor immunity after radiation have not been established. Here, we show that ionizing radiation (IR) specifically reduces BAMBI expression in immunosuppressive myeloid-derived suppressor cells (MDSCs) in both murine models and humans. Mechanistically, YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) directly binds and degrades Bambi transcripts in an N6-methyladenosine-dependent (m6A-dependent) manner, and this relies on NF-κB signaling. BAMBI suppresses the tumor-infiltrating capacity and suppression function of MDSCs via inhibiting TGF-ß signaling. Adeno-associated viral delivery of Bambi (AAV-Bambi) to the tumor microenvironment boosts the antitumor effects of radiotherapy and radioimmunotherapy combinations. Intriguingly, combination of AAV-Bambi and IR not only improves local tumor control, but also suppresses distant metastasis, further supporting its clinical translation potential. Our findings uncover a surprising role of BAMBI in myeloid cells, unveiling a potential therapeutic strategy for overcoming extrinsic radioresistance.

Integrated Clinical-Molecular Classification of Colorectal Liver Metastases: A Biomarker Analysis of the Phase 3 New EPOC Randomized Clinical Trial.

Importance: Personalized treatment approaches for patients with oligometastatic colorectal liver metastases are critically needed. We previously defined 3 biologically distinct molecular subtypes of colorectal liver metastases: (1) canonical, (2) immune, and (3) stromal. Objective: To independently validate these molecular subtypes in the phase 3 New EPOC randomized clinical trial. Design, Setting, and Participants: This retrospective secondary analysis of the phase 3 New EPOC randomized clinical trial included a bi-institutional discovery cohort and multi-institutional validation cohort. The discovery cohort comprised patients who underwent hepatic resection for limited colorectal liver metastases (98% received perioperative chemotherapy) from May 31, 1994, to August 14, 2012. The validation cohort comprised patients who underwent hepatic resection for liver metastases with perioperative chemotherapy (fluorouracil, oxaliplatin, and irinotecan based) with or without cetuximab from February 26, 2007, to November 1, 2012. Data were analyzed from January 18 to December 10, 2021. Interventions: Resected metastases underwent RNA sequencing and microRNA (miRNA) profiling in the discovery cohort and messenger RNA and miRNA profiling with microarray in the validation cohort. Main Outcomes and Measures: A 31-feature (24 messenger RNAs and 7 miRNAs) neural network classifier was trained to predict molecular subtypes in the discovery cohort and applied to the validation cohort. Integrated clinical-molecular risk groups were designated based on molecular subtypes and the clinical risk score. The unique biological phenotype of each molecular subtype was validated using gene set enrichment analyses and immune deconvolution. The primary clinical end points were progression-free survival (PFS) and overall survival (OS). Results: A total of 240 patients were included (mean [range] age, 63.0 [56.3-68.0] years; 151 [63%] male), with 93 in the discovery cohort and 147 in the validation cohort. In the validation cohort, 73 (50%), 28 (19%), and 46 (31%) patients were classified as having canonical, immune, and stromal metastases, respectively. The biological phenotype of each subtype was concordant with the discovery cohort. The immune subtype (best prognosis) demonstrated 5-year PFS of 43% (95% CI, 25%-60%; hazard ratio [HR], 0.37; 95% CI, 0.20-0.68) and OS of 63% (95% CI, 40%-79%; HR, 0.38; 95% CI, 0.17-0.86), which was statistically significantly higher than the canonical subtype (worst prognosis) at 14% (95% CI, 7%-23%) and 43% (95% CI, 32%-55%), respectively. Adding molecular subtypes to the clinical risk score improved prediction (the Gönen and Heller K for discrimination) from 0.55 (95% CI, 0.49-0.61) to 0.62 (95% CI, 0.57-0.67) for PFS and 0.59 (95% CI, 0.52-0.66) to 0.63 (95% CI, 0.56-0.70) for OS. The low-risk integrated group demonstrated 5-year PFS of 44% (95% CI, 20%-66%; HR, 0.38; 95% CI, 0.19-0.76) and OS of 78% (95% CI, 44%-93%; HR, 0.26; 95% CI, 0.08-0.84), superior to the high-risk group at 16% (95% CI, 10%-24%) and 43% (95% CI, 32%-52%), respectively. Conclusions and Relevance: In this prognostic study, biologically derived colorectal liver metastasis molecular subtypes and integrated clinical-molecular risk groups were highly prognostic. This novel molecular classification warrants further study as a possible predictive biomarker for personalized systemic treatment for colorectal liver metastases. Trial Registration: isrctn.org Identifier: ISRCTN22944367.

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.

YTHDF2 inhibition potentiates radiotherapy antitumor efficacy.

