第一部分

OSMR突变在家族性单中心型Castleman病发病中的作用机制研究

目的：单中心型Castleman病（unicentric Castleman disease, UCD）是一种罕见的淋巴增殖性疾病，其发病机制未明。自2022年8月至今，我院共接诊了3个有家族聚集倾向的CD家系，患者均为UCD，且都伴有多发性骨硬化，提示遗传因素可能参与其中。我们收集了患者及其亲属的外周血标本，利用二代测序、生物信息学等方法，根据突变注释、遗传定律、表型相关性等原理，并经一代测序验证，最终确定了共同的杂合突变OSMR (NM_003999.2): c.2195C>T (p.T732M)可能参与了UCD的发病。本研究将进一步通过体外功能和分子实验探究该突变在UCD发病中的作用机制。

方法：利用ELISA法检测患者和亲属的血清抑瘤素M（oncostatin M, OSM）水平，通过免疫组化染色明确对照和患者淋巴结中抑瘤素M受体（oncostatin M receptor, OSMR）的分布、表达水平以及OSMR下游的经典通路分子STAT3的磷酸化水平。构建点突变克隆，在内皮细胞系EA.hy926（人脐静脉细胞杂交系）中分别过表达OSMR野生型和突变型，通过转录组测序全面分析突变体的生物学效应，而后应用EDU法细胞增殖实验、划痕实验、血管形成实验探索突变细胞的增殖、迁移和成管能力，Western blot实验检测OSMR下游主要通路分子，结合转录组测序得到的差异基因，通过RT-qPCR、Western blot、ELISA等方法明确该通路可能调控的靶基因，并利用通路抑制剂验证其在相应生物学效应中的作用。

结果：家族性UCD患者均存在杂合突变OSMR p.T732M。患者血清抑瘤素M水平不高，抑瘤素M受体主要分布于淋巴结的血管内皮细胞且无高表达，而STAT3在淋巴结中的磷酸化水平显著升高。在体外实验中，转录组测序分析提示突变细胞在细胞生长、细胞周期、迁移、血管形成、细胞外基质等方面有明显富集，功能实验证实了OSMR p.T732M突变体具有促进内皮细胞系EA.hy926增殖和成管的能力。突变细胞内存在STAT3的显著激活以及细胞周期相关蛋白cyclin A2的表达上调与p21的表达下调，将突变细胞内STAT3的磷酸化抑制至与野生型细胞相同水平，会导致cyclin A2表达下降和p21表达升高，并造成细胞增殖和成管能力下降。

结论：在体外，OSMR p.T732M突变可以使受体组成型激活STAT3而促进内皮细胞的增殖和血管形成。激活的STAT3可能通过调控cyclin A2、p21的表达而影响细胞功能。OSMR突变可能通过上述机制在家族性UCD中发挥致病作用。

第二部分

单中心型Castleman病合并闭塞性细支气管炎的回顾性研究

目的：闭塞性细支气管炎（bronchiolitis obliterans, BO）是单中心型Castleman病（unicentric Castleman disease, UCD）的一种罕见且危及生命的并发症，被认为是UCD中最重要的预后不良因素和主要死亡原因，但目前缺乏相关的大宗队列研究。因此，本研究旨在探究UCD合并BO的临床特征、治疗和预后转归，以此提高对此类疾病的认识。

方法：回顾性分析了自2000年1月至2024年5月期间在****医院确诊合并BO的UCD患者的临床资料，从临床和实验室特征、治疗方案和预后展开描述，并与未合并BO的UCD患者做比较。

结果：共纳入281例诊断明确的UCD患者，其中18例（6.4%）满足BO的诊断标准。合并BO的UCD患者确诊UCD的年龄显著低于无BO的UCD患者（28.5 vs. 37.0岁，P＜0.05）。在合并BO的UCD患者中，深部淋巴结受累占比94.4%，合并PNP的占比83.3%，均显著高于其在无BO的UCD患者中的比例。在确诊BO时，第1秒用力呼气容积（forced expiratory volume in 1 s, FEV1）占预计值百分比的中位数为20.9%（10.0%-41.9%），83.3%患者患有重度BO。所有UCD患者均接受了肿瘤完整切除术。61.1%患者在手术后出现BO。合并BO的UCD患者总体生存率（overall survival, OS）低于无BO的UCD患者，5年预计OS分别为76.2%（95% CI, 47.2%-100.0%）和97.2%（95% CI, 96.0%-100.0%）。在重度BO患者中，11例未接受肺移植，其中2例死于呼吸衰竭，5年预计OS为62.5%（95.0% CI, 20.8%-100.0%）；4例接受了肺移植，术后中位随访36.5（8.1-155.2）月后，全部存活。

