A Pilot Study of Stereotactic Body Radiation Therapy Combined with Cytoreductive Nephrectomy for Metastatic Renal Cell Carcinoma

Renal cell carcinoma represents 3% of adult malignancies, and approximately one quarter of patients present with metastatic disease (1). Metastatic renal cell carcinoma (mRCC) is especially difficult to treat with a 5-year overall survival rate of approximately 10% (2). Immunotherapeutic approaches including conventional cytokine therapy (3), recently developed immune checkpoint inhibitor strategies (4), and dendritic cell vaccination (5) have shown promise in the treatment of mRCC with a subset of patients having complete, durable responses following therapy.

The emphasis of most immunotherapeutic regimens is to increase the number and improve the cytolytic function of tumor-specific T cells. This focus largely overlooks the tumor-intrinsic barriers to effective T-cell–mediated killing including T-cell trafficking to the tumor microenvironment (6), presence of immunosuppressive cells (7), and expression of immunogenic antigens by tumors cells (8). mRCC is a heavily lymphocyte infiltrated tumor type with low and heterogeneous expression of tumor-associated antigens (TAA; refs. 9, 10).

Diminished TAA expression is a well-characterized mechanism of immune evasion employed within the tumor microenvironment (8). Radiation-induced inflammation in tumors has the potential to overcome this impediment by releasing pro-inflammatory mediators, upregulating TAA, and increasing T-cell infiltration (11). A growing body of evidence has also demonstrated that sublethal doses of radiation can induce expression of calreticulin and CD80, leading to increased cytotoxic lymphocyte activity following irradiation of tumor cells (12–14). Preclinical tumor models and in vitro data utilizing other tumor types have revealed that radiotherapy can increase TAA expression (13, 15). While RCC appears resistant to conventional fractionated radiotherapy (16), recent publications have shown that high doses delivered with modern stereotactic body radiotherapy (SBRT) can produce local control with limited toxicity (17, 18). Treatment of patients with SBRT in combination with conventional high-dose IL2 treatment has demonstrated partial and complete objective responses in patients with melanoma and mRCC that correlate with increased proliferating T cells in peripheral blood (17). While dozens of clinical trials are currently investigating the combination of radiotherapy with immunotherapy for treatment of malignancies (19), the isolated contributions of high-dose radiation in preconditioning patient tumors for effective antitumor responses remain unknown.

The capacity of SBRT to control growth of primary tumors and the finding that resection of primary tumors may improve overall survival of patients with mRCC (20) led us to consider a combination of these treatments. First, we performed in vitro studies to explore the effects and timing of immune changes caused by high-dose irradiation as a surrogate for SBRT. These studies informed the design of a first in-human pilot trial to evaluate the feasibility and safety of combining single-fraction SBRT with cytoreductive nephrectomy.

These studies were performed according to a protocol approved by the Roswell Park Cancer Institute Institutional Review Board (IRB). A498 human kidney carcinoma cells (ATCC) were cultured at 37°C in 75-cm² flasks (Corning Inc.) at 200,000 cells per flask in RPMI-1640 supplemented with 0.05 mmol/L of 2-mercaptoethanol, 10% FBS (ThermoFisher Scientific), 1% penicillin/streptomycin, 1 mmol/L of sodium pyruvate, 20 mmol/L of HEPES, 1% non-essential amino acids, and 2 mmol/L of l-glutamine (Corning Inc.). Culture media were refreshed every other day. A Shepherd Mark I Model 68, 4000 Ci Cesium 137 source irradiator (J.L. Shepherd and Associates), was used to irradiate cells, with a total dose rate of 12 or 16 Gray (Gy). Cells were trypsinized with 0.125% trypsin-EDTA (ThermoFisher Scientific). ELISPOT assays were performed using NY-ESO-1–specific T-cell clones generated as described previously (21). A498 target cells previously treated with or without radiation were plated at a 1:1 ratio with NY-ESO-1–specific CD8⁺ T cells in 96-well plates (EMD Millipore) and later evaluated for IFNγ secretion using C.T.L. ImmunoSpot Analyzer (Cellular Technologies).

