中文摘要

第一部分 静脉内平滑肌瘤病临床特征分析和提出用于指导手术策略的分期分型

研究背景

静脉内平滑肌瘤病（Intravenous Leiomyomatosis, IVL）是一种罕见的、组织学良性但生物学上具有侵袭性的肿瘤，其起源于子宫平滑肌或子宫静脉壁，常可超出子宫范围，沿静脉回流方向通过子宫静脉、卵巢静脉或髂静脉延伸至下腔静脉内，进而可长入心脏和肺动脉，引起系列临床症状，严重时可因血流梗阻导致死亡。该病临床罕见，且发病隐匿，临床表现多样，缺乏特异性，容易因医疗机构对其认识不足造成临床漏诊，或者被误诊为妇科和静脉系统恶性肿瘤、血栓而延误治疗。然而，由于其罕见性，对IVL的认识主要来源于孤立的病例报告、小样本队列和有限的短期随访文献。因此，关于疾病特征、肿瘤预后的数据不足，手术经验缺乏。

目    的

总结静脉内平滑肌瘤病的临床特征、手术治疗效果和总体预后，分析影响复发或残余肿瘤进展的危险因素，并提出疾病的分期分型和相应的手术策略，为指导制定临床治疗方案提供理论依据。

方    法

回顾性收集北京协和医院于2002年1月至2022年12月间接受静脉内平滑肌瘤切除手术的患者。我们收集病例资料、围手术期参数，随访术后近远期并发症出现情况、肿瘤复发情况。我们分为完全切除和不全切除（肿瘤残余）两组分别对患者的术后结局进行分析，采用Kaplan-Meier方法、使用log-rank检验评估复发或残余进展与各种预后因素之间的关联。使用单变量Cox比例风险模型探究各种患者相关和手术相关的多变量对IVL复发或残余进展的影响。此外，我们提出新的分期分型系统，并以此来统计和归纳相关资料。

结    果

共有216名患者被纳入研究，其中I期92名（占42.6%），II期39名（占18.1%），III期76名（占35.2%），IV期9名（占4.2%）。中位随访时间为26.34个月。在此期间，18名（9.7%）接受肿瘤完全切除患者出现了复发，12名（39%）不完全切除患者出现了残余肿瘤进展。手术后的芳香化酶抑制剂（AI）辅助治疗和肿瘤在下腔静脉或心腔内的最大直径与复发或残余进展相关。而年龄以及手术后促性腺激素释放激素激动剂（GnRHa）治疗等因素和与复发或残余进展无关。在不同分期中，患者症状、手术时间、出血量等参数有统计学差异。

结    论

该研究是目前已发表文献中关于静脉内平滑肌瘤病的最大单中心报告，提供了有价值的临床特征总结、长期结局和手术技术经验。总体而言，该病手术治疗效果满意，并发症发生率低，复发少。我们提出的分类系统可以评估病变程度，有利于指导手术管理。

第二部分 单细胞测序技术揭示静脉内平滑肌瘤的肿瘤起源和发病机制

研究背景

静脉内平滑肌瘤病（Intravenous Leiomyomatosis, IVL）作为一种主要影响育龄期女性的罕见肿瘤，表现出沿血管侵袭性生长的生物学行为，体现了独特的组织生物学特征，它处于恶性肿瘤和良性肿瘤之间的交界位置，使其成为研究癌症发展的独具吸引力的研究模型。本文第一部分总结和分析了静脉内平滑肌瘤病的临床特征，但由此引出的很多问题如肿瘤来源、雌激素在肿瘤发生发展的作用以及疾病发病机制等内在病理尚未阐明。多种基于普通转录组RNA测序分析和反转录定量PCR技术的研究被开展以尝试阐明IVL潜在的分子生物机制。然而，这些研究在识别特定的细胞类型和理解IVL肿瘤异质性的复杂性方面仍然存在局限性。迄今为止，全球范围内尚无学者对静脉内平滑肌瘤病进行单细胞测序水平的研究，而单细胞分析技术对研究IVL的起源和理解细胞分子生物学水平的病理机制至关重要。

