【目的】滤泡性淋巴瘤（follicular lymphoma, FL）是目前我国较为常见的惰性非霍奇金淋巴瘤的类型之一，进展期FL被认为不可治愈，目前进展期FL患者接受标准一线治疗方案的预后各有不同，此研究拟分析进展期FL患者的临床特点、比较一线治疗方案的疗效、评价检验效能较高的预后模型以及探索预后相关生物标志物。

【方法】第一部分研究纳入了从2008年1月至2018年12月间、在中国医学科学院肿瘤医院接受了标准一线治疗的初治进展期FL患者，分析了这些患者的临床特点、一线治疗方案的疗效和生存结局，并在这些患者中进一步分析了四个预后模型（FLIPI、FLIPI2、PRIMA-PI和LDH+β2-M）的预后检验效能。第二部分研究基于一组公共数据库的RNA-seq数据，利用加权基因共表达网络分析探索了和FL发生相关的共表达网络，并对关键基因进行识别，此后在另一组独立的公共数据库RNA-seq数据中对关键基因的预后相关性进行了分析，并利用中国医学科学院肿瘤医院淋巴瘤患者血清样本库中接受一线免疫化疗的进展期FL疗前血清样本，对和预后相关的基因在蛋白表达层面进行了实验验证。

【结果】第一部分研究共纳入158例进展期FL患者进行分析，结果显示，利妥昔单抗维持治疗和更高的5年PFS率和5年OS率相关（PFS率为83.3%对比52.7%，p<0.001；OS率为97.8%对比84.1%，p=0.032），在目前广泛应用的四个预后模型中，FLIPI2在中国进展期FL患者中显示出良好的预后检验效能（C指数=0.688）。第二部分识别出了与FL发生相关的3个基因共表达模块和8个关键基因（PLA2G2D、MMP9、PTGDS、CCL19、NFIB、YAP1、RGL1和TIMP3），独立验证队列生存分析和多因素回归分析提示，CCL19和接受标准一线治疗的进展期FL的OS独立相关（HR = 0.47, 95%CI [0.25-0.86], p = 0.014）。在另一个独立队列进行酶联免疫吸附实验验证了血清CCL19浓度和预后的相关性，血清高浓度CCL19与更长的PFS（p=0.014）和OS（p=0.039）相关。

【结论】对于中国进展期FL患者，一线免疫化疗后接受利妥昔单抗维持治疗和PFS以及OS相关，应用FLIPI2能更好的进行预后判断。CCL19可能可以作为接受一线免疫化疗的进展期FL预后生物标志物。

【关键词】滤泡性淋巴瘤；治疗；预后；生物标志物；基因

第一部分

进展期滤泡性淋巴瘤患者临床特征及预后分析

【目的】FL是一类存在异质性的淋巴瘤，其预后和治疗选择也各有不同。此部分研究基于单中心真实世界患者资料，旨在分析利妥昔单抗维持治疗对中国FL患者预后的影响，并进一步评估目前的几种预后评价模型的预测效能。

【方法】本研究回顾性地纳入了从2008年1月至2018年12月间、在中国医学科学院肿瘤医院接受了标准一线治疗的初治进展期FL患者，进一步比较患者基线临床特征、长期生存结局、预后因素和四个预后模型（FLIPI、FLIPI2、PRIMA-PI和LDH+β2-M）的预测效能。

【结果】研究共纳入158例患者，按照一线标准化疗后是否接受利妥昔单抗维持治疗分为利妥昔单抗维持组和观察组，其中利妥昔单抗维持组为77例患者，观察组为81例患者。全组中位随访时间为61.0个月。结果显示，对比观察组，利妥昔单抗维持组的5年PFS率和OS率均有提高（PFS率为83.3%对比52.7%，p<0.001；OS率为97.8%对比84.1%，p=0.032）。对比其他三个预后模型，FLIPI2的C指数更高。

【结论】对于初治进展期中国FL患者，一线标准治疗后接受利妥昔单抗维持治疗和更长的PFS和OS相关。FLIPI2显示出良好预后预测能力。

【关键词】滤泡性淋巴瘤；利妥昔单抗维持治疗；预后；治疗；生存

第二部分

进展期滤泡性淋巴瘤患者生物标志物的探索

【目的】本研究致力于应用一线标准治疗方案的滤泡性淋巴瘤患者预后相关关键基因。

【方法】利用数据集GSE65135（n=24）进行差异表达基因分析。利用WGCNA构建并探索共表达网络，将共表达网络导入Cytoscape软件进行基因关联度分析以识别关键基因。进一步我们将在数据集GSE119214（n=137）进行关键基因预后验证，并在来自中国医学科学院肿瘤医院的独立患者队列（n=32）中进行ELISA实验验证。GO分析、GSEA分析和KEGG分析进一步用于探索相关基因的通路。进一步地，我们应用CIBERSORT分析探索了和关键基因相关的肿瘤浸润免疫细胞亚群的组成。

【结果】通过肿瘤组和对照组的差异基因分析，一共获得了3260个差异基因，其中上调基因为1861个，下调基因为1399个。利用WGCNA识别了8个关键基因，分别为PLA2G2D、MMP9、PTGDS、CCL19、NFIB、YAP1、RGL1和TIMP3。Kaplan-Meier分析和多因素COX回归分析提示CCL19和一线接受利妥昔单抗联合化疗的患者的OS独立相关（HR = 0.47, 95%CI [0.25-0.86], p = 0.014）。血清高浓度CCL19与更长的无进展生存期（p=0.014）和总生存期（p=0.039）相关。肿瘤浸润免疫细胞亚群分析提示CCL19的表达和单核细胞以及M1型巨噬细胞的比例呈正相关，和幼稚B细胞以及浆细胞的比例呈负相关。

【结论】CCL19的表达与一线应用利妥昔单抗联合化疗的进展期FL患者生存相关，CCL19可能能够作为接受一线免疫化疗的进展期FL的预后生物标志物。

Aim Follicular lymphoma (FL) is one of the common indolent types of non-Hodgkin lymphoma. Advanced stage FL is considered as incurable. The prognosis for advanced stage FL receiving standard first-line chemoimmunotherapy remains heterogeneous. In this study, we aimed to analyze the clinical characteristics of advanced stage FL, compare the efficacy of first-line regimen, evaluate the power of prognosis model and explore the prognosis biomarker.

