Pathological and molecular insights into intravenous leiomyomatosis: an integrative multi-omics study

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Due to its rarity, systematic studies on IVL are limited. We conducted a comprehensive multi-omics study, collecting tissues from IVL, uterine fibroid, and normal myometrium. Single-cell RNA sequencing analysis revealed a significant difference in cell composition between IVL and uterine fibroid. H&E staining demonstrated more frequent hydropic change and hyalinization, with decreased vascular density in IVL tissues compared to both normal myometrium and uterine fibroid. Proteomics analysis in 8 paired IVL and normal myometrium fresh frozen tissue identified differentially expressed proteins mainly enriched in focal adhesions and regulation of the actin cytoskeleton. The most frequently involved chromosomes included deletions in 10q22.2, 10q24.32, 13q14, and 13q21-31. Correlation analyses highlighted chromosome 10q as the most frequent cytoband, with corresponding proteins involved in regulating focal adhesions and the cytoskeleton. Integrated analysis between pathological and clinical characteristics indicated that chromosome 10q deletion and vascular morphology in IVL could serve as important markers predicting aggressive behavior. Our study illuminates the pathological and molecular changes associated with IVL, offering insights that may contribute to establishing new directions for IVL treatment. Intravenous leiomyomatosis multi-omics analysis chromosome 10q deletion vascular morphology aggressive behavior Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Intravenous leiomyomatosis (IVL) is an uncommon smooth muscle tumor characterized by histologically well-differentiated smooth muscle disease but exhibiting aggressive behavior 1 . Typically originating in the uterus, IVL extends along the venous system to the iliac vein, inferior vena cava (IVC), right heart, and occasionally the pulmonary artery, resulting in diverse clinical symptoms and, at times, sudden death. Traumatic surgery is imperative for the removal of extra-pelvic disease 2 . Two theories exist regarding the origin of IVL: one predominant theory suggests that IVL originates from uterine fibroid (UF), while the other posits that IVL stems from the smooth muscle cells (SMCs) of the vessel walls 3 – 5 . Limited research has been conducted on IVL tumorigenesis. Ordulu et al. classified IVL into cellular, vascular and usual type based on microscopic characteristics. Their array comparative genomic hybridization studies revealed frequent genetic alterations involving chromosomes 1p, 22q, 2q, 13q, and 14q 6 . Lu et al. utilized Sanger sequencing and short tandem repeat analysis, observed discordance in Mediator complex subunit 12 (MED12) mutation, microsatellite instability, and loss of heterozygosity between UF and IVL 7 . Other studies employing MED12 gene sequencing confirmed IVL as a distinct tumor from UF 8 , 9 . At the transcriptional level, Zhang et al. identified differentially expressed genes between IVL and UF, primarily enriched in the extracellular matrix, cell adhesion, and steroid hormone stimulus 10 . Despite these findings, comprehensive multi-omics studies investigating IVL tumorigenesis and development are lacking. In this investigation, we initiated single-cell RNA sequencing (scRNA-seq) to delineate the cell types in a paired set of UF and IVL fresh tissues. Subsequently, hematoxylin and eosin (H&E) as well as immunohistochemistry (IHC) staining were conducted on 23 paired IVL and normal myometrium paraffin-embedded samples. This aimed to delve deeper into the pathological changes characteristic of IVL, with results validated through an additional cohort. To unveil the molecular alterations in IVL, we employed integrated omics analyses, encompassing tandem mass tag (TMT)-based quantitative proteomics analysis and Whole-Exome Sequencing (WES). This comprehensive approach was applied to eight paired IVL samples (featuring extra-pelvic disease) and normal myometrium fresh frozen tissue. Finally, an integrated analysis was performed, bridging the gap between pathological observations and clinical characteristics. This study not only shed light on the pathological and molecular features associated with IVL tumorigenesis and tumor behavior but also charted new avenues for the treatment of this disease. Methods Ethics Our study adhered to the principles of the Declaration of Helsinki and received approval from the ethical committee of Zhongshan Hospital, Fudan University (Ethics Committee document number: B2021-488R). Written informed consent was obtained from all participating patients. Study Flowchart and Tissue Sample Collection The study's flowchart is illustrated in Fig. 1 . Patients pathologically diagnosed with IVL at Zhongshan Hospital, Fudan University, between 2014 and 2020 were included. For scRNA-seq, samples were acquired from freshly removed tissues, including one paired UF and IVL. TMT-based proteomic and WES analyses were performed on eight paired fresh frozen samples, comprising IVL lesions (both extracted from the IVC) and corresponding normal myometrium. These samples were collected during multidisciplinary surgical procedures and stored at -80°C until use. For H&E and IHC staining, 23 IVL and paired normal myometrium tissues were collected and embedded in paraffin. The validation cohort included 14 paired normal myometrium, uterine fibroid, and IVL tissues. All samples underwent reconfirmation by qualified pathologists, and comprehensive medical records were meticulously reviewed and collected. Single-cell RNA sequencing In brief, cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 1,000 cells per µL, using the Single Cell 3’ library and Gel Bead Kit v.3, following the manufacturer’s protocol. The process involved the generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification, and library construction, all in accordance with the manufacturer’s instructions. Library quantification was performed using Qubit before pooling, and the final library pool was sequenced on an Illumina Novaseq 6000 instrument. Data Processing of Single-cell RNA-seq from Chromium System The cellranger software (version 2.1.0) was utilized for mapping to the GRCh38 human genome, performing quality control, and counting reads of Ensembl genes using default parameters. Unsupervised clustering and visualization Unsupervised clustering was conducted using R with the Seurat package (version 2.2). Genes expressed in fewer than two cells were excluded. Cells with more than 200 genes and less than 10% mitochondrial genes were subjected to further processing. Subsequently, the coefficient of variation of genes was calculated using Seurat. Dimensionality reduction of the data was conducted through principal component analysis, focusing on the first 2000 genes with the highest variability. A k-nearest neighbor graph was constructed based on Euclidean distances within the space defined by the first 10 principal components. The Louvain Modularity optimization algorithm was employed to cluster the cells within the graph, and the resulting clusters