目    的

采用单细胞分析技术，探究静脉内平滑肌瘤病的起源和肿瘤微环境、特异性发病机制，增加对疾病内在机理的理解，并探索这种罕见肿瘤潜在的靶向治疗分子靶点。

方    法

收集来自7位静脉内平滑肌瘤病患者的共23个样本，其中包括19个肿瘤标本，并根据距离子宫的远近分为A段（初始段肿瘤组织）、B段（下腔静脉内的肿瘤组织）和C段（靠近心脏部分的肿瘤组织），以及2个子宫组织（N段）和2个静脉壁组织（V段）样本作为正常/“癌旁”组织对照。将样本制备成单细胞悬液，获得单细胞cDNA文库进行测序，使用生物化学分析，对细胞进行聚类和鉴定。对平滑肌细胞、间充质细胞群进行特异性分析，采用了包括细胞比例分析、差异表达基因（DEGs）分析、富集分析、数据映射、基因集评分、拷贝数变异（CNV）分析和基于线粒体基因突变的谱系追踪等多种方法进行分析，获取肿瘤内在的分子生物学信息。

结    果

我们明确了肿瘤中一组来源子宫的平滑肌细胞群（SMCs），具体表达了ESR1、PGR1和IGF1，并且发现了其在转录失调与癌症相关基因的高度富集，以及多个与癌症相关的信号通路的激活。我们还观察到了间充质细胞（MSCs）和SMCs的转化现象，揭示了MSCs在肿瘤发展中的重要作用。我们对MSCs的分析显示，在肿瘤样本中富集了几个亚群，其EGFR（表皮生长因子受体）通路激活，同时雌激素通路是失活的。

结    论

我们确认了IVL的子宫起源，发现了不依赖激素生长的平滑肌细胞亚群。我们鉴别出和细胞凋亡、血管生成以及癌化作用的基因组；发现了IGF1和EGFR（表皮生长因子受体）的激活促进肿瘤生长，这可能为这种罕见肿瘤的靶向治疗的开发提供了依据。我们的发现为认识IVL复杂的内在分子生物景观、细胞异质性、潜在治疗靶点提供了宝贵的视野。

Abstract

Part 1 Analysis of clinical characteristics of intravenous leiomyomatosis and proposing a classification system to guide surgical strategies

Background

Intravenous Leiomyomatosis (IVL) is a rare, histologically benign but biologically invasive tumor, it originates from the smooth muscle of the uterus or the wall of uterine veins. It often extends beyond the confines of the uterus, following the venous return pathway through the uterine veins, ovarian veins, or iliac veins into the inferior vena cava, and can even extend into the heart and pulmonary arteries, causing a series of clinical symptoms, which can be severe enough to lead to death. The disease is clinically rare and insidious, with diverse clinical manifestations and lacking specificity, making it prone to clinical misdiagnosis due to insufficient recognition by medical institutions, or being mistaken for gynecological and venous system malignant tumors or thrombosis, leading to delayed treatment. However, due to its rarity, understanding of IVL mainly comes from isolated case reports, small sample cohorts, and limited short-term follow-up literature. Consequently, there is insufficient data on disease characteristics and tumor prognosis, along with a lack of surgical experience.

Objectives

We described our experiences of Intravenous Leiomyomatosis (IVL) from our single center to investigate the clinical characteristics, surgical treatment outcomes, and overall prognosis of IVL. We analyzed the factors influencing recurrence or residual tumor progression and proposed a guiding classification to help for surgical management.

Materials and methods

We retrospectively collected data from patients who underwent surgical resection for intravenous leiomyomatosis (IVL) at Peaking Union Medical Hospital between January 2002 and December 2022. We gathered case information, perioperative parameters, and monitored the occurrence of postoperative complications and recurrences in the short and long term. Patients were divided into two groups based on complete resection and incomplete resection (tumor residual), and their postoperative outcomes were analyzed separately. The Kaplan-Meier method was used, and the log-rank test was employed to evaluate the association between recurrence or residual progression and various prognostic factors. Univariate Cox proportional hazard models were utilized to explore the impact of various patient-related and surgery-related variables on IVL recurrence or residual progression. Additionally, we proposed a new classification system and subsequently used it to collate and summarize relevant data.

