Radiotherapy, immunity, and immune checkpoint inhibitors.

Radiotherapy exerts immunostimulatory and immunosuppressive effects, both locally, within the irradiated tumour microenvironment, and systemically, outside the radiation field. Inspired by preclinical data that showed synergy between radiotherapy and immune checkpoint inhibitors, multiple clinical trials were initiated with the hypothesis that combined treatment with radiotherapy and immune checkpoint inhibitors could stimulate a robust systemic immune response and improve clinical outcomes. However, despite early optimism, radioimmunotherapy trials in the curative and metastatic settings have met with little success. In this Review, we summarise the immunostimulatory effects of radiotherapy that provided the theoretical basis for trials of combination radiotherapy and immune checkpoint inhibitors. We also discuss findings from clinical trials incorporating radiotherapy and immune checkpoint inhibitors and examine the success of these trials in the context of the immunosuppressive effects of radiotherapy. We conclude by highlighting targets for relieving radiotherapy-induced immunosuppression with the goal of enhancing the combined effects of radiotherapy and immune checkpoint inhibitors.

A prospective clinical and transcriptomic feasibility study of oral-only hormonal therapy with radiation for unfavorable prostate cancer in men 70 years of age and older or with comorbidity.

BACKGROUND: Androgen deprivation therapy (ADT) improves outcomes in unfavorable-risk prostate cancer (PCa) treated with radiation therapy (RT). It was hypothesized that replacing luteinizing hormone-releasing hormone (LHRH) agonists with a 5-α-reductase inhibitor (5-ARI) would improve hormonal health-related quality of life (HRQOL) without differentially suppressing androgen-responsive (AR) gene expression. METHODS: Patients with localized unfavorable-risk PCa, aged ≥70 years or Charlson Comorbidity Index score ≥2 were treated with oral ADT (oADT), consisting of 4 months of bicalutamide, a 5-ARI, and RT at 78 Gy. The primary end point was Expanded Prostate Cancer Index Composite HRQOL at 6 months ≤30%, and improvement compared with a synchronous standard of care (SOC) cohort receiving 4 months of bicalutamide and long-term LHRH agonist with RT. RNA sequencing was performed from matched pre-/post-ADT prostate tumor biopsies in a subset of men. Differential gene and pathway expressional changes were examined using gene set enrichment. RESULTS: Between 2011 and 2018, 40 and 30 men were enrolled in the oADT and SOC cohorts, respectively. Median follow-up was 40 months. Those with ≤30% decline in hormonal HRQOL at 6 months was 97% (oADT) and 93% (SOC). The average 6-month hormonal decline was 1% (oADT) versus 12% (SOC; P = .04). The 4-year freedom from biochemical failure was 88% (oADT) versus 81% (SOC; P = .48). RNA sequencing (n = 9) showed similar numbers of downregulated and upregulated genes between the treatment groups (fold-change = 2; false-discovery rate-adjusted P ≤ .05). Both treatments comparably decreased the expression of 20 genes in canonical androgen receptor signaling. CONCLUSIONS: For men with PCa undergoing RT, oral versus standard ADT may improve 6-month QOL and appears to have a similar impact on androgen-responsive gene expression.

Treatment of Cancer with Radio-Immunotherapy: What We Currently Know and What the Future May Hold.

Radiotherapy and immunotherapy are most effective as cancer therapies in the setting of low-volume disease. Although initial studies of radio-immunotherapy in patients with metastatic cancer have not confirmed the efficacy of this approach, the role of radio-immunotherapy in patients with limited metastatic burden is unclear. We propose that further investigation of radio-immunotherapy in metastatic patients should focus upon patients with oligometastatic disease.

Integration of radiotherapy and immunotherapy for treatment of oligometastases.

Metastasis is the leading cause of cancer-related mortality and remains one of the prevailing challenges in cancer treatment. Most patients with metastatic disease are treated with systemic agents, which prolong survival and improve symptoms but are typically not curative. The oligometastatic hypothesis challenges the perspective that metastasis is an invariably disseminated process, and proposes a biological spectrum of metastatic virulence. Mounting evidence supports the idea that patients with numerically and spatially restricted sites of metastases, termed oligometastases, can achieve prolonged survival following metastasis-directed therapies, such as surgery or radiotherapy. Improvements in clinical and molecular staging of metastatic disease, as well as integration of effective systemic therapies with localised interventions, might achieve better outcomes for patients with diverse metastatic states. In this Series paper, we propose a rationale for the integration of immune checkpoint inhibitors with radiotherapy to advance the potential for effective treatment along the spectrum of disease, with emphasis on how immunotherapy can potentiate radiotherapy treatment in the oligometastatic setting.

Oligometastatic prostate cancer: Reality or figment of imagination?

