5 Stereotactic Body Radiation Therapy Enriches Mediators of Humoral Immune Activity in Metastatic Renal Cell Carcinoma

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Abstract Background Stereotactic body radiation therapy (SBRT) has demonstrated efficacy in local control of renal cell carcinoma (RCC). SBRT has also been shown to induce tumor associated antigen generation alongside effector T cell expansion and activation in the tumor microenvironment (TME) of metastatic RCC (mRCC), suggesting a potential role as an immunomodulatory adjuvant for improving immune checkpoint blockade (ICB) efficacy (Singh et al. CCR. 2017; Chow et al. JITC. 2023). However, the impact of SBRT on humoral immunity, which has consistently been associated with ICB sensitivity in mRCC, remains uncharacterized. Methods Primary tumors from patients with treatment-naïve mRCC were resected four weeks after receiving 15 Gy SBRT, processed to single cell suspension, and cryopreserved (NCT01892930). RNA sequencing (RNAseq) was performed on SBRT-treated and clinicopathologically matched non-SBRT-treated (control) RCC tumors. Gene set enrichment analysis and xCell deconvolution were performed between groups. The B cell receptor (BCR) repertoire was reconstructed using the TRUST4 pipeline. Single cell RNAseq (scRNAseq) was performed on SBRT-treated and control RCC tumors; B cells were computationally extracted for downstream analyses. Per-sample results were compared between groups using Mann-Whitney U Test. Full spectrum flow cytometry using a 17-marker panel designed to interrogate B cell populations was also performed. The impact of B cell subtypes on objective response to ICB was assessed using immune deconvolution of tumor transcriptomes from the IMmotion151 and HCRN-GU16-260 trial cohorts. Results In total, n = 8 SBRT-treated and n = 8 control primary RCC tumors were analyzed by bulk RNAseq. Of 31 msigdb pathways with a normalized enrichment score >3 (favoring SBRT), ten directly pertained to B cell maturation, B cell signaling, or immunoglobulin production (all padj<1x10-25). Additionally, two tertiary lymphoid structure signatures – Imprint and 12 Chemokine – were also significantly enriched in SBRT vs. control (NES=2.77 and 1.75, padj=2.3x10-11 and 0.03, respectively). Immune deconvolution demonstrated enrichment of total B cell populations (p = 0.002) and class-switched memory B cells (p = 0.010) in SBRT-treated tumors, paralleling a non-significant increase in total IGH clonotypes per sample identified through BCR repertoire reconstruction in SBRT vs control (p = 0.16). SBRT-treated (n = 4) and control (n = 2) RCC tumors underwent scRNAseq, totaling n = 37,029 cells. B cells were identified by lineage marker expression. The relative abundance of plasma cells amongst B lymphocytes was increased in SBRT-treated tumors (15.3% vs. 6.5%). Differential expression analysis revealed significant enrichment of plasma cell marker genes and immunoglobulin-encoding genes in SBRT-treated B cells by scRNAseq. Flow cytometry performed on n = 5 SBRT-treated and n = 8 control RCC tumors revealed significant increases in the relative abundance of antibody-producing CD45+ CD19+ B cells in SBRT-treated tumors (CD38+ IgD-; p = 0.006) whereas the abundance of naïve B cells (CD38- IgD+ CD21+) was significantly enriched in control tumors (p = 0.029). Within IMmotion151 (n = 757 tumors), memory, class-switched memory, and plasma B cell subsets were significantly increased in patients achieving an objective response to atezolizumab/bevacizumab compared to atezolizumab/bevacizumab non-responders (p = 0.008, 0.009, and 0.051, respectively). Additionally, these subsets were also significantly enriched in atezolizumab/bevacizumab responders compared to sunitinib responders (p = 0.001, 0.012, and 0.032, respectively). Within HCRN-GU16-260 (n = 70 tumors), class-switched memory B cell scores were again significantly enriched in patients achieving an objective response to nivolumab compared to non-responders (p = 0.020). Conclusions SBRT results in enriched humoral immune maturity in the RCC TME. Furthermore, the abundance of the mature B cell subtypes heightened in the TME following SBRT is associated with responsiveness to ICB in patients with mRCC. These results suggest SBRT promotes immunostimulatory activity and ultimately may improve responses to ICB therapy. Prospective trials seek to assess this concept in RCC. DOD CDMRP Funding yes