Supplementary Material for: A novel signature based on m6A RNA methylation regulators reveals distinct prognostic subgroups and associates with tumor immunity of patients with pancreatic neuroendocrine neoplasms

Introduction: The RNA N6-methyladenosine (m6A) regulators play a crucial role in tumorigenesis and could be indicators of prognosis and therapeutic targets in various cancers. However, the expression status and prognostic value of m6A regulators have not been studied in pancreatic neuroendocrine neoplasms (PanNENs). We aimed to investigate the expression patterns and prognostic value of m6A regulators and assess their correlations with immune checkpoints and infiltrates in PanNENs. Methods: Immunohistochemistry was performed for 15 m6A regulators and immune markers using tissue microarrays obtained from 183 patients with PanNENs. The correlation between m6A protein expression and clinicopathological parameters with recurrence-free survival (RFS) was examined using a random survival forest, Cox regression model and survival tree analysis. Results: Among the 15 m6A proteins, high expression of YTHDF2 and HNRNPC was found to be significantly associated with recurrence and served as independent risk factors. High YTHDF2 expression was associated with higher number of CD3+ T cells, whereas high HNRNPC expression was significantly correlated with the expression of PD-L1. A YTHDF2-based signature was determined, including five patterns from survival tree analysis: patients with the LNnegYTHDF2high signature had a 5-year RFS rate of 92.1%, whereas patients with LNposTumorSize＜2.5cm signature had the worst 5-year RFS rate of 0%. The area under receiver operating characteristic curve was 0.870 for the YTHDF2-based signature. The C-index was 0.978, suggesting good discrimination ability; the risk score of recurrence served as an independent prognostic factor indicating shorter RFS. Conclusions: YTHDF2 appears to serve as a promising prognostic biomarker and therapeutic target. A YTHDF2-based signature can identify distinct subgroups, which may be helpful to strategize personalized postoperative monitoring.

引言：RNA N6-甲基腺苷（RNA N6-methyladenosine，m6A）调控因子在肿瘤发生过程中发挥关键作用，可作为多种癌症的预后指标与治疗靶点。然而，胰腺神经内分泌肿瘤（pancreatic neuroendocrine neoplasms，PanNENs）中m6A调控因子的表达状态与预后价值尚未得到相关研究。本研究旨在探讨m6A调控因子的表达模式及其预后价值，并评估其与PanNENs中免疫检查点及免疫浸润的相关性。方法：本研究使用183例PanNENs患者的组织微阵列，对15种m6A调控因子及免疫标志物开展免疫组化检测。采用随机生存森林、Cox回归模型及生存树分析，探究m6A蛋白表达与临床病理参数及无复发生存期（recurrence-free survival，RFS）之间的关联。结果：在15种m6A蛋白中，YTHDF2与HNRNPC的高表达被证实与肿瘤复发显著相关，且为独立危险因素。YTHDF2高表达与CD3+ T细胞数量增多呈正相关，而HNRNPC高表达则与PD-L1的表达显著相关。本研究构建了基于YTHDF2的预后模型，经生存树分析得到5个患者亚型：携带LNnegYTHDF2high亚型的患者5年无复发生存率为92.1%，而携带LNposTumorSize＜2.5cm亚型的患者5年无复发生存率仅为0%，预后最差。基于YTHDF2的预后模型的受试者工作特征曲线下面积为0.870，一致性指数（C-index）为0.978，表明其具备良好的区分能力；复发风险评分可作为独立预后因素，提示患者无复发生存期更短。结论：YTHDF2有望成为极具前景的预后生物标志物与治疗靶点。基于YTHDF2的预后模型可识别不同的患者亚组，有助于制定个性化的术后监测策略。