We report results of a single-center, IRB-approved pilot study (ClinicalTrials.gov identifier: NCT01892930). All patients provided written informed consent, and the study was conducted in accordance to all applicable local regulatory requirements and laws. Eligible patients were at least 18 years of age, had mRCC, and were deemed fit for cytoreductive surgery to the primary tumor site. The exclusion criteria included: any chemotherapy, any radiotherapy, or investigational agent within 30 days prior to enrollment or radiation to the primary tumor at any point prior to treatment.

Patients with mRCC received 15-Gy SBRT to the primary tumor on day 1 followed by nephrectomy on day 29 (±3 days). This dose was chosen on the basis of our recent experience with this treatment being well-tolerated in patients with inoperable RCC (18). Patients were followed for protocol-related toxicity 30 ± 3 days following surgery or until resolution of any toxicity. Thereafter, patients were followed as clinically indicated. An interim analysis for safety was conducted after the first five patients were treated. The study was then allowed to proceed. The data safety monitoring board of Roswell Park Cancer Institute reviewed this study annually.

SBRT was delivered as previously described (18). Briefly, a stereotactic body immobilization system (BodyFIX, Medical Intelligence) was used with a 4-dimensional CT to assess tumor motion with respiration. Tumor delineation was carried out in all respiratory phases, creating an internal target volume, and then a 5-mm isotropic expansion was used to create a planning target volume (PTV). Treatment was carried out with a Varian True-Beam linear accelerator (Varian Medical Systems). Volumetric modulated arc therapy with 6-MV photons was used. On the day of treatment, cone-beam CT was obtained following setup on the treating machine, and appropriate shifts were made.

Archival tumor specimens were obtained from patients who underwent cytoreductive nephrectomy for RCC and had given written consent for research use of a portion of the pathologic specimen. An independent broker, a person not associated with this research study, linked demographic and follow-up data with specimens to allow analysis. Archival specimens utilized in flow cytometric experiments were from nine women and seven men, resected between 2011 and 2015. Median age for this group was 60 years (range, 41–72 years; Supplementary Table S1). The control cohort utilized in immunohistochemical (IHC) analysis consisted of two women and nine men with a median age of 69 years (range, 45–80 years; Supplementary Table S1).

Under sterile conditions, RCC patient tumor samples were minced into small pieces and exposed to collagenase/hyaluronidase (collagenase 1 mg/mL, hyaluronidase 0.1 mg/mL, Sigma-Aldrich) digestive solution for 120 minutes. Samples were filtered through 100-μm cell strainers, washed with PBS, and stored at −80°C.

Cells were isolated, washed in PBS, and stained with Zombie UV Fixable Viability Kit for 10 minutes (Cat# 423108 2.5:100, BioLegend). Flow cytometric analysis was performed after staining with antibodies (Supplementary Table S2). NY-ESO-1 was detected with rat anti-mouse IgG1 secondary antibody (cat# 550083 PE 1:100, BD Biosciences). For intracellular staining, cells were washed and incubated with Fixation/Permeabilization Solution Kit (Cat# 554714, BD Biosciences). Cells were stained for expression of intracellular NY-ESO-1 and labeled with PE secondary antibody and fixed in 2% formaldehyde solution.

Flow cytometric analysis was performed on BD LSR Fortessa flow cytometer (BD Biosciences). For adjustment, control cells were stained with each individual fluorochrome to serve as compensation controls. The outcome was analyzed using FlowJo_V10 cytometry software.

IHC assays for Ki67 & CD8 double stain, FOXP3, CD68, PD-L1, CD31, and vimentin were performed by the Pathology Network Shared Resource at Roswell Park Cancer Institute (see Supplementary Table S3 for antibody information). Sections (4–5 μm) were placed on charged slides and dried at 60°C for 1 hour, then cooled to room temperature, deparaffinized in three changes of xylene, and rehydrated using graded alcohols. For antigen retrieval, slides were submerged in either Target Retrieval Solution, pH 9 (Dako) or EDTA buffer, pH 8 (ThermoFisher Scientific), and heated in a steamer (Supplementary Table S3), followed by a cooldown at room temperature for 20 minutes. Endogenous peroxidases were quenched with aqueous 3% H₂O₂ for 10 minutes and washed with PBS/T. Slides were loaded onto a DAKO autostainer (Dako), and serum-free protein block was applied for 5 minutes, blown off, and then primary antibody was applied (Supplementary Table S3). An irrelevant isotype-matched antibody was applied on replicate slides instead of primary antibody as a negative control. The EnVision+ Peroxidase system or G2 System/AP (Dako), PowerVision system (Leica), and/or the Vectastain Elite ABC kit (Vector Laboratories) and corresponding chromogens were used for visualization (Supplementary Table S3). Finally, slides were counterstained with hematoxylin, dehydrated, cleared, and cover slipped.