Results

A total of 216 patients were included, with 92 (42.6%) in stage I, 39 (18.1%) in stage II, 76 (35.2%) in stage III, 9 (4.2%) in stage IV. The median follow-up was 26.34 months, during which 18 (9.7%) complete resection patients had recurrence and 12 (39%) incomplete resection patients had progression. Adjuvant therapy of aromatase inhibitor (AI) after surgery and the maximal diameter of tumor thrombus were associated with recurrence or residual progression. Whereas age and gonadotropin-releasing hormone agonist (GnRHa) after surgery were not associated factors. Significant statistical differences were observed in certain parameters such as patient symptoms, surgical duration, and blood loss among different stages.

Conclusions

This study represents the largest single-center report on intravenous leiomyomatosis (IVL) published to date, offering valuable summaries of clinical characteristics, long-term outcomes, and surgical expertise. Overall, surgical treatment of this condition appears to yield satisfactory results with low complication rates and minimal recurrence. The classification system we proposed enables the assessment of lesion severity, thereby aiding in surgical management guidance.

Part 2 Single-cell Sequencing Unveils Tumor Origin and Pathogenesis in Intravenous Leiomyomatosis

Background

Intravenous leiomyomatosis (IVL) is a rare neoplasm primarily affecting women of reproductive age, it exhibits biologically aggressive behavior characterized by vascular invasion. IVL displays unique histological and clinical traits, occupying a hybrid position between cancers and benign neoplasms, which renders it an attractive model for studying cancer development. The first part of this study summarizes and analyzes the clinical characteristics of IVL, raising questions about its tumor origin, the role of estrogen in tumor occurrence and development, and underlying pathogenic mechanisms that remain unclear. Various studies employing RNA sequencing analysis and reverse transcription-quantitative PCR have endeavored to elucidate the molecular pathological mechanisms underlying IVL. However, these studies still have limitations in identifying specific cell types and understanding the complexity of IVL tumor heterogeneity. To date, there has been no single-cell sequencing study of IVL conducted globally, and single-cell analysis techniques are crucial for investigating the origin of IVL and understanding the pathological mechanisms at the cellular and molecular levels.

Objectives

By employing single-cell analysis techniques, we aim to investigate the origin of intravenous leiomyomatosis (IVL), as well as the tumor microenvironment and specific pathogenic mechanisms underlying the disease. Additionally, we explore the potential targeted therapy of this rare tumor.

Materials and methods

We presented a comprehensive single-cell RNA sequencing analysis of 23 samples from 7 IVL patients. This included 19 tumor biopsy samples segmented by distance from the uteris into segment A (initial tumor tissue), segment B (tumor tissue in IVC), and segment C (tumor tissue near heart), as well as two uteri (segment N) and two venous wall (segment V) samples as normal/adjacent tissue controls. Single-cell suspensions were prepared from these samples, and single-cell cDNA libraries were sequenced. Through biochemical analysis, cells were clustered and identified. Specific analysis of smooth muscle cells (SMCs) and mesenchymal cells (MSCs) populations was conducted, employing various methods including cell proportion analysis, differential gene expression (DEGs) analysis, enrichment analysis, data mapping, gene set scoring, copy number variation (CNV) analysis, and lineage tracing based on mitochondrial gene mutations, to obtain molecular insights into the tumor's biology.

Results

We identified a subset of smooth muscle cells (SMCs) derived from the uterus, specifically expressing ESR1, PGR1, and IGF1, with high enrichment of transcriptional misregulation in cancer genes and activation of several cancer-related pathways. We also observed a transformation in mesenchymal stem cells (MSCs) and SMCs, highlighting the role of MSCs in tumor development. Our analysis of MSCs revealed several subsets enriched in tumor samples with activation of the EGFR (Epidermal Growth Factor Receptor) pathway while inactivation of the Estrogen pathway.

Conclusions

We confirmed the uterine origin of IVL and identified subsets of smooth muscle cells that grow independently of hormones. We identified genomic factors associated with cell apoptosis, angiogenesis, and carcinogenesis. Furthermore, we discovered that activation of IGF1 and EGFR (Epidermal Growth Factor Receptor) promotes tumor growth, providing a basis for the development of targeted therapies for this rare tumor. Our findings offer valuable insights into the complex molecular landscape, cellular heterogeneity, and potential therapeutic targets of IVL.