The term "oligometastatic prostate cancer" refers to a heterogeneous group of disease states currently defined solely on the basis of clinical features. Oligorecurrent disease, de novo oligometastases, and oligoprogressive disease likely have unique biologic underpinnings and natural histories. Evidence suggesting the existence of a subset of patients who harbor prostate cancer with limited metastatic potential currently includes disparate and overwhelmingly retrospective reports. Nevertheless, emerging prospective data have corroborated the "better-than-expected," retrospectively observed outcomes, particularly in the setting of oligorecurrent prostate cancer. Improved functional imaging with prostate-specific membrane antigen-targeted strategies may enhance the identification of patients with oligometastatic prostate cancer in the short term. In the long term, refinement of the oligometastatic case definition likely will require biologic risk-stratification schemes. To determine optimal treatment strategies and identify patients most likely to benefit from metastasis-directed therapy, future efforts should focus on conducting high-quality, prospective trials with much-needed molecular correlative studies.

RIG-I-like receptor LGP2 protects tumor cells from ionizing radiation.

An siRNA screen targeting 89 IFN stimulated genes in 14 different cancer cell lines pointed to the RIG-I (retinoic acid inducible gene I)-like receptor Laboratory of Genetics and Physiology 2 (LGP2) as playing a key role in conferring tumor cell survival following cytotoxic stress induced by ionizing radiation (IR). Studies on the role of LGP2 revealed the following: (i) Depletion of LGP2 in three cancer cell lines resulted in a significant increase in cell death following IR, (ii) ectopic expression of LGP2 in cells increased resistance to IR, and (iii) IR enhanced LGP2 expression in three cell lines tested. Studies designed to define the mechanism by which LGP2 acts point to its role in regulation of IFNß. Specifically (i) suppression of LGP2 leads to enhanced IFNß, (ii) cytotoxic effects following IR correlated with expression of IFNß inasmuch as inhibition of IFNß by neutralizing antibody conferred resistance to cell death, and (iii) mouse embryonic fibroblasts from IFN receptor 1 knockout mice are radioresistant compared with wild-type mouse embryonic fibroblasts. The role of LGP2 in cancer may be inferred from cumulative data showing elevated levels of LGP2 in cancer cells are associated with more adverse clinical outcomes. Our results indicate that cytotoxic stress exemplified by IR induces IFNß and enhances the expression of LGP2. Enhanced expression of LGP2 suppresses the IFN stimulated genes associated with cytotoxic stress by turning off the expression of IFNß.

Clinical and molecular markers of long-term survival after oligometastasis-directed stereotactic body radiotherapy (SBRT).

BACKGROUND: The selection of patients for oligometastasis-directed ablative therapy remains a challenge. The authors report on clinical and molecular predictors of survival from a stereotactic body radiotherapy (SBRT) dose-escalation trial for oligometastases. METHODS: Patients who had from 1 to 5 metastases, a life expectancy of >3 months, and a Karnofsky performance status of >60 received escalating SBRT doses to all known cancer sites. Time to progression, progression-free survival, and overall survival (OS) were calculated at the completion of SBRT, and clinical predictors of OS were modeled. Primary tumor microRNA expression was analyzed to identify molecular predictors of OS. RESULTS: Sixty-one evaluable patients were enrolled from 2004 to 2009. The median follow-up was 2.3 years for all patients (range, 0.2-9.3 years) and 6.8 years for survivors (range, 2.0-9.3 years). The median, 2-year, and 5-year estimated OS were 2.4 years, 57%, and 32%, respectively. The rate of progression after SBRT was associated with an increased risk of death (hazard ratio [HR], 1.44; 95% confidence interval [CI], 1.24-1.82). The time from initial cancer diagnosis to metastasis (HR, 0.98; 95% CI, 0.98-0.99), the time from metastasis to SBRT (HR, 0.98; 95% CI, 0.98-0.99), and breast cancer histology (HR, 0.12; 95% CI, 0.07-0.37) were significant predictors of OS. In an exploratory analysis, a candidate classifier using expression levels of 3 microRNAs (miR-23b, miR-449a, and miR-449b) predicted survival among 17 patients who had primary tumor microRNA expression data available. CONCLUSIONS: A subset of oligometastatic patients achieves long-term survival after metastasis-directed SBRT. Clinical features and primary tumor microRNA expression profiling, if validated in an independent dataset, may help select oligometastatic patients most likely to benefit from metastasis-directed therapy. Cancer 2016;122:2242-50. © 2016 American Cancer Society.

Clinical and molecular correlates of tumor aneuploidy in metastatic non-small cell lung cancer.

Recent studies have linked elevated tumor aneuploidy to anti-tumor immune suppression and adverse survival following immunotherapy. Herein, we provide supportive evidence for tumor aneuploidy as a biomarker of response to immunotherapy in patients with non-small cell lung cancer (NSCLC). We identify a dose-response relationship between aneuploidy score and patient outcomes. In two independent NSCLC cohorts (n = 659 patients), we demonstrate a novel association between elevated aneuploidy and non-smoking-associated oncogenic driver mutations. Lastly, we report enrichment of TERT amplification and immune-suppressive phenotypes of highly aneuploid NSCLC. Taken together, our findings emphasize a potentially critical role for tumor aneuploidy in guiding immunotherapy treatment strategies.