Slides were digitally scanned using Aperio ScanScope (Aperio Technologies, Inc.) with 20 × bright-field microscopy. Once scanned, Aperio ImageScope version 11.2.0.780 (Aperio Technologies, Inc.) was used to view images for analysis. Slide image data fields were populated; images were examined for quality and amended as necessary. Annotation layers were created for each image representing tumor and interface regions with analysis performed on an average of four fields per tumor. Areas of target cells for image analysis were circled with care taken to avoid areas with staining artifacts or red blood cells using the negative free-form pen.

Proliferating (Ki67⁺) CD8⁺ T cells were quantified using Aperio's Image analysis tools as follows. Ki67 is expected to be present in the nuclei of many cell types in the tumor microenvironment. Genie software was, therefore, first trained to recognize CD8⁺ T lymphocytes to limit Ki67 analysis to this cell population. Once training was complete, the fully trained Genie was saved and applied as a classifier prior to the basic nuclear algorithm. This combined approach provided the total number of CD8⁺ lymphocytes and the percentage of Ki67⁺, proliferating CD8⁺ T cells within the area of analysis.

The primary purpose of this study was to demonstrate the feasibility of conducting a nephrectomy in patients receiving SBRT for mRCC. Eligible patients who had nephrectomy were considered evaluable for statistical analysis. SBRT complication rates were quantified as the binomial proportion of evaluable patients with at least one surgical complication related to SBRT. The plausible upper limit for the true SBRT complication rate was estimated using a one-sided 95% Clopper–Pearson confidence interval. Comparisons of SBRT-treated patient specimens with archival samples were performed using independent-sample Student t tests. Overall survival was defined as the number of months between SBRT initiation and death from any cause. Patients who were still alive at the time of analysis were censored at the date of last follow-up. The overall survival distribution was described using the Kaplan–Meier estimator. Throughout, P < 0.05 was deemed statistically significant. The P values have not been adjusted for multiplicity. All statistical analyses were done using PRISM v6.07 (GraphPad Software).

Initial in vitro studies focused on the sensitivity of human RCC A498 cells to radiation-induced immunomodulation. We found a higher percentage of tumor cells expressed calreticulin, CD80, and HSP70 on live A498 cells following treatment with either 12 or 16 Gy compared with untreated RCC cells (Fig. 1A). These changes were consistent when evaluating mean fluorescence intensity (MFI; Supplementary Fig. S1A) and occurred as early as 24 hours after radiation treatment. A498 displayed an increased percentage of cells expressing CD86 and increased CD54 MFI (Supplementary Fig. S1B) following radiation. Irradiation of A498 cells also increased the percentage of cells expressing TAA as early as 24 hours (NY-ESO-1), with all TAA (CA9, 5T4, MUC-1, and intracellular NY-ESO-1) expressed at higher levels at 96 hours posttreatment with either radiation dose (Fig. 1B). MFIs of CA9, MUC-1, and intracellular NY-ESO-1 were also increased on A498 cells treated with radiation (Supplementary Fig. S2A). Previous studies showing calreticulin binds NY-ESO-1 protein at the cell surface (22), led us to evaluate membrane expression of NY-ESO-1. We determined that radiation increased surface expression of NY-ESO-1 as early as 72 hours after treatment (Supplementary Fig. S2B). Importantly, as studies have shown radiation can increase autofluorescence (23), we found a minimal contribution of nonspecific staining for any isotype control antibody and fluorochrome combination (Supplementary Fig. S2C).