结论：合并BO的UCD患者具有独特的临床特征。完整切除肿瘤既无法缓解BO，也不能阻止BO进展。对于重度BO患者而言，肺移植可能是改善预后的关键治疗手段。

第三部分

西罗莫司治疗复发/难治性特发性多中心型Castleman病的疗效和安全性

目的：特发性多中心型Castleman病（idiopathic multicentric CD, iMCD）是Castleman病的一种临床亚型，患者通常有全身性炎症和多器官功能障碍。虽然白细胞介素-6（interleukin-6, IL-6）抑制剂是iMCD的一线治疗，而约半数的iMCD患者对IL-6抑制治疗无反应，且部分患者最终仍会有疾病复发，总体预后较差。对于复发/难治性iMCD（relapsed/refractory iMCD, R/R iMCD）患者的治疗方案尚未形成共识，值得进一步探索。已有研究发现，除了IL-6通路，抑瘤素M受体（oncostatin M receptor, OSMR）在CD淋巴结中是高表达的；在部分iMCD患者血清中，也有检测到抗抑瘤素M（oncostatin M, OSM）的自身抗体，而哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）亦是OSM/OSMR促炎信号通路的下游分子，且mTOR在iMCD中是显著激活的。西罗莫司通过抑制mTOR而发挥免疫抑制作用，是潜在的iMCD治疗药物。因此，本研究旨在探究西罗莫司治疗R/R iMCD患者的疗效和安全性。

方法：回顾性分析了自2019年10月至2023年12月在****医院接受包含西罗莫司方案治疗的R/R iMCD患者，描绘了其临床特征、治疗方案和有效率、药物安全性、预后等。

结果：本研究共纳入了26例接受含西罗莫司方案治疗的R/R iMCD患者，其开始使用西罗莫司治疗的中位年龄为40.5（23.0-60.0）岁，既往治疗线数的中位数为2（1-5）。15例（57.7%）患者接受了西罗莫司单药治疗，11例（42.3%）患者接受了联合治疗。18例（69.2%）患者获得了症状和生化指标的改善，达到至少总体部分缓解的中位时间为1.9（0.5-14.6）月。从开始西罗莫司治疗的中位随访时间为11.7（1.6-50.7）月，至下一线治疗的中位时间为46.2个月。末次随访时无患者死亡，大多数患者病情处于持续缓解状态，并在继续接受西罗莫司治疗。R/R iMCD患者对西罗莫司耐受良好，不良反应轻微。

结论：西罗莫司是一种方便的口服药物，对于治疗R/R iMCD有效且安全。

Part I
The mechanism of OSMR mutation in the pathogenesis of familial unicentric Castleman disease

Objective: Unicentric Castleman disease (UCD) is a rare lymphoproliferative disorder and the pathogenesis of it remains unclear. Since August 2022, our hospital has admitted a total of 3 CD families with a tendency of familial aggregation. All the patients are UCD subtype, and they are all accompanied by multiple osteosclerosis, suggesting that genetic factors may be involved in the pathogenesis. We collected peripheral blood samples from patients and their relatives, used next-generation sequencing and bioinformatics and finally determined that the shared heterozygous mutation OSMR (NM_003999.2): c.2195C>T (p.T732M) in UCD patients, which was verified by Sanger sequencing, may be involved in the pathogenesis of UCD based on principles of mutation annotation, genetic laws, phenotypic correlation, etc. This study will further explore the mechanism of this mutation in the pathogenesis of UCD through in vitro functional and molecular experiments.

Methods: The serum oncostatin M (OSM) levels of patients and their relatives were detected by ELISA. Immunohistochemical staining was used to clarify the localization and expression levels of oncostatin M receptor (OSMR), and the phosphorylation level of STAT3, a molecule in the classical pathway downstream of OSMR, in the lymph nodes of the control group and patients. Point mutation clones were constructed, and the wild-type and mutant OSMR were overexpressed in endothelial cell line EA.hy926 (human umbilical vein endothelial cell hybrid line), respectively. The biological characteristics of the mutants were comprehensively analyzed by transcriptome sequencing. Subsequently, the EDU cell proliferation assay, scratch assay, and tube formation assay were applied to explore the proliferation, migration, and tube formation capabilities of the mutant cells. Western blot was used to detect the main downstream pathway molecules of OSMR. Combined with the differentially expressed genes obtained from transcriptome sequencing, methods including RT-qPCR, Western blot, and ELISA were used to identify the potential target genes regulated by this pathway, and pathway inhibitors were utilized to validate their roles in the biological effects.

Results: Familial UCD patients had the heterozygous mutation OSMR p.T732M. The serum oncostatin M levels in patients were not high. Oncostatin M receptor was mainly distributed in the vascular endothelial cells of the lymph nodes without high expression, while the phosphorylation level of STAT3 in the lymph nodes was significantly increased. In vitro, transcriptome sequencing analysis suggested that mutant cells were significantly enriched in cell growth, cell cycle, migration, tube formation, and extracellular matrix. Functional experiments confirmed that the OSMR p.T732M mutant had the ability to promote proliferation and angiogenesis of endothelial cell line EA.hy926. There was significant activation of STAT3 in mutant cells, as well as upregulation of cyclin A2 expression and downregulation of p21 expression. Inhibiting the phosphorylation level of STAT3 in mutant cells to the same level as wild-type cells resulted in a decrease in cyclin A2 expression and an increase in p21 expression, as well as a decrease in proliferation and tube formation ability of the mutant cells.