Sharing the Burden: The Case for Definitive Local Therapy in Place of Immune Checkpoint Blockade for Patients With a Low-Volume Burden of Metastatic Disease.

COMMENTARY: Sharing the burden of low-volume metastatic cancer between ICB and local treatments.

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 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.

Safety of combined ablative radiotherapy and immune checkpoint inhibitors in three phase I trials.

BACKGROUND: Stereotactic body radiotherapy (SBRT) is safe and effective for treatment of extracranial metastatic disease, but its safety when combined with immune checkpoint inhibitors (ICI) has not yet been comprehensively reported. Here we report adverse events (AEs) associated with combined SBRT and ICI using prospectively-collected data on patients in three trials investigating multi-site SBRT combined with ICI. METHODS: Patients were included from three prospective trials of ICI (pembrolizumab; nivolumab/urelumab or nivolumab/cabiralizumab; nivolumab/ipilimumab) with SBRT to 1-4 sites. AEs were recorded prospectively using the CTCAE v4.0. Survival was analyzed using Kaplan-Meier method with a 90-day landmark. Association of patient characteristics with cumulative incidence of AEs was assessed using Fine-Gray regression. RESULTS: 213 patients were included, with a median follow-up of 10 months. Over the follow-up period, 50 % and 27 % of patients experienced at least one grade ≥ 2 or grade ≥ 3 AE, respectively. Cumulative incidences of grade ≥ 2 and grade ≥ 3 AEs at 6 months were 47 % and 23 %, respectively. Three grade 5 AEs rated "possibly" related to treatment occurred outside the 90-day dose-limiting toxicity window. Landmarked survival analysis of patients with or without grade ≥ 3 AEs showed no significant difference in progression-free or overall survival. Dual-agent ICI was significantly associated with grade ≥ 3 AE. CONCLUSION: This analysis features the largest prospectively evaluated cohort of patients treated with combination ablative SBRT and ICI to date and provides context for future trial design. We conclude that multi-site SBRT and ICI can be safely co-administered when SBRT is delivered with prioritization of normal tissue constraints.

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.

DARES: A Phase II Trial of Durvalumab and Ablative Radiation in Extensive-Stage Small Cell Lung Cancer.

BACKGROUND: Immunotherapy in combination with chemotherapy is first-line treatment for patients with extensive-stage small-cell lung cancer (ES-SCLC). Growing evidence suggests that radiation, specifically stereotactic body radiation therapy (SBRT), may enhance the immunogenic response as well as cytoreduce tumor burden. The primary objective of the study is to determine the progression free survival for patients with newly diagnosed ES-SCLC treated with combination multisite SBRT and chemo-immunotherapy (carboplatin, etoposide, and durvalumab). METHODS: This is a multicenter, single arm, phase 2 study. Patients with treatment-naïve, ES-SCLC will be eligible for this study. Patients will receive durvalumab 1500mg IV q3w, carboplatin AUC 5 to 6 mg/mL q3w, and etoposide 80 to 100 mg/m2 on days 1 to 3 q3w for four cycles, followed by durvalumab 1500mg IV q4w until disease progression or unacceptable toxicity. Ablative radiation will be delivered 1 to 4 extracranial sites in 3 or 5 fractions, determined by location, during cycle 2. The primary endpoint is progression-free survival, measured from day 1 of chemoimmunotherapy. Secondary endpoints include grade ≥3 toxicity by CTCAE v5.0 within three months of RT, overall survival, response rate, time to second line systemic therapy, and time to new distant progression. CONCLUSIONS: Now that immunotherapy is an established part of ES-SCLC management, it is important to further optimize its use and effect. This study will investigate the progression-free survival of combined SBRT and chemo-immunotherapy in patients with ES-SCLC. In addition, the data from this study may further inform the immunogenic role of SBRT with chemo-immunotherapy, as well as identify clinical, biological, or radiomic prognostic features.

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.

YTHDF1 loss in dendritic cells potentiates radiation-induced antitumor immunity via STING-dependent type I IFN production.

RNA N6-methyladenosine (m6A) reader YTHDF1 is implicated in cancer etiology and progression. We discovered that radiotherapy (RT) increased YTHDF1 expression in dendritic cells (DCs) of PBMCs from cancer patients, but not in other immune cells tested. Elevated YTHDF1 expression of DCs was associated with poor outcomes in patients receiving RT. We found that loss of Ythdf1 in DCs enhanced the antitumor effects of ionizing radiation (IR) via increasing the cross-priming capacity of DCs across multiple murine cancer models. Mechanistically, IR upregulated YTHDF1 expression in DCs through STING-IFN-I signaling. YTHDF1 in turn triggered STING degradation by increasing lysosomal cathepsins, thereby reducing IFN-I production. We created a YTHDF1 deletion/inhibition prototype DC vaccine, significantly improving the therapeutic effect of RT and radio-immunotherapy in a murine melanoma model. Our findings reveal a new layer of regulation between YTHDF1/m6A and STING in response to IR, which opens new paths for the development of YTHDF1-targeting therapies.

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.

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.

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.

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.