To directly assess whether TAA-specific CD8⁺ T cells were more responsive to irradiated RCC tumor cells expressing higher levels of TAA, we performed co-culture experiments with NY-ESO-1–specific CD8⁺ T cells and evaluated IFNγ secretion by ELISPOT. In these experiments, more antigen-specific CD8⁺ T cells secreted IFNγ when cultured with A498 cells that were irradiated 96 hours prior to co-culture (Fig. 1C).

A total of 16 patients were enrolled between July 2013 and November 2015 (Table 1). The median age at diagnosis was 63.9 years (range, 52–75 years). Patients were treatment-naïve prior to SBRT therapy, but many received systemic treatment following nephrectomy (Table 1). SBRT was successfully performed in all patients per study specifications. Patient #2 (intracranial bleeding from a small, previously unknown brain metastasis) died prior to surgery. This prompted the requirement of brain imaging prior to SBRT to rule out immune-mediated intracranial side effects. Patient #11 had known spinal disease (outside of SBRT field), developed cord compression 12 days following SBRT, and therefore did not have nephrectomy. She died 12 months after SBRT. The remaining 14 patients underwent nephrectomies. Twelve patients had clear cell RCC, whereas patient #4 was found to have a high-grade papillary urothelial carcinoma and patient #8 showed chromophobe RCC. Tumor type can influence TAA expression and infiltration. Therefore, we limited our remaining analysis to patients with clear cell histology.

The median planning target volume (PTV) for SBRT was 441 cc (range, 37–1,241 cc) for clear cell patients. Respiratory gating to treat the tumor only at prespecified points in the respiratory cycle and active breath hold techniques were both allowed. A 15-Gy dose was prescribed to the PTV. The maximum percent dose 2 cm from the PTV ranged from 57 to 89 and generally increased with increasing PTV size. The ratio of the intermediate dose spillage at 50% of the prescription dose volume and the PTV (R50%) was measured to describe the dose gradient. The average R50% was 3.67 (range, 2.97–4.32.). Two weeks after SBRT, CT scans were performed. There were no significant changes from baseline in any scan.

Intraoperatively, there was a noticeable degree of perinephric fibrosis. This made the dissection of the retroperitoneal space a bit more challenging at times. Kocherizing the duodenum was difficult for patient #1, but no intraoperative complications were encountered.

Three patients had partial nephrectomies and 11 had total nephrectomies. Blood loss averaged 100 cc. The planned type of nephrectomy was not changed following SBRT. There were no post-surgical complications in the 14 patients who received surgery. Using a one-sided 95% Clopper–Pearson confidence limit, the true SBRT complication rate is likely less than 0.23.

Treatment-related adverse events are shown (Table 2). One patient had a grade III anemia. The most common adverse events were nausea and vomiting occurring in 44% and 38% of patients, respectively. Of the 16 patients, four (25%) suffered grade II acute toxicity. One- and 2-year overall survival estimates are 71% and 48%, respectively, and all patients still alive have been followed for a minimum of 16 months (Table 1).

CD45 and viability stains were utilized throughout analysis to identify live tumor cells (CD45⁻) or leukocytes (CD45⁺) within single-cell suspensions (Supplementary Fig. S3A and S3B). Tumor cell surface expression of calreticulin was increased in samples resected from patients with clear cell RCC treated by SBRT (Fig. 2A). The expression of surface molecules CD80, HSP70, MHC I, and CD86 were unchanged when comparing SBRT-treated patient tumor cells with the archival cohort (Fig. 2B and C; Supplementary Fig. S4A and S4B). CD54⁺ (ICAM-1) expression was decreased in SBRT-treated samples compared with controls (Supplementary Fig. S4C).

Analysis of TAA expression (CA9, MUC1, 5T4, and NY-ESO-1) by flow cytometry showed increased expression in SBRT-treated specimens compared with the control cohort, in terms of both the percentage of tumor cells expressing TAA and the expression level of TAA (Fig. 3A–D). Further analysis demonstrated that surface expression of NY-ESO-1 was increased in SBRT samples compared with archived control samples (Supplementary Fig. S4D).