were visualized using t-distributed Stochastic Neighbor Embedding (tSNE) projection. Cells expressing high levels of genes encoding hemoglobin were removed from the analysis. Marker gene identification and cell-type annotation The 'bimod' test implemented in the Seurat FindMarkers function was utilized to compute the differential expression of each cluster. Genes exhibiting a log2 average expression difference of 0.585 and a significance level of P < 0.05 were designated as marker genes. Canonical markers of established cell types were employed to annotate cell clusters. Seurat-Bimod statistical test was used to find differentially expressed genes between each group of cells and other groups of cells (FDR ≤ 0.05 and |log2 Fold Change| ≥ 1.5). The TopGO R package was utilized for Gene Ontology (GO) enrichment analysis of these significantly differentially expressed genes, while the Hypergeometric test in R was employed for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. GO terms and KEGG pathways that were significantly enriched were identified based on a threshold FDR (adjusted P-value) ≤ 0.05. TMT Protein Labeling and Bioinformatics Analysis Following the extraction of total protein from the samples, a portion of the protein was utilized for determining concentration and SDS-PAGE analysis, while another portion underwent trypsin hydrolysis and labeling. Equal amounts of each labeled protein sample were amalgamated for chromatographic separation, facilitating liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The qualitative and quantitative data obtained were subjected to analysis. Expression level analysis and functional analysis were conducted subsequent to quality evaluation and preprocessing. The functional annotation encompassed the use of several common data analysis tools, including GO and KEGG pathway analysis. Interaction analysis was then executed for the differentially expressed proteins (DEPs) identified. Furthermore, correlation analysis, expression pattern clustering, and Venn analysis were performed, and heat maps were generated to enhance data interpretation and visualization. Whole Exosome Sequencing Qualified DNA samples underwent random fragmentation into 150 to 220 bp fragments using Covaris. Subsequently, the Agilent SureSelect Human All Exon V6 kit was employed for library construction and capture. The library assembly involved various steps, including DNA end-joining, addition of polyA tails, incorporation of sequencing adapters, purification, magnetic bead capture, PCR amplification, and other processes. A thorough re-evaluation of raw data quality was conducted, considering parameters such as error rate, data volume, comparison rate, and coverage. The resulting high-quality sequences were aligned to the reference genome to detect sample variation information, which was subsequently analyzed. Integrated Multi-Omics Analysis of IVL To harmonize data across multiple platforms, we compiled a list of all genes exhibiting copy number variation (CNV) and their associated proteins. Correlation (Pearson P-value) was calculated, with significance set at P < 0.05. Enrichment analyses, including GO and KEGG pathway analyses, were performed for positively correlated CNVs/proteins. In exploring the potential molecular mechanisms of IVL tumorigenesis, we enriched the frequency of chromosome positions and analyzed the corresponding proteins within the altered CNVs/proteins. H&E and IHC staining Archival paraffin blocks from 23 IVL patients, along with paired normal myometrium samples, were meticulously chosen for this study. Sections of 4 µm thickness were obtained from the paraffin-embedded samples, and subsequent H&E and IHC staining procedures were conducted on these slides. For H&E staining, microscopic characteristics were thoroughly examined in each slide, encompassing the proportion of hydropic change and hyalinization, as well as vascular densities in the tumor parenchyma. Furthermore, all IVL cases were classified based on cellular, vascular, and usual morphology. The extent of these morphological features was scored as minimal ( 25%) following a prior study 6 . In the case of IHC staining, the final protein expression score was derived by multiplying the percentage of positive cells (PPC) with the intensity of staining (IS). PPC categories included: 0 = < 10% of positive cells, 1 = 10–25%, 2 = 25–50%, 3 = 50–75%, and 4 = ≥ 75% of positive cells. IS was classified as 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The cumulative score was then categorized as “low” for scores ranging from 0 to 3, “medium” for scores between 4 and 7, and “high” for scores from 8 to 12. Random fields under the 100x microscope were selectively chosen for vessel calculation. The specific protocol for this study is summarized in the Supplemental Methods. Results IVL and UF Exhibit Profound Differences in Cellular Composition through scRNA-seq To delve into the cellular heterogeneity between IVL and UF, scRNA-seq profiles were generated from a paired IVL and UF tissue sample. The results, illustrated in Figure 2A, unveiled the existence of 22 distinct clusters among high-quality cells. These 22 clusters were further categorized into eight subpopulations, encompassing fibroblasts, SMCs, endothelial cells, macrophages, T cells, monocytes, mast cells, and progenitor cells (Figure 2B). Examining the distribution in Figure 2C and Figure 2D, fibroblasts dominated the cellular landscape of the IVL lesion, constituting 84.73% of the identified cells. SMCs and endothelial cells accounted for 4.38% and 2.37%, respectively. In contrast, the UF lesion exhibited a distinct composition, with endothelial cells comprising 56.64% and SMCs making up 37.02%, while fibroblasts constituted only 5.35%. These findings underscore a substantial cellular component disparity between IVL and UF, characterized by reduced levels of SMCs and a markedly increased prevalence of fibroblasts in IVL. Moreover, the observation of limited endothelial cells in IVL suggests a potential hypovascular characteristic. The heat map in Figure 2E summarizes the top five expressed genes in each cluster. In conclusion, the single-cell analysis underscores the profound differences in cellular components between IVL and UF, emphasizing the necessity for a comprehensive exploration of the pathological and molecular changes associated with IVL. Pathomorphological alterations in IVL Subsequently, we scrutinized the pathomorphological distinctions between IVL and normal myometrium in a cohort of 23 IVL patients. The clinical attributes of these patients are succinctly outlined in Supplementary Table 1. Macroscopically, IVL exhibited a "worm-like" tissue appearance, softer in texture compared to both normal myometrium and UF (Figure 3A). Histologically, IVL was characterized as a benign smooth muscle tumor located within venous vascular spaces (Figure 3B). Pathomorphologically, numerous IVL tissues displayed hydropic changes and areas of hyalinization in proximity to the tumor parenchyma (Figure 3C/D). In rare instances, adipose tissue was observed in IVL, accompanied by focal hemorrhage and neutrophil infiltration (Figure 3E/F). Vascular morphology exhibited variability, with some instances featuring large vessels characterized by thick vascular walls (Figure 3G). Among the 23 IVL cases in our study, the prevailing morphology was vascular (16/23, 69.6%), followed by the cellular (2/23, 8.7%) and usual (2/23, 8.7%) types. Two patients exhibited a combination of cellular and vascular types, while one patient presented with both vascular and usual types. Utilizing these morphological characteristics, we initially compared the hydropic change, hyalinization, and vascular density in tumor parenchyma between IVL and normal myometrium. In IVL, 8 (34.8%), 10 (43.5%), and 5 (21.7%) patients exhibited hydropic changes as minimal, focal, and diffused, respectively, whereas only 4 patients (3 minimal and 1 focal) showed hydropic changes in normal myometrium. Hyalinization manifested as minimal, focal, and diffused in 7 (30.4%), 11 (47.8%), and 5 (21.7%) patients in IVL, respectively, while only 2 patients (1 minimal and 1 focal) displayed hydropic changes in normal myometrium. The findings imply that the proportions of hydropic change and hyalinization were significantly higher in IVL (Figure 3C/D). The median blood vessel counts in IVL and normal myometrium tissues were 10 (range 3 to 25) and 20 (range 11 to 30), respectively, suggesting that blood vessel density in IVL was lower than that in normal myometrium (P < 0.001) (Figure 3H). To further investigate whether these morphological characteristics differed between IVL and UF, 14 paired normal myometrium, UF, and IVL tissues were selected. The results also indicated that hydropic change and hyalinization were more frequently observed in IVL tissue than in both normal myometrium and uterine fibroids. Conversely, the vascular density was decreased in IVL tissues compared to both normal myometrium and uterine fibroids (Figure 3I). In summary, when compared with normal myometrium and uterine fibroids, the proportions of hydropic change and hyalinization areas were significantly increased, and the vascular density was decreased in IVL tissues. Proteomic alterations between IVL and paired normal myometrium To delve deeper into the molecular changes within IVL, we conducted TMT-based proteomic analysis and WES on eight paired IVL and normal myometrium tissues. The clinical characteristics of these eight patients are summarized in Supplementary Table 2. A total of 263 DEPs were identified, with 133 proteins (50.6%) being upregulated and 130 downregulated (Figure 4A). The volcano plot and heat map illustrate the distribution of these DEPs (Figure 4B/C and Supplementary Table 3). Functional annotation analysis using GO was performed to gain a better understanding of the biological functions of the DEPs. As depicted in Figure 4D, the most downregulated GO terms included cell-matrix adhesion, focal adhesion, and actin cytoskeleton, while the most upregulated GO terms included complement activation and extracellular exosome. Furthermore, KEGG enrichment analysis indicated that the DEPs are primarily involved in the regulation of actin cytoskeleton, extracellular matrix (ECM)-receptor interaction, and complement and coagulation cascades (Figure 4E and Supplementary Tables 4/5). The downregulated proteins in the protein-protein interaction (PPI) network included vinculin (VCL), ACTIN1, and ACTA2 (α-SMA), while the upregulated proteins included C3, C4B, APOA1, APOB, and FN1 (Figure 4F). Vinculin, a cytoskeletal protein associated with cell-cell and cell-matrix junctions, plays a crucial role in cell morphology and locomotion. To further validate the results of the proteomic analysis, IHC staining of Vinculin was performed on the 23 paired IVL and normal myometrium paraffin-embedded tissues. As shown in Figure 4G, the expression of Vinculin was significantly lower in IVL than in normal myometrium. Genetic variants between IVL and paired normal myometrium WES was conducted to investigate the genetic variants between IVL and the corresponding normal myometrium. As illustrated in Figure 5A, a total of 381 mutations were identified, with nonsynonymous mutations constituting the most frequent type (61.4%), followed by synonymous mutations (24.4%). Excluding synonymous mutations, a total of 239 potential driver mutations were identified, with a median of 25 mutations in each sample. The prominently mutated genes included PKD1, CAPN 15, ZNF90, SNAPC4, and MUC4 (Figure 5B), with a mutation frequency of 25.0% (2/8). Details of each mutated gene mentioned above are summarized in Supplementary Table 6. Subsequently, we analyzed CNVs between the two groups. The cytobands of somatic CNVs for each IVL sample are depicted in Figure 5C. As indicated, the most frequent deletions in chromosomes were concentrated in 10q22.2, 10q24.32, 13q14, and 13q21-31, with a frequency of 37.5%. The top CNV cytobands of gain were observed in 8q24, 11q13, 12q24, 19q13, and 20q13 (Figure 5D). Correlation analysis of TMT-based proteomics and WES To consolidate the findings from both proteomic and WES analyses, we conducted Pearson correlation analysis. The P-value was 0 (Figure 6A). GO analyses were subsequently employed to unravel the potential functions of these 47 CNVs-DEPs. As depicted in Figure 6B, ECM organization, cell-matrix adhesion, focal adhesion, and integrin binding were the primary enrichments. Subsequently, we delved into the cytoband distributions corresponding to these 47 altered CNVs-DEPs. Figure 6C reveals that the most frequent cytoband was located on chromosome 10q, with a frequency of 21.3% (10/47). Upon comparing protein quantitation between IVL and the paired normal myometrium, we identified significant differences in three out of the 10 proteins. The expression levels of GLUD1, PDLIM1, and SLK were notably decreased in IVL (Figure 6D and Figure 6E). Significantly, both PDLIM1 and SLK play roles in the regulation of focal adhesion. Intriguingly, in the TMT-based proteomic analysis, we observed that the gene for vinculin, a cytoskeletal protein enriched in focal adhesions, is also located on chromosome 10q. These findings suggest that a chromosome 10q deletion in IVL may lead to the downregulation of several proteins involved in the regulation of the actin cytoskeleton and focal adhesion. Integrated analysis of clinical and pathological characteristics To explore potential correlations between clinical characteristics and pathological changes, we scrutinized data from 23 IVL patients, stratified by the extent of tumor disease (as illustrated in Table 1). Among the 15 aggressive IVL patients (with extra-pelvic disease), 26.7% exhibited diffuse hyalinization, with an equal percentage showing hydropic changes. These manifestations were observed in 12.5% of patients with intra-pelvic disease. In the subgroup of eight patients with intra-pelvic disease, Vinculin high expression was identified in 37.5% of cases, while the proportion of Vinculin high expression was 13.3% (2/15) in aggressive IVL cases. Despite the small sample size limiting our ability to draw definitive conclusions about the relationship between IVL tumor extent and vinculin IHC staining score, the