Characterization of resistance to tumor-localized interleukin-2 and interleukin-12 therapy in pet dogs with advanced malignant melanoma

Pet dogs with oral melanoma were treated in a dose-escalation trial with collagen-anchored interleukin-2 and interleukin-12 following a single 9Gy dose of radiation therapy. Dogs that progressed during, or after, treatment and were ultimately euthanized had tissue collected from primary and metastatic tumors to analyze gene expression and assess mechanisms of resistance to this therapy. Fifteen client-owned dogs with malignant melanoma (n=14 oral melanoma, n=1 periocular melanoma) were recruited to receive intratumoral doses of interleukin-2 and interleukin-12 collagen-binding fusion proteins at different dose levels. The first cohort (1x, n=3) received IL-2 (17.4 ug/kg) and IL-12 (2.08 ug/kg); the second cohort (2x, n=6) received 2x the initial dose (i.e. 34.8 ug/kg IL-2, 4.16 ug/kg IL-12) and was the expansion cohort after encountering toxicity at higher doses; the third cohort (3.3x, n=4) received 3.3x the initial dose (i.e. 57.4 ug/kg IL-2; 6.86 ug/kg IL-12), and the final cohort (5x, n=2) received 5x the initial dose (i.e. 87 ug/kg IL-2; 10.4 ug/kg IL-12). Up to 6 doses of cytokine were provided at 2-week intervals and were dosed after a single 9Gy focal radiation dose to the primary tumor. Dogs were periodically examined following treatment to monitor responses, and dogs that were ultimately euthanized due to cancer progression (n=8, 53.3%) had their primary and metastatic tumors collected for analysis. Tumor tissue was formalin-fixed and paraffin-embedded prior to RNA extraction (Qiagen FFPE RNEasy) and QC on Bioanalyzer (Agilent). Gene expression was analyzed through hybridization to the Nanostring Canine IO nCounter panel set probes, with quantification by Nanostring Digital Analyzer. Expression data was analyzed using Nanostring nSolver software to examine modes of tumor resistance to the RT + IL-2 and IL-12 therapy.