Conclusions: In vitro, the OSMR p.T732M mutation constitutively activates STAT3, promoting endothelial cell proliferation and angiogenesis. Activated STAT3 may affect cellular function by regulating the expression of cyclin A2 and p21. The OSMR mutation may exert pathogenic effects in familial UCD through the aforementioned mechanisms.

Part Ⅱ

Unicentric Castleman disease complicated with bronchiolitis obliterans

A retrospective study

Objective: Bronchiolitis obliterans (BO) is a rare and life-threatening complication of unicentric Castleman disease (UCD), and is considered the most important poor prognostic factor and the leading cause of death in UCD. However, currently, there is a lack of large-scale cohort studies. Therefore, this study aims to explore the clinical characteristics, treatment, and outcomes of UCD complicated with BO, in order to improve the understanding of the diseases.

Methods: We conducted a retrospective study of the clinical data of UCD patients complicated with BO at **** Hospital from January 2000 to May 2024. Clinical and laboratory characteristics, treatment options, and prognosis were shown and compared with UCD patients without BO.

Results: A total of 281 patients diagnosed with UCD were included in the analysis and 18 patients (6.4%) fulfilled the criteria for BO. UCD patients complicated with BO had a significantly lower age of UCD diagnose than UCD without BO group (28.5 vs. 37.0 years，P＜0.05). Of all UCD patients complicated with BO, 94.4% had deep lymph nodes involvement and 83.3% had PNP, which were both significantly higher than that of UCD without BO cases. Median forced expiratory volume in 1 s of predicted was 20.9% (10.0%-41.9%) at BO diagnosis and 83.3% patients had severe BO. All patients underwent complete tumor resection for UCD. 61.1% patients developed BO after surgery. UCD patients complicated with BO had a poorer overall survival (OS) than UCD without BO patients, and the estimated 5-year OS was 76.2% (95% CI, 47.2%-100.0%) and 97.2% (95% CI, 96.0%-100.0%), respectively. Among severe BO, 11 patients did not receive lung transplantation and 2 of them died of respiratory failure, with 62.5% (95% CI, 20.8%-100.0%) of estimated 5-year OS. 4 patients underwent lung transplantation and all survived with a median postoperative follow-up of 36.5 months (8.1-155.2).  

Conclusions: UCD patients complicated with BO had distinct identity. Complete resection could neither alleviate BO nor prevent BO progression. Lung transplantation is probably a vital treatment for improving prognosis for patients with severe BO.

Part III

The efficacy and safety of sirolimus in the treatment of relapsed/refractory idiopathic multicentric Castleman disease

Objective: Idiopathic multicentric Castleman disease (iMCD) is a clinical subtype of Castleman disease. Patients usually present with systemic inflammation and multi-organ dysfunction. Although interleukin-6 (IL-6) inhibitors have been recommended as the first-line treatment for iMCD, approximately half of iMCD patients do not respond to IL-6 inhibition therapy, moreover the disease eventually will relapse in some patients, with a poor prognosis. There is no consensus on the treatment regimen for patients with relapsed/refractory iMCD (R/R iMCD), which is worthy of further exploration. Previous studies have shown that in addition to the IL-6 pathway, oncostatin M receptor (OSMR) is highly expressed in the lymph nodes of CD patients. Autoantibodies against oncostatin M (OSM) have also been detected in the serum of some iMCD patients. Moreover, the mammalian target of rapamycin (mTOR) is also a downstream molecule of the pro-inflammatory signaling pathway of OSM/OSMR, and mTOR is significantly activated in iMCD. Sirolimus exerts an immunosuppressive effect by inhibiting mTOR and is a potential therapeutic drug. Therefore, this study aims to investigate the efficacy and safety of sirolimus in the treatment of R/R iMCD patients.

Methods: Patients with R/R iMCD who received sirolimus-containing regimen in **** Hospital from October 2019 to December 2023 were retrospectively analyzed. Their clinical characteristics, treatment regimens, response rates, drug safety, and prognosis were described.

Results: A total of 26 R/R iMCD patients who received sirolimus-containing regimen were included in this study. The median age at sirolimus initiation was 40.5 years (23.0-60.0), and the median prior treatment lines was 2 (1-5). 15 patients (57.7%) received sirolimus monotherapy, and 11 patients (42.3%) received combination therapy. Symptomatic and biochemical improvements were achieved in 18 patients (69.2%), and the median time to at least overall partial response was 1.9 months (0.5-14.6). The median follow-up time from sirolimus initiation was 11.7 months (1.6-50.7), and the median time to the next-line treatment was 46.2 months. No patients died at the last follow-up. Most patients were in continuous remission and continued to receive sirolimus treatment. Sirolimus is well tolerated, with mild adverse effects.

Conclusions: Sirolimus is a convenient oral therapy that is effective and safe for patients with R/R iMCD.