Immune-checkpoint blockade (ICB) has shown significant efficacy across various tumor types. However, tumors with low intraepithelial T-cell infiltration, often referred to as “cold” tumors, are expected to yield poor responsiveness to ICB. We investigated the potential of low-dose radiotherapy (LDRT) to enhance ICB responses in 25 patients with multimetastatic immune-excluded solid tumors through a multi-cohort phase I clinical trial (RACIN). Primary endpoint was to determine the safety and tolerability of the combination of a backbone treatment, comprising nivolumab, ipilimumab, aspirin/celecoxib, and low-dose cyclophosphamide (Cy) in association with escalated LDRT. Secondary endpoints included among others disease control rate (DCR) and overall survival (OS). Exploratory endpoints included biomarkers and molecular correlates of response. The combination treatment showed a manageable safety profile, with Grade 3 or higher adverse events in 12% to 21% of patients across cohorts. The overall DCR was 42%. Progression free survival (PFS) across all cohorts was 2.1 months (95% C.I.: 1.8 – 4.2 months), with the highest PFS observed in cohort 1 which received 0.5 Gy (5.7 months, (95% C.I.: 1.9 - 11.3 months). Median OS was 14.0 months (95% CI: 8.5–24.6 months), with one patient with ovarian cancer still maintaining a complete response at three years follow-up. Site-paired tumor biopsies collected for each patient at baseline and after LDRT +/− Cy enabled the comprehensive characterization of the dynamics of excluded tumor microenvironments (TME) at the single cell level. Response to LDRT and ICB was associated with DNA damage and repair responsiveness and the presence of detectable intratumoral PD1+CD8+ tumor infiltrating lymphocytes (TILs) at baseline. Our data revealed that LDRT amplified CD8+ TIL functionality in responding patients offering mechanistic insights on how LDRT improves ICB effectiveness. In contrast, we observed a radiosensitivity of TILs in tumors of non-responders. Detailed single cell immune profiling before LDRT also highlighted a lack of key immune stimulatory myeloid cells that can therefore limit ICB efficacy in excluded tumors. Collectively, this study represents the most comprehensive profiling of longitudinal samples of cancer patients treated with LDRT. Our findings highlight several genetic, transcriptomic and TME parameters associated with response to combinatorial LDRT and ICB in advanced immune-excluded solid cancers, generating rationale for their validation in larger cohorts.

One sentence summary Combinatorial treatment with low-dose radiotherapy and immune checkpoint blockade in patients with immune-excluded tumors enhances CD8⁺ T cell functionality, particularly in those with DNA repair deficiencies, offering new biomarkers for patient selection.

FH reports academic grants from Prostate Cancer Foundation, Bristol-Myers-Squibb, San Salvatore Foundation, and Loterie Romande. Research support from Accuray Inc, Bioprotect, Nanobiotix, Astra Zeneca, Esai, MSD, Seagen and non-financial support from Roche ImFlame cooperative group. FH has received honoraria for consultations from Johnson and Johnson. FH reports scientific relationships with: European Organization for Research and Treatment of Cancer (EORTC) Gynecology Cancer Group (chairman, secretary, and treasurer), ESMO Scientific Committee member for drug development, ASTRO scientific committee member annual meeting, ESTRO scientific committee member annual meeting. DDL reports research grants from Hoffman La Roche and 10xGenomics. MO reports honoraria for consultation from Pfizer. SZ is an employee of Bayer U.S. LLC, Research & Development, Pharmaceuticals. MI has received honoraria for consultations from Astra Zeneca, Bristol-Myers-Squibb. AST reports honoraria for lectures, presentations, or educational events (paid to institution) from AstraZeneca, Daiichi Sankyo, Novartis, Gilead, Seagen; reports support for attending meetings and/or travel from Roche, Eli Lilly, Daiichi Sankyo, Gilead and Pfizer.

NCT03728179

This work was supported by the Ludwig Institute for Cancer Research, and grants from Bristol-Myers Squibb, the Prostate Cancer Foundation Challenge Award (18CHAL08), and the Cancera, Biltema and Paul Matson Foundations. This Research Project was partially supported by the Spanish Society of Medical Oncology (SEOM).

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Ethics committee of Centre Hospitalier Universitaire Vaudois (Lausanne University Hospital), Lausanne, Switzerland gave ethical approval for this work.

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