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Purpose: While stereotactic body radiotherapy (SBRT) can reduce tumor volumes in patients with metastatic renal cell carcinoma (mRCC), little is known regarding the immunomodulatory effects of high-dose radiation in the tumor microenvironment. The main objectives of this pilot study were to assess the safety and feasibility of nephrectomy following SBRT treatment of patients with mRCC and analyze the immunological impact of high-dose radiation.Experimental Design: Human RCC cell lines were irradiated and evaluated for immunomodulation. In a single-arm feasibility study, patients with mRCC were treated with 15 Gray SBRT at the primary lesion in a single fraction followed 4 weeks later by cytoreductive nephrectomy. RCC specimens were analyzed for tumor-associated antigen (TAA) expression and T-cell infiltration. The trial has reached accrual (ClinicalTrials.gov identifier: NCT01892930).Results: RCC cells treated in vitro with radiation had increased TAA expression compared with untreated tumor cells. Fourteen patients received SBRT followed by surgery, and treatment was well-tolerated. SBRT-treated tumors had increased expression of the immunomodulatory molecule calreticulin and TAA (CA9, 5T4, NY-ESO-1, and MUC-1). Ki67+ -proliferating CD8+ T cells and FOXP3+ cells were increased in SBRT-treated patient specimens in tumors and at the tumor–stromal interface compared with archived patient specimens.Conclusions: It is feasible to perform nephrectomy following SBRT with acceptable toxicity. Following SBRT, patient RCC tumors have increased expression of calreticulin, TAA, as well as a higher percentage of proliferating T cells compared with archived RCC tumors. Collectively, these studies provide evidence of immunomodulation following SBRT in mRCC. Clin Cancer Res; 23(17); 5055–65. ©2017 AACR.

Supplementary figure S3 shows gating strategy and isotype staining of live tumor cells used for patient samples.

Supplementary table 3 shows immunohistochemical antibodies and dilutions.

Supplementary figure S5 shows PD-1+ CD4+ T cells and CD68+ macrophages are increased in SBRT-treated specimens compared to nephrectomy controls.

Supplementary figure S2 shows immunomodulation of tumor cells following high-dose radiation.

Supplementary figure S4 shows tumors resected from RCC patients treated with SBRT have decreased CD54 and increased surface NY-ESO-1 expression.

Supplementary figure S1 shows radiation induces changes in surface expression of immunogenic molecules.

Supplementary table S2 shows flow cytometry antibodies and dilutions.

Purpose: While stereotactic body radiotherapy (SBRT) can reduce tumor volumes in patients with metastatic renal cell carcinoma (mRCC), little is known regarding the immunomodulatory effects of high-dose radiation in the tumor microenvironment. The main objectives of this pilot study were to assess the safety and feasibility of nephrectomy following SBRT treatment of patients with mRCC and analyze the immunological impact of high-dose radiation. Experimental Design: Human RCC cell lines were irradiated and evaluated for immunomodulation. In a single-arm feasibility study, patients with mRCC were treated with 15 Gray SBRT at the primary lesion in a single fraction followed 4 weeks later by cytoreductive nephrectomy. RCC specimens were analyzed for tumor-associated antigen (TAA) expression and T-cell infiltration. The trial has reached accrual (ClinicalTrials.gov identifier: NCT01892930). Results: RCC cells treated in vitro with radiation had increased TAA expression compared with untreated tumor cells. Fourteen patients received SBRT followed by surgery, and treatment was well-tolerated. SBRT-treated tumors had increased expression of the immunomodulatory molecule calreticulin and TAA (CA9, 5T4, NY-ESO-1, and MUC-1). Ki67+ -proliferating CD8+ T cells and FOXP3+ cells were increased in SBRT-treated patient specimens in tumors and at the tumor–stromal interface compared with archived patient specimens. Conclusions: It is feasible to perform nephrectomy following SBRT with acceptable toxicity. Following SBRT, patient RCC tumors have increased expression of calreticulin, TAA, as well as a higher percentage of proliferating T cells compared with archived RCC tumors. Collectively, these studies provide evidence of immunomodulation following SBRT in mRCC. Clin Cancer Res; 23(17); 5055–65. ©2017 AACR.

542 Background: Radiation increases the immunogenicity of many cancers. We have previously shown increased immunogenicity of RCC with cell line irradiation. Herein, we describe the effect of irradiation of RCC cell lines on the expression of TAAs as well as several molecules involved in CD8+ T Cell (CTL) mediated tumor cytotoxicty and the in vitro effect of tumor irradiation on NY-ESO-1 specific CTL clones. Methods: Six RCC cell lines (ACHN, 786-0, 769-P, Caki-1, A-498, A-704) were cultured and exposed to 0, 12 or 16 Gy of ionizing radiation. Cells were isolated at 2, 24 and 48 hours after irradiation. Expression of cell surface molecules (CD95, CD54, Calreticulin, NYESO-1, CA-9, 5T4, MUC-1 and HSP-70) was assessed using multi-color flow cytometry. Results are reported as mean relative increase from control (dose 0 Gy, time 2 hours post irradiation). In a second experiment, NY-ESO-1 specific CTL clones were cultured in the presence of A-498 cells that were subjected to radiation under the above noted conditions and assessed using an ELISPOT assay. Negative control consisted of only CD8+ T Cells. Results: The ELISPOT experiment demonstrated stimulation of NY-ESO-1-specific CTL by irradiated A-498 cells. Negative control showed no stimulation, with only 4 spots. An increase in spots was observed from 79 and 82 spots at 2 hours post-irradiation to 166 and 205 spots at 48 hours post-irradiation for 0 Gy and 16 Gy, respectively. Conclusions: Sublethal irradiation of human RCC cell lines increases their immunogenicity, provoking TAA-specific T cell stimulation. [Table: see text]