To evaluate CD8⁺ T-cell infiltration within intratumoral and peritumoral regions following SBRT treatment, we performed two-color IHC staining of nephrectomy specimens for CD8⁺ T cells and Ki67 as a marker of proliferation. We observed no differences in CD8⁺ T-cell infiltration within the tumor or at the tumor–stromal interface following SBRT compared with control nephrectomy specimens (Fig. 4A and B). However, when we focused our analysis on proliferating cells, we observed that the density of Ki67⁺ CD8⁺ cells was increased in SBRT samples compared with control (Fig. 4A and B). Similar to our findings by IHC that CD8⁺ density was not increased, characterization of the T-cell infiltrate by flow cytometry demonstrated no change in the number of CD4⁺ or CD8⁺ T cells in resected tumors (Supplementary Fig. S5A). However, the percentage of CD4⁺ T cells that expressed the immune checkpoint protein PD-1 was increased in SBRT-treated specimens (Supplementary Fig. S5A). Further investigation into the immune cell infiltrate showed increased FOXP3⁺ cell and CD68⁺ macrophage accumulation in SBRT-treated samples (Fig. 4C; Supplementary Fig. S5B).

We performed additional analysis to investigate the effects of SBRT on the tumor stroma. We evaluated vascular density in SBRT-treated tumors and observed no difference (Supplementary Fig. S5C). However, expression of vimentin, a common fibroblast marker which can be expressed on endothelial cells and RCC, was increased in SBRT samples compared with archived controls (Supplementary Fig. S5D). IHC analysis of patient biopsy samples showed no change in PD-L1 expression or any correlation of PD-L1 with Ki67⁺ CD8⁺ density (Supplementary Fig. S5E) as previously described (24).

This unique prospective pilot trial demonstrates that single-fraction, 15-Gy SBRT followed by nephrectomy for mRCC: (i) is feasible and well-tolerated, (ii) induced tumor-intrinsic changes (increased calreticulin and TAA expression), and (iii) increased the percentage of proliferating CD8⁺ T cells in primary tumors.

Overall, SBRT followed by nephrectomy was safe. The single grade III treatment-related adverse event occurred in a patient with a pre-existing history of anemia who received a transfusion following SBRT. Grade II acute toxicity occurred in only 25% of patients. There were no grade III or higher surgical complications, and the qualitative clinical assessment of the surgeons was that SBRT did not make surgery appreciably more difficult. There are two published reports, with a total of four patients, of histologic examination of kidney tissue following SBRT. Onishi and colleagues reported autopsy data on a patient treated with 60 Gy in 10 fractions who died 2.5 years later of unrelated causes showing that some viable renal cancer cells remained (25). Ponski and colleagues performed surgery in three patients 8 weeks after 16 Gy was delivered in four fractions and showed a complete response in one patient (26). Consistent with our findings that SBRT prior to surgery was well-tolerated, there were no acute or late toxicities within the first year of follow-up.

However, it must be noted that two patients (intracranial bleed and cord compression) on this trial did not receive their planned surgery because of tumor progression soon after, but far distant from the irradiated area. These may have been random events of disease progression and were officially coded as such. Yet, the patient with initial cord compression from known vertebral body metastases remained alive with good function for one year following neurosurgical decompression. Notably, similar events (described as pseudo-progression) have been seen with nivolumab in a small number of patients with mRCC (27) and a variety of other immune agents as summarized by Chiou and Burrato (28). It is therefore also possible that disease progression events noted in this trial are partly the result of swelling from infiltration of immune cells.

SBRT has been shown, in preclinical models, to induce both stimulating and suppressive immune effects within the tumor microenvironment (7). The balance between stimulation and suppression may depend on tumor type, radiation dose, and number of fractions (14), as well as the time of observation. Clinically, a wide range of SBRT fractions (1–10) and doses (18–60 Gy) for inoperable RCC have been reported in the literature (29). In this study, 15 Gy in one fraction was utilized due to three factors: (i) clinical experience with this dose being well-tolerated in our inoperable patients (18), (ii) radiobiological data suggesting vascular effects with this dose (30), and (iii) in vitro experiments (Fig. 1) showing that this dose has immunomodulatory effects.