results suggest that lower vinculin protein expression levels may be positively correlated with a more advanced tumor stage. Table 1. Clinical characteristics and pathological changes in 23 IVL patients Case Tumor extension Age (y) hydropic change Hyalinization Vinculin expression Morphology 1 Intrapelvic 47 minimal minimal middle cellular 2 Intrapelvic 51 diffuse focal low vascular 3 Intrapelvic 37 focal minimal Low usual 4 Intrapelvic 47 focal focal middle cellular, vascular 5 Intrapelvic 47 minimal minimal high vascular 6 Intrapelvic 52 focal focal low cellular, vascular 7 Intrapelvic 59 minimal diffuse high vascular 8 Intrapelvic 34 minimal focal high vascular 9 Inferior Vena Cava 37 diffuse diffuse low vascular 10 Inferior Vena Cava 44 focal minimal high cellular 11 Inferior Vena Cava 48 diffuse focal high vascular 12 Inferior Vena Cava 50 focal focal middle vascular 13 Heart 30 diffuse focal low vascular 14 Heart 42 diffuse minimal low vascular 15 Heart 43 minimal focal low vascular 16 Heart 45 focal minimal low vascular, usual 17 Heart 47 minimal focal middle vascular 18 Heart 50 focal focal low vascular 19 Heart 55 minimal diffuse low usual 20 Heart 59 minimal minimal low vascular 21 Heart 62 focal diffuse middle vascular 22 Lung 45 focal diffuse low vascular 23 Lung 47 focal focal middle vascular Among eight non-aggressive IVL patients, cellular, vascular, and usual morphology were observed in 25%, 50%, and 12.5% of patients, respectively. Two patients exhibited both cellular and vascular features. In the group of 15 aggressive IVL patients, a total of 80% were categorized as having vascular morphology, with one patient displaying both vascular and usual features. These findings imply that vascular morphology constitutes a substantial proportion in aggressive IVL cases. Discussion IVL exhibits a higher degree of fibrosis and hyalinization compared to normal myometrium and UF In a recent study involving scRNA-seq analysis of five unpaired UF and normal myometrium samples, the cellular composition showed no significant differences between the two groups 11 . In our study, utilizing UF tissue as a control, we observed a striking difference in cellular composition between one paired IVL and UF tissue through scRNA-seq. Specifically, there was a notable decrease in SMCs and an increase in fibroblasts in IVL. These findings were corroborated by our proteomic analysis, demonstrating a significant decrease in α-SMA (SMCs marker) and an increase in FN1 (fibroblasts marker) in IVL tissue. It's worth noting that although we initially dissected three paired UF and IVL fresh tissues for scRNA-seq, only one was successfully tested due to the low percentage of tumor cells in IVL tissue. The other two attempts failed because of inadequate cellular numbers, potentially linked to the increased fibrotic and hyalinized nature of IVL tissue. HE staining revealed a significant increase in the proportions of hydropic change and hyalinization areas in IVL tissues compared to normal myometrium. While hydropic change and hyalinization are features also found in UF, we conducted a comparative analysis to discern differences between IVL and UF in terms of these histological features. The results further indicated a higher prevalence of hydropic change and hyalinization in IVL compared to both normal myometrium and UF. Given that hyalinization involves the transformation of a hard, cellular tumor into softer, acellular material, our findings provide insights into the clinical observation that IVL is noticeably softer than both normal myometrium and UF. One of the primary functions of fibroblasts is the synthesis of collagen proteins 12 , 13 . Collagen, a significant component of the extracellular matrix, contributes to the formation of a three-dimensional meshwork, providing physical support to cells and maintaining structural integrity in tissues 14 . Beyond its role in structural support, collagen plays a crucial role in various cellular processes, including cell migration, adhesion, angiogenesis, tissue development, and repair. Studies by Sahai E have highlighted that during migration, cancer cells utilize collagen fibers as tracks to leave the primary tumor, emphasizing the modulatory role of collagen in cell behavior 15 . Additionally, collagen has been identified as a novel driver of tumor invasion, promoting local invasion and metastasis 16 , 17 . In our study, fibroblasts were predominantly identified in IVL. Upon comparing IVL with normal myometrium tissues and UF, we observed higher proportions of hyalinization areas. These areas, characterized by the replacement of smooth muscle cells with collagen, exhibited a similar transformation in blood vessels within regions of hyaline necrosis 18 . Proteomic analysis further revealed an enrichment of upregulated proteins in the collagen-containing extracellular matrix. Consequently, we deduced that fibroblasts in IVL contribute to the increased production of collagen tissue, potentially correlating with the invasiveness of the tumor. In summary, our findings lead us to conclude that IVL exhibits more fibrotic and hyalinization features than normal myometrium and UF. These characteristics may be associated with the tumor development observed in IVL. IVL exhibits a poorer blood supply compared to normal myometrium and and UF Our scRNA-seq results indicated significantly lower proportions of endothelial cells in IVL compared to UF. Additionally, HE staining results confirmed a lower blood vessel density in IVL compared to normal myometrium and UF. These findings suggest that IVL experiences inadequate blood supply, potentially explaining the higher frequency of hyalinization in IVL. Insufficient blood supply within the myoma is recognized as a key factor contributing to hyalinization. Interestingly, our results diverge from a previous study suggesting that IVL tumors possess a strong ability to promote angiogenesis. The earlier study proposed that IVL tumors exhibit upregulated expression of proangiogenic factors, including GATA1, LIF, CXCL8, SH2D2A, et al 19 . To further investigate angiogenesis-associated pathway changes in IVL, we conducted a thorough analysis by identifying enriched angiogenesis-related terms in the GO analysis based on our proteomic data. DEPs associated with GO: 0016525 (negative regulation of angiogenesis) demonstrated significant upregulation in IVL. Notably, APOH (Apolipoprotein H; fold change = 1.72), THBS1 (Thrombospondin 1; fold change = 1.79), HRG (Histidine Rich Glycoprotein; fold change = 1.60), THBS4 (Thrombospondin 4; fold change = 2.19), and THBS2 (Thrombospondin 2; fold change = 1.68) exhibited substantial increases in expression. Conversely, DEPs associated with GO: 0045766 (positive regulation of angiogenesis) displayed significant downregulation in IVL. Noteworthy examples include HSPB1 (Heat Shock Protein Family B Member 1; fold change = 0.58), RRAS (RAS Related; fold change = 0.59), and AQP1 (Aquaporin 1; fold change = 0.61), all showing marked decreases in expression. Additionally, proteins involved in the general process of angiogenesis (GO: 0001525) underwent significant changes in IVL, with MYH9 (Myosin Heavy Chain 9; fold change = 0.66), FN1 (Fibronectin 1; fold change = 1.93), TGFBI (Transforming Growth Factor Beta Induced; fold change = 1.72), and APOD (Apolipoprotein D; fold change = 1.58) exhibiting altered expression levels. Collectively, these results align with our earlier findings, supporting the notion that IVL experiences a poor blood supply in comparison to normal myometrium. However, to validate and further elucidate this conclusion, additional studies are warranted. Dysregulation of focal adhesion and the actin cytoskeleton appears to be closely associated with the development of IVL. Our TMT-based proteomic analysis revealed that the most significantly downregulated signaling pathway was related to the regulation of the actin cytoskeleton and focal adhesion. Notably, key actin cytoskeletal proteins such as vinculin, α-actinin 1, Filamin A, and others exhibited downregulation. Focal adhesions, crucial for ECM interactions, play a fundamental role in maintaining tissue homeostasis and have been implicated in cancer development and pathogenesis 20 , 21 . The disassembly of focal adhesions in cells has been linked to cytoskeletal transmission, promotion of epithelial-mesenchymal transition, and induction of cell migration 22 – 24 , all of which contribute to cancer formation and progression. A prior study conducted RNA-seq to explore differentially expressed genes between IVL and normal uterine muscle tissues. Consistent with our proteomic findings, the results from this study also suggested that IVL may disrupt the homeostasis of gene networks involved in ECM and cytoskeleton regulation 10 . These collective observations underscore the potential significance of focal adhesion and actin cytoskeletal dysregulation in the pathogenesis of IVL, emphasizing the need for further investigation into these molecular mechanisms. Chromosome 10q deletions and vascular morphology could be important markers predicting the aggressive behavior of IVL WES outcomes revealed an enrichment of chromosome deletions at specific loci, including 10q22.2, 10q24.32, 13q14, and 13q21-31, within IVL tissues. Correlation analyses unveiled that the deletion of chromosome 10q in IVL might lead to the downregulation of proteins crucial in the regulation of the actin cytoskeleton and focal adhesion, which could play a significant role in IVL tumorigenesis. The validity of these findings was substantiated through vinculin IHC staining. Additionally, our investigation unveiled a correlation between lower vinculin protein expression and the distinct vascular morphology with the aggressive behavior observed in IVL. Chromosome 10q deletions have been implicated in various diseases, including cutaneous T-cell lymphoma 25 , low-grade gliomas 26 , and prostate cancer 27 . A prior study by Ordulu et al. further supported the association of a 10q deletion in IVL lesions 6 , 28 . In Ordulu's research, hierarchical clustering analysis identified three groups, with all IVL instances in group 3 (n = 5) exhibiting a deletion on 10q. Notably, 80% of IVL cases in group 3 displayed aggressive behavior. In our study, the removal of all eight IVL tissues for TMT-based proteomic and WES from the inferior vena cava already signaled their aggressive nature. Furthermore, Ordulu's study highlighted that 80% of cases with a vascular morphology among the 5 IVL instances associated with recurrence, aligning with our results where 80% (12 out of 15) of aggressive IVL patients exhibited vascular morphology. Collectively, these findings strongly suggest that chromosome 10q deletions and vascular morphology serve as crucial markers for predicting the aggressive behavior of IVL. Conclusion Our study sought to investigate the pathological and molecular alterations in IVL patients through a comprehensive multi-omics approach. However, the study is subject to several limitations that warrant attention in future research. Firstly, the impact of our findings is constrained by the availability of only one paired IVL and UF sample for scRNA-seq. Secondly, to prevent the overinterpretation of IVL origin, further validation of WES results, including genetic variations, is essential with a larger sample size. Lastly, additional basic research is imperative to enhance our understanding of the origin and development of IVL. In summary, this pioneering study conducted an integrated analysis, incorporating scRNA-seq, H&E, and IHC staining, TMT-based quantitative proteomics analysis, and WES of IVL. Our investigation unveiled both the pathomorphological and molecular characteristics in IVL, paving the way for new avenues in basic research and clinical treatment for IVL. Abbreviations IVL Intravenous leiomyomatosis IVC: Inferior vena cava UF Uterine fibroid SMCs Smooth muscle cells MED12 Mediator complex subunit 12 scRNA-seq Single-cell RNA sequencing H&E Hematoxylin and eosin IHC Immunohistochemistry TMT Tandem mass tag WES Whole-Exome Sequencing tSNE t-distributed Stochastic Neighbor Embedding GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes DEPs Differentially expressed proteins CNV Copy number variation PPC Percentage of positive cells IS Intensity of staining ECM Extracellular matrix Declarations Author Contributions Jiarong Zhang, Yingyong Hou and Chunsheng Wang designed the study; Sheng Yin, Peipei Shi, Jing Han, Aimin Ren, Li Ma, Wenbin Tang, Hua Li, Wenxue Liu, Sihui Yu and Tingting Li collected the data; Sheng Yin, Peipei Shi and Jing Han analyzed the data; Sheng Yin and Peipei Shi wrote the original draft; Jiarong Zhang revised the manuscript. All authors approved the final paper. Funding This study was funded by the Zhongshan Development Program (XK-066). Availability of data and materials The primary data of genetics and proteomics has been deposited to the GSA-human database (https://ngdc.cncb.ac.cn/gsa-human/; ID: PRJCA015471) and OMIX (https://ngdc.cncb.ac.cn/omix/; OMIX003497), which is available from the corresponding author on request. Ethics approval and consent to participate The study was approval by the ethical committee of Zhongshan Hospital, Fudan University (Ethics Committee document number: B2021-488R). Written informed consent was obtained from all participating patients. Consent for publication Not applicable. Competing interests All authors declare no competing interests. Acknowledge We thank all the patients enrolled in this study. References CP HL. K. Intravenous leiomyomatosis with cardiac extension: tumor thrombectomy through an abdominal approach. J Vasc Surg. 2000;31(35):1046–51. 10.1067/mva.2000.10460 . Li H, et al. 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MED12 exon 2 mutation is uncommon in intravenous leiomyomatosis: clinicopathologic features and molecular study. Hum Pathol. 2020;99:36–42. 10.1016/j.humpath.2020.03.011 . Matsubara A, et al. Prevalence of MED12 mutations in uterine and extrauterine smooth muscle tumours. Histopathology. 2013;62:657–61. 10.1111/his.12039 . Zhang X, et al. Identification of the molecular relationship between intravenous leiomyomatosis and uterine myoma using RNA sequencing. Sci Rep. 2019;9:1442. 