Purpose: In NABUCCO, safety and efficacy of preoperative ipilimumab plus nivolumab were assessed in stage III urothelial cancer. Encouraging responses were achieved and ipilimumab 3 mg/kg (ipilimumab-high) appeared more effective than ipilimumab 1 mg/kg (ipilimumab-low). We explored ipilimumab plus nivolumab response biomarkers and tumor micro environment (TME) treatment dynamics.

Experimental design: Baseline FFPE tumor tissue was analyzed using PD-L1 immunohistochemistry (n=51), whole-exome and transcriptome sequencing (both n=53) and correlated with response. Baseline infiltration of CD8+ T-cells (n=51) and at cystectomy (n=42) was examined. ScRNAseq of CD3+ T-cells was conducted on on-treatment resection tissue of two responders to ipilimumab-high to explore characteristics of CD8+ T-cells within the TME.

Results: High tumor mutational burden (TMB) and PD-L1 positivity were associated with response to ipilimumab plus nivolumab. Non-responding patients exhibited increased expression of a TGFβ signature. We observed increased transcription of the g2m checkpoint and e2f target in responders to ipilimumab-high and enhanced transcription of IFN-α and -γ hallmarks in responders to ipilimumab-low. CD8+TCF7+ T-cells accumulated in the TME of responders to ipilimumab-high. ScRNAseq of CD8+TCF7+ T-cells demonstrated enhanced expression of IL7R, CCR7, GPR15, XCL1, SELL and LEF1.

Conclusions: Our data indicate that TMB, PD-L1 and TGFβ are potential biomarkers for response to ipilimumab plus nivolumab in stage III urothelial cancer. An inflammatory TME might be relevant for responding to ipilimumab-low. We found that in responders to ipilimumab-high, TCF7+CD8+ T-cells accumulated in the TME. ScRNAseq in two responders suggested that TCF7+CD8+ T-cells express genes associated with immunological memory formation and T-cell homing.

目的： 在NABUCCO研究中，评估了新辅助伊匹单抗联合尼伏鲁马布在III期尿路上皮癌中的安全性和有效性。结果显示令人鼓舞的反应，并且3毫克/千克剂量的伊匹单抗（高剂量）似乎比1毫克/千克剂量（低剂量）更有效。我们探索了伊匹单抗和尼伏鲁马布应答生物标志物以及肿瘤微环境（TME）治疗动态。

实验设计： 使用PD-L1免疫组化分析基线FFPE肿瘤组织（n=51），并进行全外显子测序和转录组测序（两者均为n=53），并与反应相关联。检查了基线CD8+ T细胞浸润（n=51）以及在膀胱切除术时的浸润情况（n=42）。对两个高剂量伊匹单抗应答者的治疗期间切除组织进行了CD3+ T细胞scRNAseq，以探索TME内CD8+ T细胞的特点。

结果： 肿瘤突变负荷（TMB）和PD-L1阳性与伊匹单抗联合尼伏鲁马布的反应相关。未应答患者表现出TGFβ信号通路表达增加。我们观察到高剂量伊匹单抗应答者的g2m检查点和e2f目标转录上调，低剂量伊匹单抗应答者的IFN-α和-γ特征转录增强。在对高剂量伊匹单抗有反应的患者中，CD8+TCF7+ T细胞在TME内累积。对两个高剂量应答者进行CD8+TCF7+ T细胞scRNAseq表明IL7R、CCR7、GPR15、XCL1、SELL和LEF1表达增强。

结论： 我们的数据表明，TMB、PD-L1和TGFβ可能是III期尿路上皮癌中伊匹单抗联合尼伏鲁马布应答的潜在生物标志物。炎症性TME可能与低剂量伊匹单抗反应相关。我们发现，在对高剂量伊匹单抗有反应的患者中，TCF7+CD8+ T细胞在TME内累积。两个应答者的scRNAseq表明TCF7+CD8+ T细胞表达与免疫记忆形成和T细胞归巢相关的基因。