Data Sheet 2_Phase 2 trial (NCI-COTC030) of adjuvant inhaled recombinant human IL-15 combined with amputation and adjuvant chemotherapy in dogs with appendicular osteosarcoma.pdf

BackgroundWe have previously shown inhaled IL-15 is associated with anti-tumor responses in dogs with metastatic osteosarcoma (OSA) and melanoma. We evaluated inhaled IL-15 combined with amputation and chemotherapy for localized canine OSA eligible for treatment with curative intent.
MethodsIn a multicenter COTC phase-II-trial for dogs with limb OSA, we hypothesized 2 weeks of inhaled rhIL-15 after amputation and prior to chemotherapy would reduce the risk of metastatic failure at the completion of chemotherapy from a historical rate of 40% to 20%. Using a 2-sided alpha of 0.05, we planned an accrual of 40 dogs to test this hypothesis with 80% power. We performed immune correlative assays and sequencing of peripheral blood mononuclear cells (PBMCs) and primary amputation specimens.
ResultsUnexpectedly, disease-free survival and overall survival were statistically inferior for dogs in the intent-to-treat population compared to a well-validated historical control cohort, so the trial was halted for futility. Cytotoxicity assays of PBMCs showed significant decreases after both surgery and chemotherapy with an overall decrease from the start to end of therapy (-18.2 ± 16.1%, P<0.001). Some dogs demonstrated positive fold change in PBMC cytotoxicity, which correlated significantly with improved dog survival (P = 0.004, r=0.62). Although plasma concentrations of key cytokines varied markedly with no significant differences between disease-free and metastatic-failure patients, inflammatory cytokines such as IL-6 showed absolute increases post-amputation and post-chemotherapy, correlating with decreases in cytotoxicity. Tumor sequencing data reproduced immune signatures as observed in both human and canine cohorts, and PBMC single cell sequencing data showed that gene expression profiles of NK and T cells were significantly different between short and long disease-free interval subjects.
ConclusionsInhaled rhIL-15 combined with amputation and chemotherapy is associated with worse outcomes in dogs with OSA. Correlative assays suggest significant immunological effects of amputation and chemotherapy on immune responses. These data have important implications on novel immunotherapy strategies involving multimodality approaches including surgery and chemotherapy.

研究背景
我们此前已证实，吸入式IL-15可在转移性骨肉瘤（OSA）与黑色素瘤患犬中诱导抗肿瘤应答。本研究针对可接受根治性治疗的局部性犬类OSA患者，评估吸入式IL-15联合截肢术与化疗的治疗效果。

研究方法
本研究为针对肢体OSA患犬的多中心COTC二期临床试验。我们假设，在截肢术后、化疗前给予2周的吸入式重组人IL-15（recombinant human IL-15, rhIL-15），可将化疗结束时的转移性失败风险从历史40%降至20%。本研究采用双侧检验水准α=0.05，计划纳入40只患犬以获得80%的检验效能来验证该假设。我们对外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）以及截肢术中获取的原发肿瘤标本进行了免疫相关分析与测序。

研究结果
出乎意料的是，与经过充分验证的历史对照队列相比，意向治疗人群中患犬的无病生存期与总生存期均显著更差，因此试验因无效性提前终止。PBMC细胞毒性实验结果显示，手术与化疗后PBMC的细胞毒性均显著降低，治疗全程整体下降幅度为-18.2±16.1%（P<0.001）。部分患犬的PBMC细胞毒性呈现正向倍数变化，该变化与患犬生存期延长显著相关（P=0.004，r=0.62）。尽管关键细胞因子的血浆浓度波动明显，且无病患者与转移性失败患者之间无显著差异，但IL-6等炎症细胞因子在截肢术后与化疗后均出现绝对水平升高，且该变化与细胞毒性降低相关。肿瘤测序数据重现了在人类与犬类队列中均观察到的免疫特征；PBMC单细胞测序数据显示，无病间期长短不同的受试者之间，自然杀伤细胞（natural killer cells, NK）与T细胞的基因表达谱存在显著差异。

研究结论
吸入式rhIL-15联合截肢术与化疗，与犬类OSA患者的不良预后相关。相关分析结果表明，截肢术与化疗对免疫应答存在显著的免疫学影响。本研究数据为包含手术与化疗在内的多模态新型免疫治疗策略提供了重要参考。