Methods Part one of the study included patients with treatment-naïve advanced stage FL who were treated in Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College between January 2008 and December 2018. We analyzed the clinical characteristics, first-line regimens and survival outcomes of these patients. Furthermore, we compared the power of four common FL prognosis model in this cohort (FLIPI, FLIPI2, PRIMA-PI and LDH+β2-M). In part two of the study, we explored the FL-related co-expression network utilizing weighted gene co-expression network analysis and recognized hub genes utilizing the RNA-seq data from public database. In addition, we explored the potential prognosis biomarker in another independent cohort containing RNA-seq data and validate the protein expression of the recognized prognosis biomarker in a patient cohort containing pre-treatment plasma samples of advanced stage FL patients from our center.

Result A total of 158 advanced stage FL patients were enrolled in the study of part one. Results showed that rituximab maintenance was associated with higher 5-year progression-free survival rate (83.3% versus 52.7%, p<0.001) and 5-year overall survival rate (97.8% versus 84.1%, p=0.032). Among the prognosis models, FLIPI2 showed promising power in advanced stage FL (C-index = 0.688). In part two of the study, we recognized three FL-related gene co-expression modules and eight hub genes (PLA2G2D, MMP9, PTGDS, CCL19, NFIB, YAP1, RGL1 and TIMP3). Kaplan-Meier analysis and multivariate COX analysis indicated that CCL19 was associated with OS in patients with advanced stage FL receiving standard first-line chemoimmunotherapy (HR = 0.47, 95%CI [0.25-0.86], p = 0.014). In the enzyme-linked immune sorbent assay (ELISA) experiment, higher level of plasma CCL19 concentration was associated with longer progression-free survival (p=0.014) and overall survival (p=0.039).

Conclusion For Chinese advanced stage FL patients, rituximab maintenance after first-line chemoimmunotherapy is associated with better PFS and OS, and FLIPI2 is a powerful prognosis model for these patients. CCL19 could be a potential prognosis biomarker for advanced stage FL patients receiving first-line chemoimmunotherapy.

Keywords follicular lymphoma, treatment, prognosis, biomarker, gene, chemokine

Part 1 Clinical characteristics and prognosis analysis of advanced stage follicular lymphoma

Aim The prognosis and treatment options for FL remain heterogenous. This study aimed to analyze the prognostic impact of rituximab maintenance and evaluate the prognostic models in Chinese FL patients.

Methods We retrospectively evaluated patients with treatment-naïve advanced stage FL who were treated in our center between January 2008 and December 2018. We compared the baseline characteristics, long-term survival outcomes, prognostic factors, and the performance of four common prognostic models (i.e., FLIPI, FLIPI2, PRIMA-PI, and LDH+β2-M) among them.

Results A total of 158 patients were included in this study, with 77 patients in rituximab maintenance group and 81 patients in observation group. Patients in rituximab maintenance group showed significantly higher 5-year progression-free survival rate (83.3% vs 52.7%, p<0.001) and overall survival rate (97.8% vs 84.1%, p=0.032) than the patients in observation group. FLIPI2 showed a more discriminating C index than the other three models.

Conclusion This study showed that rituximab maintenance after chemoimmunotherapy induction can prolong both progression-free survival and overall survival in patients with advanced-stage FL, and FLIPI2 is a promising prognostic model.

Keywords follicular lymphoma; rituximab maintenance; prognosis; treatment; survival

Part 2 Exploration of prognostic biomarker for follicular lymphoma utilizing weighted gene co-expression network analysis

Aims This study aimed to recognize the hub genes associated with prognosis in FL treated with first-line rituximab combined with chemotherapy.

Method RNA sequencing data of dataset GSE65135 (n=24) were included in differentially expressed genes analysis. The coexpression network was constructed by weighted gene co-expression network analysis and hub genes was identified by Cytoscape. Validation of hub genes expression and prognosis were applied in dataset GSE119214 (n=137) and independent patient cohort from Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (n=32), respectively, by analyzing RNA-seq expression data and serum protein concentration quantified by ELISA. GSEA, GO and KEGG pathway enrichments analysis were performed. CIBERSORT was applied for tumor-infiltrating immune cells subset analysis.

Results A total of 3260 differential expression genes were obtained, with 1861 genes upregulated and 1399 genes downregulated. Using WGCNA, eight hub genes, PLA2G2D, MMP9, PTGDS, CCL19, NFIB, YAP1, RGL1, and TIMP3 were identified. Kaplan-Meier analysis and multivariate COX regression analysis indicated that CCL19 independently associated with OS for FL patients treated with rituximab and chemotherapy (HR = 0.47, 95%CI [0.25-0.86], p = 0.014). Higher serum CCL19 concentration was associated with longer PFS (p=0.014) and OS (p=0.039). TIICs subset analysis showed that CCL19 expression had a positive correlation with monocytes and macrophages M1, and a negative correlation with naïve B cells and plasma cells.

Conclusion CCL19 expression was associated with survival outcomes and might be a potential prognostic biomarker for FL treated with first-line chemoimmunotherapy.