Experiments performed in vitro found significant upregulation of both immunomodulatory markers (calreticulin, CD80, and HSP70, Fig. 1A) and TAAs (MUC1, NY-ESO-1, CA9, and 5T4, Fig. 1B) following radiation. These findings are consistent with data that radiation-induced tumor cell death can promote antitumor immunity through surface expression of calreticulin and secretion of immunogenic proteins (31). Radiotherapy has been shown to increase expression of calreticulin (12, 32) while enhancing expression of costimulatory molecules (15, 33) and TAA (15) on live tumor cells. The finding of increased IFN γ secretion from NY-ESO-1–specific T cells following co-culture with an irradiated human renal tumor cell line (Fig. 1C) is consistent with data showing that irradiation can improve tumor cell sensitivity to effector CD8⁺ T-cell–mediated cytolysis (34).

Consistent with our in vitro findings that surface expression of calreticulin can be induced on RCC cells surviving radiation treatment (Fig. 1A), we found an increase in calreticulin expression on samples resected from patients treated by SBRT (Fig. 2A). Contrary to our in vitro data, the expression of previously identified radio-inducible surface molecules CD80, HSP70, MHC I, (33, 35, 36), as well as CD86 and PD-L1 were unchanged when comparing SBRT-treated patient tumor cells with the archival cohort (Fig. 2B and C; Supplementary Figs. S4A and S4B and S5E). Unexpectedly, expression of CD54 (ICAM-1) was decreased on SBRT-treated human tumor (Supplementary Fig. S5C), which is in contrast to previous reports showing that radiation can increase CD54 expression on tumor cell lines (15).

Immunostimulation results from two basic mechanisms in preclinical models: (i) direct killing of tumor cells by radiation increases the availability and cross-presentation of TAA to CD8⁺ T cells (12, 15) and (ii) immune changes resulting from cytokine production, particularly IFNγ. Radiation doses as low as 10 Gy have been shown to increase TAA expression in numerous cell lines (15, 34), but few studies have addressed TAA expression in human tumors treated in vivo (37). Furthermore, previous work utilizing conventional radiation has shown RCC to be refractory to treatment (16). We selected the TAAs CA9, MUC-1, 5T4, and NY-ESO-1 which are overexpressed in RCC patient tumors compared with normal tissues (38–40) and were increased by radiation in our in vitro studies (Fig. 1B).

Our finding of NY-ESO-1 and MUC-1 upregulation in mRCC patient tumors following 15-Gy SBRT (Fig. 3) is supported by published in vitro studies showing an upregulation of these TAAs in multiple non-RCC cell lines following ionizing radiation (13, 37). Similarly, CA9 was found to be elevated following SBRT in our study (Fig. 3). This upregulation is a novel finding and is particularly significant, as CA9 is a known prognostic factor in RCC; in a meta-analysis of 15 studies with 2,611 patients with RCC, low expression was associated with poor disease-specific survival (41).

We observed higher levels of proliferating CD8⁺ T cells and FOXP3⁺ T regulatory cells (Tregs) without changes in overall CD8⁺ T-cell numbers or tumor size from baseline, suggesting the presence of additional mechanisms preventing CD8⁺ T-cell infiltration and cytolytic function (Fig. 4B and C; Supplementary Fig. S5A). Lower intratumoral ratios of CD8⁺ T cells to Tregs may indicate poor prognosis (42, 43), providing rationale for strategic inhibition of Tregs with SBRT to promote antitumor responses. Studies have shown increased intratumoral proliferating CD8⁺ T cells in patients who respond to immune checkpoint inhibitors (24), providing additional support for a combined SBRT and immune checkpoint blockade strategy which has been the subject of numerous investigations (43, 44). Previous studies demonstrating that a single dose of radiation can induce durable immune responses to antigen that are still observed weeks after treatment (45) also suggest that increased Ki67⁺ CD8⁺ intratumoral T cells may indicate a sustained systemic immune response.

