Center for Gynecologic Oncology Amsterdam (CGOA), Amsterdam University Medical Center (UMC), Cancer Center Amsterdam (CCA), Vrije Universiteit Amsterdam, Amsterdam, Netherlands.
Department of Medical Oncology Amsterdam UMC, Cancer Center Amsterdam (CCA), Vrije Universiteit Amsterdam, Amsterdam, Netherlands.
Front Immunol. 2020 Dec 17;11:596825. doi: 10.3389/fimmu.2020.596825. eCollection 2020.
PD-1/PD-L1 immune checkpoint inhibitors show potential for cervical cancer treatment. However, low response rates suggest that patient selection based on PD-L1 protein expression is not optimal. Here, we evaluated different PD-L1 detection methods and studied transcriptional regulation of PD-L1/PD-L2 expression by The Cancer Genome Atlas (TCGA) mRNAseq analysis. First, we determined the copy number of the PD-L1/PD-L2 locus by fluorescence hybridization (FISH), PD-L1 mRNA expression by RNA hybridization (RNAish), and PD-L1/PD-L2 protein expression by immunohistochemistry (IHC) on tissue microarrays containing a cohort of 60 patients. Additionally, distribution of PD-L1/PD-L2 was visualized based on flow cytometry analysis of single-cell suspensions (n = 10). PD-L1/PD-L2 locus amplification was rare (2%). PD-L1 mRNA expression in tumor cells was detected in 56% of cases, while 41% expressed PD-L1 protein. Discordant scores for PD-L1 protein expression on tumor cells between cores from one patient were observed in 27% of cases. Interestingly, with RNAish, PD-L1 heterogeneity was observed in only 11% of the cases. PD-L2 protein expression was found in 53%. PD-L1 mRNA and protein expression on tumor cells were strongly correlated (p < 0.001). PD-L1 and PD-L2 protein expression showed no correlation on tumor cells (p = 0.837), but a strong correlation on cells in stromal fields (p < 0.001). Co-expression of PD-L1 and PD-L2 on macrophage-like populations was also observed with flow cytometry analysis. Both PD-L1 and PD-L2 TCGA transcript levels strongly correlated in the TCGA data, and both PD-L1 and PD-L2 strongly correlated with interferon gamma (IFNG) expression/transcript levels (p < 0.0001). Importantly, patients with high PD-L1/PD-L2/IFNG transcript levels had a survival advantage over patients with high PD-L1/PD-L2 and low IFNG expression. Based on these findings, we conclude that PD-L1/PD-L2 expression in cervical cancer is mainly associated with interferon induction and not gene amplification, which makes FISH unsuitable as biomarker. The heterogeneous PD-L1 and PD-L2 expression patterns suggest IHC unreliable for patient selection. RNAish, in conjunction with interferon signaling evaluation, seems a promising technique for immune checkpoint detection. These results warrant further investigation into their prognostic and predictive potential.
PD-1/PD-L1 免疫检查点抑制剂在宫颈癌治疗中显示出潜力。然而，低反应率表明基于 PD-L1 蛋白表达的患者选择并不理想。在这里，我们评估了不同的 PD-L1 检测方法，并通过癌症基因组图谱（TCGA）mRNAseq 分析研究了 PD-L1/PD-L2 表达的转录调控。首先，我们通过荧光杂交（FISH）确定 PD-L1/PD-L2 基因座的拷贝数，通过 RNA 杂交（RNAish）确定 PD-L1 mRNA 表达，通过免疫组织化学（IHC）在包含 60 例患者的组织微阵列上检测 PD-L1/PD-L2 蛋白表达。此外，还基于单细胞悬液的流式细胞术分析（n=10）可视化 PD-L1/PD-L2 的分布。PD-L1/PD-L2 基因座扩增很少见（2%）。在 56%的病例中检测到肿瘤细胞中 PD-L1 mRNA 的表达，而 41%的病例表达 PD-L1 蛋白。在一个患者的核心之间观察到肿瘤细胞上 PD-L1 蛋白表达的不一致评分在 27%的病例中。有趣的是，使用 RNAish，仅在 11%的病例中观察到 PD-L1 异质性。PD-L2 蛋白表达在 53%的病例中发现。肿瘤细胞上的 PD-L1 mRNA 和蛋白表达呈强相关性（p<0.001）。肿瘤细胞上 PD-L1 和 PD-L2 蛋白表达无相关性（p=0.837），但在基质场的细胞中呈强相关性（p<0.001）。通过流式细胞术分析还观察到巨噬细胞样群体上 PD-L1 和 PD-L2 的共表达。在 TCGA 数据中，PD-L1 和 PD-L2 TCGA 转录水平强烈相关，PD-L1 和 PD-L2 均与干扰素γ（IFNG）表达/转录水平强烈相关（p<0.0001）。重要的是，具有高 PD-L1/PD-L2/IFNG 转录水平的患者的生存优势超过了具有高 PD-L1/PD-L2 和低 IFNG 表达的患者。基于这些发现，我们得出结论，宫颈癌中 PD-L1/PD-L2 的表达主要与干扰素诱导有关，而不是基因扩增，这使得 FISH 不适合作为生物标志物。PD-L1 和 PD-L2 表达模式的异质性表明 IHC 不适合用于患者选择。RNAish 结合干扰素信号转导评估，似乎是一种有前途的免疫检查点检测技术。这些结果表明需要进一步研究其预后和预测潜力。