10.1038/s41598-018-37452-3 . Goad J, et al. Single-cell sequencing reveals novel cellular heterogeneity in uterine leiomyomas. Hum Reprod. 2022;37:2334–49. 10.1093/humrep/deac183 . Kalluri R, Zeisberg M. Fibroblasts in cancer. Nat Rev Cancer. 2006;6:392–401. Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol. 2002;3:349–63. De Martino D, Bravo-Cordero JJ. Collagens in Cancer: Structural Regulators and Guardians of Cancer Progression. Cancer Res. 2023;83:1386–92. 10.1158/0008-5472.CAN-22-2034 . Sahai E, et al. Simultaneous imaging of GFP, CFP and collagen in tumors in vivo using multiphoton microscopy. BMC Biotechnol. 2005;5:14. Wishart AL, et al. Decellularized extracellular matrix scaffolds identify full-length collagen VI as a driver of breast cancer cell invasion in obesity and metastasis. Sci Adv. 2020;6. 10.1126/sciadv.abc3175 . Graf F, Horn P, Ho AD, Boutros M, Maercker C. The extracellular matrix proteins type I collagen, type III collagen, fibronectin, and laminin 421 stimulate migration of cancer cells. FASEB J. 2021;35:e21692. 10.1096/fj.202002558RR . Robboy SJ, Bentley RC, Butnor K, Anderson MC. Pathology and pathophysiology of uterine smooth-muscle tumors. Environ Health Perspect. 2000;108(Suppl 5):779–84. Wang W, et al. Intravenous leiomyomatosis is inclined to a solid entity different from uterine leiomyoma based on RNA-seq analysis with RT-qPCR validation. Cancer Med. 2020;9:4581–92. 10.1002/cam4.3098 . Pickup MW, Mouw JK, Weaver VM. The extracellular matrix modulates the hallmarks of cancer. EMBO Rep. 2014;15:1243–53. 10.15252/embr.201439246 . Grashoff C, et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature. 2010;466:263–6. 10.1038/nature09198 . Mouneimne G, et al. Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion. Cancer Cell. 2012;22:615–30. 10.1016/j.ccr.2012.09.027 . Zhang H, et al. Loss of profilin 2 contributes to enhanced epithelial-mesenchymal transition and metastasis of colorectal cancer. Int J Oncol. 2018;53:1118–28. 10.3892/ijo.2018.4475 . Schumacher S, Vazquez Nunez R, Biertümpfel C, Mizuno N. Bottom-up reconstitution of focal adhesion complexes. Febs j. 2022;289:3360–73. 10.1111/febs.16023 . Cristofoletti C, et al. Comprehensive analysis of PTEN status in Sezary syndrome. Blood. 2013;122:3511–20. 10.1182/blood-2013-06-510578 . van Thuijl HF, et al. Spatial and temporal evolution of distal 10q deletion, a prognostically unfavorable event in diffuse low-grade gliomas. Genome Biol. 2014;15:471. 10.1186/s13059-014-0471-6 . Mao X, et al. Distinct genomic alterations in prostate cancers in Chinese and Western populations suggest alternative pathways of prostate carcinogenesis. Cancer Res. 2010;70:5207–12. 10.1158/0008-5472.Can-09-4074 . Buza N, et al. Recurrent chromosomal aberrations in intravenous leiomyomatosis of the uterus: high-resolution array comparative genomic hybridization study. Hum Pathol. 2014;45:1885–92. Supplementary Files Supplementarys.docx Cite Share Download PDF Status: Published Journal Publication published 26 Feb, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 05 May, 2024 Reviewers invited by journal 28 Apr, 2024 Editor assigned by journal 04 Apr, 2024 First submitted to journal 02 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4210065","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":296421237,"identity":"491d6390-4d96-4733-bfad-de45d79ac164","order_by":0,"name":"Sheng 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05:24:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4210065/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4210065/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12967-024-05919-9","type":"published","date":"2025-02-26T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55796395,"identity":"7cef209d-eaa7-4480-a3b1-ad4bdbc9814c","added_by":"auto","created_at":"2024-05-03 11:02:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":293982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the experimental design\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4210065/v1/1a57c132923e8217f81c9893.png"},{"id":55796396,"identity":"27eb0727-db75-47cd-a373-e11f80d4cde4","added_by":"auto","created_at":"2024-05-03 11:02:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4732388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSingle-cell atlas of IVL and UF.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) tSNE plot illustrating clusters of high-quality cells from IVL and UF.\u003c/p\u003e\n\u003cp\u003e(B) Identification of cell lineages based on gene expression.\u003c/p\u003e\n\u003cp\u003e(C) Annotation of cell clusters per sample.\u003c/p\u003e\n\u003cp\u003e(D) Distribution of cells per sample.\u003c/p\u003e\n\u003cp\u003e(E) Heatmap of gene expression analyzed by single-cell RNA sequencing, presenting key markers for each cell type.\u003c/p\u003e\n\u003cp\u003eSMC, Smooth Muscle Cell.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4210065/v1/7417546df31a40485fa9bc8a.png"},{"id":55796723,"identity":"277ff99a-dc0a-4490-8d6c-1c49d0886a6c","added_by":"auto","created_at":"2024-05-03 11:10:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6529800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathological characteristics in IVL.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Gross appearance of IVL.\u003c/p\u003e\n\u003cp\u003e(B) Typical microscopic image of IVL. An intravascular tumour plug.\u003c/p\u003e\n\u003cp\u003e(C) Edema change in IVL. The proportions of the edema area between normal myometrium and IVL.\u003c/p\u003e\n\u003cp\u003e(D) Hyaline degeneration in IVL. The proportions of the hyaline degeneration between normal myometrium and IVL.\u003c/p\u003e\n\u003cp\u003e(E) The adipose tissue in IVL.\u003c/p\u003e\n\u003cp\u003e(F) Focal hemorrhage and neutrophils in IVL.\u003c/p\u003e\n\u003cp\u003e(G) Thick vascular wall in IVL.\u003c/p\u003e\n\u003cp\u003e(H) Vascular number between normal myometrium and IVL.\u003c/p\u003e\n\u003cp\u003e(I) The proportions of the edema area, hyaline degeneration and vascular number between N, UF and IVL.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4210065/v1/06566040fd0184be1f21a50e.png"},{"id":55796399,"identity":"4efedf90-58aa-4257-8b61-42a7940e150a","added_by":"auto","created_at":"2024-05-03 11:02:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3602515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the proteomics of IVL patients.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Visualization of 263 identified proteins, with orange indicating upregulated proteins and green indicating downregulated proteins.\u003c/p\u003e\n\u003cp\u003e(B) Volcano Plot depicting significantly upregulated (red dots) and downregulated (blue dots) proteins, with gray dots representing proteins with no significant difference. The x-axis represents the fold change (Log2) between two groups, and the y-axis represents the p-value (-Log10) to illustrate the significance of differences. The top 10 upregulated and downregulated DEPs are labeled.\u003c/p\u003e\n\u003cp\u003e(C) Heatmap for hierarchical clustering analysis of DEPs. Each row represents a protein, and each column indicates a sample. Colors indicate expression levels (red for high, purple for low). Violin plots depict the probability distribution of expression values. \"+\" denotes the median value, and the vertical axis represents protein expression.