RNA N6-methyladenosine (m6A) modification is implicated in cancer progression. However, the impact of m6A on the antitumor effects of radiotherapy and the related mechanisms are unknown. Here we show that ionizing radiation (IR) induces immunosuppressive myeloid-derived suppressor cell (MDSC) expansion and YTHDF2 expression in both murine models and humans. Following IR, loss of Ythdf2 in myeloid cells augments antitumor immunity and overcomes tumor radioresistance by altering MDSC differentiation and inhibiting MDSC infiltration and suppressive function. The remodeling of the landscape of MDSC populations by local IR is reversed by Ythdf2 deficiency. IR-induced YTHDF2 expression relies on NF-κB signaling; YTHDF2 in turn leads to NF-κB activation by directly binding and degrading transcripts encoding negative regulators of NF-κB signaling, resulting in an IR-YTHDF2-NF-κB circuit. Pharmacological inhibition of YTHDF2 overcomes MDSC-induced immunosuppression and improves combined IR and/or anti-PD-L1 treatment. Thus, YTHDF2 is a promising target to improve radiotherapy (RT) and RT/immunotherapy combinations.

Oligometastases: Characterizing the Role of Epigenetic Regulation of Epithelial-Mesenchymal Transition.

The "oligometastasis" hypothesis proposes that metastases exist as a spectrum and are not always disseminated. According to this theory, a subset of patients with metastatic disease could benefit from aggressive local therapies. However, the identification of patients most likely to exhibit an oligometastatic phenotype remains challenging. Recent literature focusing on basic and translational studies has identified novel epigenetic regulators of epithelial-mesenchymal transition (EMT) and the emergence of a spectrum of metastatic behavior. Herein, we review these scientific advances and suggest that the spectrum of metastatic virulence produced by these epigenetic mechanisms broadly contributes to the emergence of clinically evident "oligometastases." Epigenetic regulation of EMT programs can result in a spectrum of cell trajectories (e.g., quasi-mesenchymal and highly mesenchymal states) with differential propensity to develop metastases. We propose that quasi-mesenchymal cell states may be associated with a polymetastatic phenotype, whereas highly mesenchymal cell states may be associated with a more oligometastatic phenotype. The mechanisms governing epigenetic regulation of EMT and its array of intermediate states are multifaceted and may contribute to the development of the metastatic spectrum observed clinically. Within this context, translational studies that support the role of EMT and its epigenetic regulation are discussed. Continued translation of these mechanistic discoveries into novel biomarkers may help optimally select patients most likely to exhibit an oligometastatic phenotype and benefit from aggressive local therapies, such as surgery, radiotherapy, and other ablative procedures.

STK11 Inactivation Predicts Rapid Recurrence in Inoperable Early-Stage Non-Small-Cell Lung Cancer.

PURPOSE: Molecular factors predicting relapse in early-stage non-small-cell lung cancer (ES-NSCLC) are poorly understood, especially in inoperable patients receiving radiotherapy (RT). In this study, we compared the genomic profiles of inoperable and operable ES-NSCLC. MATERIALS AND METHODS: This retrospective study included 53 patients with nonsquamous ES-NSCLC (stage I-II) treated at a single institution (University of Chicago) with surgery (ie, operable; n = 30) or RT (ie, inoperable; n = 23) who underwent tumor genomic profiling. A second cohort of ES-NSCLC treated with RT (Stanford, n = 39) was included to power clinical analyses. Prognostic gene alterations were identified and correlated with clinical variables. The primary clinical end point was the correlation of prognostic genes with the cumulative incidence of relapse, disease-free survival, and overall survival (OS) in a pooled RT cohort from the two institutions (N = 62). RESULTS: Although the surgery cohort exhibited lower rates of relapse, the RT cohort was highly enriched for somatic STK11 mutations (43% v 6.7%). Receiving supplemental oxygen (odds ratio [OR] = 5.5), 20+ pack-years of tobacco smoking (OR = 6.1), and Black race (OR = 4.3) were associated with increased frequency of STK11 mutations. In the pooled RT cohort (N = 62), STK11 mutation was strongly associated with inferior oncologic outcomes: 2-year incidence of relapse was 62% versus 20% and 2-year OS was 52% versus 85%, remaining independently prognostic on multivariable analyses (relapse: subdistribution hazard ratio = 4.0, P = .0041; disease-free survival: hazard ratio, 6.8, P = .0002; OS: hazard ratio, 6.0, P = .022). STK11 mutations were predominantly associated with distant failure, rather than local. CONCLUSION: In this cohort of ES-NSCLC, STK11 inactivation was associated with poor oncologic outcomes after RT and demonstrated a novel association with clinical hypoxia, which may underlie its correlation with medical inoperability. Further validation in larger cohorts and investigation of effective adjuvant systemic therapies may be warranted.

