Supplementary Material for: Characterization of pleural mesothelioma by hierarchical clustering analyses using immune cells within tumor microenvironment.

Introduction: Over the past decade, classifications using immune cell infiltration have been applied to many types of tumors; however, mesotheliomas have been less frequently evaluated. Methods: In this study, 60 well-characterized pleural mesotheliomas (PMs) were evaluated immunohistochemically for the characteristics of immune cells within tumor microenvironment (TME) using 10 immunohistochemical markers CD3, CD4, CD8, CD56, CD68, CD163, FOXP3, CD27, PD-1 and TIM-3. For further characterization of PMs, hierarchical clustering analyses using these 10 markers were performed. Results: Among the immune cell markers, CD3 (P < 0.0001), CD4 (P = 0.0016), CD8 (P = 0.00094), CD163+ (P = 0.042) and FOXP3+ (P = 0.025) were significantly associated with unfavorable clinical outcome. Immune checkpoint receptor expressions on tumor-infiltrating lymphocytes such as PD-1 (P = 0.050), CD27 (P = 0.014) and TIM-3 (P = 0.0098) were also associated with unfavorable survival. Hierarchical clustering analyses identified three groups showing specific characteristics and significant associations with patient survival (P = 0.011): the highest number of immune cells (ICHigh); the lowest number of immune cells, especially CD8+ and CD163+ cells (ICLow); and intermediate number of immune cells (ICInt). ICHigh tumors showed significantly higher expression of PD-L1 (P = 0.00038). Cox proportional hazard model identified ICHigh [hazard ratio (HR) = 2.90] and ICInt (HR = 2.97) as potential risk factors compared with ICLow. Tumor CD47 (HR = 2.36), tumor CD70 (HR = 3.04) and tumor PD-L1 (HR = 3.21) expressions were also identified as potential risk factors for PM patients. Conclusion: Our findings indicate immune checkpoint and/or immune cell-targeting therapies against CD70-CD27 and/or CD47-SIRPA axes may be applied for PM patients in combination with PD-L1-PD-1 targeting therapies in accordance with their tumor immune microenvironment characteristics.

引言：近十余年间，基于免疫细胞浸润的分型策略已广泛应用于多种肿瘤的研究中，但间皮瘤的相关评估却相对少见。方法：本研究选取60例特征明确的胸膜间皮瘤（pleural mesotheliomas, PM）样本，采用10种免疫组化标记物——CD3、CD4、CD8、CD56、CD68、CD163、FOXP3、CD27、PD-1及TIM-3，对其肿瘤微环境（tumor microenvironment, TME）内的免疫细胞特征进行免疫组化分析。为进一步对胸膜间皮瘤进行分型，本研究基于上述10种标记物开展了层级聚类分析。结果：在免疫细胞标记物中，CD3（P < 0.0001）、CD4（P = 0.0016）、CD8（P = 0.00094）、CD163+（P = 0.042）及FOXP3+（P = 0.025）的表达与不良临床结局显著相关。肿瘤浸润淋巴细胞上的免疫检查点受体表达，如PD-1（P = 0.050）、CD27（P = 0.014）及TIM-3（P = 0.0098），同样与不良生存预后相关。层级聚类分析鉴定出3个具有特定特征且与患者生存显著相关（P = 0.011）的亚组：免疫细胞丰度最高组（immune cell high, ICHigh）、免疫细胞丰度最低组（尤其CD8+及CD163+细胞，immune cell low, ICLow）以及免疫细胞丰度中等组（immune cell intermediate, ICInt）。ICHigh亚组肿瘤的PD-L1表达水平显著更高（P = 0.00038）。Cox比例风险模型分析显示，与ICLow亚组相比，ICHigh组[风险比（hazard ratio, HR）= 2.90]与ICInt组（HR = 2.97）均为潜在的预后风险因素。肿瘤CD47（HR = 2.36）、肿瘤CD70（HR = 3.04）及肿瘤PD-L1（HR = 3.21）的表达同样被鉴定为胸膜间皮瘤患者的潜在预后风险因素。结论：本研究结果提示，针对CD70-CD27及/或CD47-SIRPA通路的免疫检查点或免疫细胞靶向治疗，可结合PD-L1-PD-1靶向治疗方案，根据患者的肿瘤免疫微环境特征应用于胸膜间皮瘤患者。