\u003c/p\u003e\n\u003cp\u003e(D) GO Pathway Analyses of the top 15 enriched terms, classified by biological process (green), cellular component (blue), and molecular function (red).\u003c/p\u003e\n\u003cp\u003e(E) Top 20 enriched KEGG terms, categorized into cellular processes (green), environmental information processing (purple), human disease (orange), and organismal systems (yellow).\u003c/p\u003e\n\u003cp\u003e(F) PPI Network of the top 25 DEPs. DEPs are represented as dots, and edges between proteins indicate potential interactions. Dot size corresponds to combined interaction scores identified by Cytoscape.\u003c/p\u003e\n\u003cp\u003e(G) Vinculin staining in IVL and normal myometrium.\u003c/p\u003e\n\u003cp\u003e(H) Vinculin IHC staining score analysis.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4210065/v1/f5c4d93dfa757025dea55c8e.png"},{"id":55796401,"identity":"650d6472-2302-4a26-946c-929df4c41e9f","added_by":"auto","created_at":"2024-05-03 11:02:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3707055,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenomic profiles based on WES.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Representation of different mutational events in all IVL lesions.\u003c/p\u003e\n\u003cp\u003e(B) Identification of the top 5 mutated genes corresponding to 8 samples.\u003c/p\u003e\n\u003cp\u003e(C) Characteristics of genome-wide Copy Number Variations (CNVs) distribution in 8 samples, with a pooled analysis at the bottom. Red, blue, and green indicate amplification, deletion, and no change, respectively. X-axis represents chromosome cytoband, and Y-axis represents copy ratio (log2).\u003c/p\u003e\n\u003cp\u003e(D) Overall chromosomal distribution of CNVs, where blue represents deletion and red represents amplification.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4210065/v1/a5f10845daca05fb7a98db3c.png"},{"id":55796397,"identity":"cfd26746-5d08-4f64-902d-a888fb0bc3ea","added_by":"auto","created_at":"2024-05-03 11:02:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1436052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation analysis of TMT-based Quantitative Proteomics and whole-exosome sequencing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Heatmap illustrating 47 positively related CNVs-DEPs.\u003c/p\u003e\n\u003cp\u003e(B) Identification of the top 30 enriched GO terms related to CNVs-DEPs.\u003c/p\u003e\n\u003cp\u003e(C) Distribution of cytobands associated with CNVs-DEPs.\u003c/p\u003e\n\u003cp\u003e(D) Presentation of 10 CNVs-DEPs with significant protein quantitation.\u003c/p\u003e\n\u003cp\u003e(E) Quantification of the expression of three proteins (GLUD1, PDLIM1, and SLK) between IVL and normal myometrium.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4210065/v1/05b839e425bab695b5bc3032.png"},{"id":77622911,"identity":"e9586130-84f9-481d-95c8-db50509eeb08","added_by":"auto","created_at":"2025-03-03 16:10:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":24620719,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4210065/v1/2ff1371d-33fc-4f1b-a4ec-16178e61077c.pdf"},{"id":55796402,"identity":"4eceb913-0382-4abe-8511-ae2e0c49fb52","added_by":"auto","created_at":"2024-05-03 11:02:20","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":39109,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarys.docx","url":"https://assets-eu.researchsquare.com/files/rs-4210065/v1/452e431e7a568e25b0af5f3a.docx"}],"financialInterests":"","formattedTitle":"Pathological and molecular insights into intravenous leiomyomatosis: an integrative multi-omics study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIntravenous leiomyomatosis (IVL) is an uncommon smooth muscle tumor characterized by histologically well-differentiated smooth muscle disease but exhibiting aggressive behavior \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Typically originating in the uterus, IVL extends along the venous system to the iliac vein, inferior vena cava (IVC), right heart, and occasionally the pulmonary artery, resulting in diverse clinical symptoms and, at times, sudden death. Traumatic surgery is imperative for the removal of extra-pelvic disease \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTwo theories exist regarding the origin of IVL: one predominant theory suggests that IVL originates from uterine fibroid (UF), while the other posits that IVL stems from the smooth muscle cells (SMCs) of the vessel walls \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Limited research has been conducted on IVL tumorigenesis. Ordulu et al. classified IVL into cellular, vascular and usual type based on microscopic characteristics. Their array comparative genomic hybridization studies revealed frequent genetic alterations involving chromosomes 1p, 22q, 2q, 13q, and 14q \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Lu et al. utilized Sanger sequencing and short tandem repeat analysis, observed discordance in Mediator complex subunit 12 (MED12) mutation, microsatellite instability, and loss of heterozygosity between UF and IVL \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Other studies employing MED12 gene sequencing confirmed IVL as a distinct tumor from UF \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. At the transcriptional level, Zhang et al. identified differentially expressed genes between IVL and UF, primarily enriched in the extracellular matrix, cell adhesion, and steroid hormone stimulus \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Despite these findings, comprehensive multi-omics studies investigating IVL tumorigenesis and development are lacking.\u003c/p\u003e \u003cp\u003eIn this investigation, we initiated single-cell RNA sequencing (scRNA-seq) to delineate the cell types in a paired set of UF and IVL fresh tissues. Subsequently, hematoxylin and eosin (H\u0026amp;E) as well as immunohistochemistry (IHC) staining were conducted on 23 paired IVL and normal myometrium paraffin-embedded samples. This aimed to delve deeper into the pathological changes characteristic of IVL, with results validated through an additional cohort. To unveil the molecular alterations in IVL, we employed integrated omics analyses, encompassing tandem mass tag (TMT)-based quantitative proteomics analysis and Whole-Exome Sequencing (WES). This comprehensive approach was applied to eight paired IVL samples (featuring extra-pelvic disease) and normal myometrium fresh frozen tissue. Finally, an integrated analysis was performed, bridging the gap between pathological observations and clinical characteristics. This study not only shed light on the pathological and molecular features associated with IVL tumorigenesis and tumor behavior but also charted new avenues for the treatment of this disease.