Synergizing radiotherapy and immunotherapy: Current challenges and strategies for optimization.

Numerous clinical studies are investigating the integration of radiotherapy and immune checkpoint inhibitors (ICI) in the management of advanced or metastatic solid cancers based on preclinical evidence demonstrating a synergistic interaction between these treatments. However, it remains unclear how to optimally integrate these therapeutic modalities in the treatment of cancer patients. Beyond disease-specific factors there exists numerous unanswered questions regarding optimal sequencing of radiation and ICI, as well as, radiation dosing and target selection. Here, we examine the available clinical evidence for combination radiation and ICI approaches and propose strategies to expand investigations of the potential synergy in cancer patients.

Phase 1 Randomized Trial of Stereotactic Body Radiation Therapy Followed by Nivolumab plus Ipilimumab or Nivolumab Alone in Advanced/Unresectable Hepatocellular Carcinoma.

PURPOSE: Immunotherapy has emerged as a promising therapeutic option for advanced or unresectable hepatocellular carcinoma (HCC). However, survival remains poor with only a subset of patients deriving benefit. This trial investigated the safety and efficacy of stereotactic body radiation therapy (SBRT) with immunotherapy in HCC. METHODS AND MATERIALS: In this multicenter phase 1 randomized trial, patients with advanced or unresectable HCC received liver SBRT (40 Gy in 5 fractions) followed by either nivolumab alone or nivolumab plus ipilimumab. The primary endpoint was dose-limiting toxicity occurring within 6 months of SBRT. Secondary endpoints included overall response rate, progression-free survival, overall survival (OS), distant disease control, and local control of the irradiated tumor. Disease status and response endpoints were assessed radiographically every 8 weeks until progression or initiation of nonprotocol therapy. Response was determined using both RECIST (Response Evaluation Criteria in Solid Tumors) 1.1 and iRECIST. RESULTS: Fourteen patients were enrolled across 3 centers. Thirteen patients were evaluated for study endpoints. The study was closed early because of slow accrual. The median follow-up time was 42.7 months. Dose-limiting toxicities within 6 months occurred in 2 (15.4%) of 13 patients: 1 of 6 patients in the nivolumab arm (16.7%; 90% confidence interval [CI], 0.9%-58.2%) and 1 of 7 patients in the nivolumab plus ipilimumab arm (14.3%; 90% CI, 0.7%-52.1%). Grade 3 adverse events occurred in 8 (61.6%), 5 (71.4%), and 3 (50.0%) patients in the overall nivolumab plus ipilimumab and nivolumab cohorts. Grade 3 hepatotoxicity occurred in 4 (30.8%), 3 (42.9%), and 1 (16.7%) patients in the respective cohorts. Clinical outcomes favored the nivolumab plus ipilimumab arm compared with nivolumab alone, including an overall response rate of 57% (4 of 7 patients; 90% CI, 23%-87%) versus 0% (0 of 6 patients; 90% CI, 0%-39%), median progression-free survival of 11.6 months (90% CI, 4.5 months to not reached) versus 2.7 months (90% CI, 1.3-4.7 months), and median OS of 41.6 months (90% CI, 4.5 months to not reached) versus 4.7 months (90% CI, 2.0-16.2 months) (all P < .05). With combination immunotherapy, 3-year OS was 57% (90% CI, 23%-81%), with 2 patients alive after 42.7 months without progression and negative PET. CONCLUSIONS: In this first prospective trial investigating the combination of SBRT and immunotherapy for HCC, multimodal therapy demonstrated acceptable safety. SBRT with nivolumab plus ipilimumab compared favorably to outcomes of immunotherapy alone and warrants further investigation.

Highly aneuploid non-small cell lung cancer shows enhanced responsiveness to concurrent radiation and immune checkpoint blockade.

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.

Tumor aneuploidy predicts survival following immunotherapy across multiple cancers.

Tumor mutational burden (TMB) has emerged as a promising biomarker of immunotherapy response across multiple cancer types; however, clinical outcomes among patients with low TMB tumors are heterogeneous. Herein, we demonstrate that tumor aneuploidy provides independent prognostic value among patients with lower TMB (<80th percentile) tumors treated with immunotherapy. A higher aneuploidy score is associated with poor prognosis following immunotherapy among tumors with low TMB, but not those with high TMB. Importantly, aneuploidy scores can be calculated from existing clinical targeted sequencing infrastructure, facilitating deployment of aneuploidy scores as a clinical biomarker.