Abstract
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Objectives: Administration of programmed cell death ligand 1 (PD-L1) blocking treatment has shown improvement in efficacy of ionizing radiation (IR) through a cytotoxic T cell-dependent mechanism. Moreover, elevated PD-L1 expression has been seen in the literature after IR treatment as an adaptive resistance mechanism.1,2 Despite renal cell carcinoma's (RCC) notorious resistance to conventional radiation therapy, a recent study reported that stereotactic ablative radiotherapy (SABR) had promising control over the metastatic RCC.3,4 Owing to RCC's immunogenic nature, using SABR and immune check-point inhibitors as a combination therapy might synergistically accentuate the therapeutic effect. As such, we reason that immuno-positron emission tomography (iPET) with zirconium-89 (89Zr) labeled atezolizumab (ATZ, anti-PD-L1 antibody) could be used as a non-invasive imaging tool to evaluate this therapeutic effect by assessing the dynamic changes in PD-L1 expression. In this preliminary study, we tested this aforementioned application of iPET after SABR treatment in patient derived xenograft (PDX) mouse models of RCC.
Methods: ATZ was conjugated with deferoxamine (DFO) chelator at a molar ratio of 1 ATZ:1.9 DFO and radiolabeled with 89Zr at a specific activity of ~2 mCi/mg. Stability in rat serum and immunoreactivity by Lindmo assay were measured for 89Zr-DFO-ATZ. SCID/NOD mice implanted with RCC PDXs (PD-L1 expression: 30 - 55%) received tumor-targeted SABR treatment (25 Gy, single fraction, n = 8). Three hours after the treatment, the mice received intravenous injection of ~100 µCi of 89Zr-DFO-ATZ. iPET/CT imaging was performed on the mice from day 2 to day 6 post-injection (p.i.). iPET/CT imaging was also performed on another 3 mice without SABR treatment which were saved as controls. Tumors were excised after imaging and subject to immunohistochemical (IHC) analyses to test for PD-L1 expression. iPET imaging data analysis and IHC assays were double-blinded. Statistical analyses were performed by un-paired t tests, one-tailed, assuming unequal variances.
Results: 89Zr-DFO-ATZ was radiolabeled with high (>95%) radiochemical purity and immunoreactivity (86.2 ± 4%, n = 6). 89Zr-DFO-ATZ was stable for 6 days (>80%) in serum at 37ºC. iPET imaging detected a significantly greater mean tumor uptake for the mice after SABR treatment (range: 4.0 - 6.4% ID/g) than that for the controls (range: 3.8 - 4.8% ID/g) from 2 through 6 days p.i. (p-value < 0.05). The radiated mice showed elevated PD-L1 expression (55 - 80%, n = 7; 30%, n = 1) compared to the control mice (40 - 55%, n = 2; 75%, n = 1) by IHC analyses. Excluding the outliers from each group, PD-L1 expression in the PDX models demonstrated a direct correlation with iPET imaging on day 4 p.i. of 89Zr-DFO-ATZ (r = 76%, r-square = 0.57, p < 0.018).
Conclusions: Using iPET with 89Zr-DFO-ATZ, we observed a significant elevation in the PD-L1 expression in PDX RCC mouse models after radiation treatment. Acknowledgements: This work was supported by a grant from the Cancer Prevention and Research Institute of Texas (RP110771), a Career Enhancement Program Award from the NCI’s Kidney Cancer SPORE at UT Southwestern (P50CA196516), and the Dr. Jack Krohmer Professorship Funds. References: 1. Deng L, Liang H, Burnette B, et. al., J Clin Invest. 124(2): 687-95 (2014); 2. Weichselbaum RR, Liang H, Deng L, et. al., Nat Rev Clin Oncol. 14(6): 365-379 (2017); 3. Walshaw RC, Honeychurch J, and Illidge TM, Br J Radiol.89(1066): 20160472 (2016); 4. Wang CJ, Christie A, Lin MH, et. al., Int. J Radiat Oncol Biol Phys, 98(1), 91-100 (2017)