Gene expression analysis and IHC studies have characterized RCC as a heavily lymphocyte-infiltrated tumor type with high levels of cytolytic activity (10, 46). However, unlike other tumor types, abundant CD8⁺ T-cell infiltration is associated with shorter overall survival in RCC (10). Conversely, a high proliferative capacity of intratumoral CD8⁺ T cells, evaluated by Ki67 staining, is associated with longer survival in RCC (10). Park and colleagues found a similar increase in tumor-reactive CD8⁺ T cells in murine models of renal cell cancer and melanoma treated with SBRT; moreover, they found that this effect was enhanced in mice treated with PD-1 blockade and in PD-1 knockout mice (47). Recent human studies have shown that higher levels of intratumoral proliferating T cells are prognostic indicators for response to immune checkpoint inhibition strategies. Immune checkpoint blockade has also been shown to increase intratumoral proliferating T cells in responding patients (24, 43). Interestingly, tyrosine kinase inhibitors may also improve the proliferative capacity of intratumoral lymphocytes in patients with RCC (48) and thus may provide another treatment option to bolster endogenous antitumor immune responses.

A significant limitation of this study is the single observation of patient tumors 4 weeks after SBRT treatment. This snapshot of the tumor microenvironment following radiation does not reflect the dynamic process that may have occurred earlier. As others have noted in preclinical murine models and in vitro studies, increased immune infiltration and expression of immunomodulatory molecules including MHC I occurs early following radiation before returning to baseline levels (11, 33). Furthermore, while FOXP3⁺ Treg cell and macrophage infiltration were increased at this time point, these cells may be functioning in an anti-inflammatory role weeks after a local immune response was triggered. The observation of the tumor microenvironment at this single time point (28 days after SBRT) does not allow this investigation to delineate early and late responses to treatment and the plasticity of the immune cell subsets within the tumor microenvironment. Specifically, intratumoral macrophages could have protumorigenic, angiogenic, and immunosuppressive function within RCC tumors leading to immune dysfunction (49, 50). Conversely, macrophages within RCC can also promote cytotoxic function (50). The impact of radiation on the behavior of these cells within the RCC lesions is an open area of investigation. The extension of our findings on immunomodulation to patient survival are also limited by the number of patients, lack of a pretreatment biopsy for comparison, as well as the varied treatments following enrollment in this trial (Table 1).

In conclusion, this trial is the first to demonstrate that nephrectomy following SBRT to the primary tumor is safe and feasible. These data demonstrate for the first time that SBRT can make significant changes to the immune landscape within primary RCC lesions, which may impact the efficacy of immunotherapies.

No potential conflicts of interest were disclosed.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Conception and design: A.K. Singh, T.B. Winslow, L. Heit, G.W. Warren, W. Bshara, T. Schwaab, J.B. Muhitch

Development of methodology: A.K. Singh, T.B. Winslow, M. Habiby Kermany, V. Goritz, L. Heitt, G.W. Warren, W. Bshara, T. Schwaab, J.B. Muhitch

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.K. Singh, T.B. Winslow, M. Habiby Kermany, V. Goritz, L. Heitt, G.W. Warren, W. Bshara, K. Odunsi, T. Schwaab, J.B. Muhitch

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.K. Singh, T.B. Winslow, M. Habiby Kermany, V. Goritz, L. Heit, A. Miller, N.C. Hoffend, L.C. Stein, L. Kumaraswamy, G.W. Warren, W. Bshara, K. Odunsi, S.I. Abrams, T. Schwaab, J.B. Muhitch

Writing, review, and/or revision of the manuscript: A.K. Singh, T.B. Winslow, M. Habiby Kermany, N.C. Hoffend, L. Kumaraswamy, G.W. Warren, W. Bshara, K. Odunsi, S.I. Abrams, T. Schwaab, J.B. Muhitch

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.K. Singh, L. Kumaraswamy, W. Bshara, K. Odunsi, J. Matsuzaki, T. Schwaab, J.B. Muhitch

Study supervision: A.K. Singh, W. Bshara, K. Odunsi, T. Schwaab, J.B. Muhitch

The authors would like to thank Angela Omilian for assistance during setup of human IHC studies and Michelle Appenheimer for critical reading of this article.

Research reported here was supported by the National Center for Advancing Translational Sciences of the NIH under award number UL1TR001412 to the University at Buffalo. This work also supported by National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Cancer Institute's Flow and Image Cytometry as well as Biostatistics Shared Resources. Research funding also provided by NIH R01CA140622 (S.I. Abrams), the Sklarow Foundation, Elsa Kreiner Memorial Fund, Fraternal Order of Eagles, and RPCI Friends of Urology (T. Schwaab and J.B. Muhitch).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.