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics\u003c/h2\u003e \u003cp\u003e Our study adhered to the principles of the Declaration of Helsinki and received approval from the ethical committee of Zhongshan Hospital, Fudan University (Ethics Committee document number: B2021-488R). Written informed consent was obtained from all participating patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStudy Flowchart and Tissue Sample Collection\u003c/h2\u003e \u003cp\u003eThe study's flowchart is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Patients pathologically diagnosed with IVL at Zhongshan Hospital, Fudan University, between 2014 and 2020 were included. For scRNA-seq, samples were acquired from freshly removed tissues, including one paired UF and IVL. TMT-based proteomic and WES analyses were performed on eight paired fresh frozen samples, comprising IVL lesions (both extracted from the IVC) and corresponding normal myometrium. These samples were collected during multidisciplinary surgical procedures and stored at -80\u0026deg;C until use. For H\u0026amp;E and IHC staining, 23 IVL and paired normal myometrium tissues were collected and embedded in paraffin. The validation cohort included 14 paired normal myometrium, uterine fibroid, and IVL tissues. All samples underwent reconfirmation by qualified pathologists, and comprehensive medical records were meticulously reviewed and collected.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSingle-cell RNA sequencing\u003c/h2\u003e \u003cp\u003eIn brief, cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 1,000 cells per \u0026micro;L, using the Single Cell 3\u0026rsquo; library and Gel Bead Kit v.3, following the manufacturer\u0026rsquo;s protocol. The process involved the generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification, and library construction, all in accordance with the manufacturer\u0026rsquo;s instructions. Library quantification was performed using Qubit before pooling, and the final library pool was sequenced on an Illumina Novaseq 6000 instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData Processing of Single-cell RNA-seq from Chromium System\u003c/h2\u003e \u003cp\u003eThe cellranger software (version 2.1.0) was utilized for mapping to the GRCh38 human genome, performing quality control, and counting reads of Ensembl genes using default parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eUnsupervised clustering and visualization\u003c/h2\u003e \u003cp\u003eUnsupervised clustering was conducted using R with the Seurat package (version 2.2). Genes expressed in fewer than two cells were excluded. Cells with more than 200 genes and less than 10% mitochondrial genes were subjected to further processing. Subsequently, the coefficient of variation of genes was calculated using Seurat. Dimensionality reduction of the data was conducted through principal component analysis, focusing on the first 2000 genes with the highest variability. A k-nearest neighbor graph was constructed based on Euclidean distances within the space defined by the first 10 principal components. The Louvain Modularity optimization algorithm was employed to cluster the cells within the graph, and the resulting clusters were visualized using t-distributed Stochastic Neighbor Embedding (tSNE) projection. Cells expressing high levels of genes encoding hemoglobin were removed from the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMarker gene identification and cell-type annotation\u003c/h2\u003e \u003cp\u003eThe 'bimod' test implemented in the Seurat FindMarkers function was utilized to compute the differential expression of each cluster. Genes exhibiting a log2 average expression difference of 0.585 and a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were designated as marker genes. Canonical markers of established cell types were employed to annotate cell clusters. Seurat-Bimod statistical test was used to find differentially expressed genes between each group of cells and other groups of cells (FDR\u0026thinsp;\u0026le;\u0026thinsp;0.05 and |log2 Fold Change| \u0026ge; 1.5). The TopGO R package was utilized for Gene Ontology (GO) enrichment analysis of these significantly differentially expressed genes, while the Hypergeometric test in R was employed for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. GO terms and KEGG pathways that were significantly enriched were identified based on a threshold FDR (adjusted P-value)\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTMT Protein Labeling and Bioinformatics Analysis\u003c/h2\u003e \u003cp\u003eFollowing the extraction of total protein from the samples, a portion of the protein was utilized for determining concentration and SDS-PAGE analysis, while another portion underwent trypsin hydrolysis and labeling. Equal amounts of each labeled protein sample were amalgamated for chromatographic separation, facilitating liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The qualitative and quantitative data obtained were subjected to analysis. Expression level analysis and functional analysis were conducted subsequent to quality evaluation and preprocessing. The functional annotation encompassed the use of several common data analysis tools, including GO and KEGG pathway analysis. Interaction analysis was then executed for the differentially expressed proteins (DEPs) identified. Furthermore, correlation analysis, expression pattern clustering, and Venn analysis were performed, and heat maps were generated to enhance data interpretation and visualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWhole Exosome Sequencing\u003c/h2\u003e \u003cp\u003eQualified DNA samples underwent random fragmentation into 150 to 220 bp fragments using Covaris. Subsequently, the Agilent SureSelect Human All Exon V6 kit was employed for library construction and capture. The library assembly involved various steps, including DNA end-joining, addition of polyA tails, incorporation of sequencing adapters, purification, magnetic bead capture, PCR amplification, and other processes. A thorough re-evaluation of raw data quality was conducted, considering parameters such as error rate, data volume, comparison rate, and coverage. The resulting high-quality sequences were aligned to the reference genome to detect sample variation information, which was subsequently analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIntegrated Multi-Omics Analysis of IVL\u003c/h2\u003e \u003cp\u003eTo harmonize data across multiple platforms, we compiled a list of all genes exhibiting copy number variation (CNV) and their associated proteins. Correlation (Pearson P-value) was calculated, with significance set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Enrichment analyses, including GO and KEGG pathway analyses, were performed for positively correlated CNVs/proteins. In exploring the potential molecular mechanisms of IVL tumorigenesis, we enriched the frequency of chromosome positions and analyzed the corresponding proteins within the altered CNVs/proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E and IHC staining\u003c/h2\u003e \u003cp\u003eArchival paraffin blocks from 23 IVL patients, along with paired normal myometrium samples, were meticulously chosen for this study. Sections of 4 \u0026micro;m thickness were obtained from the paraffin-embedded samples, and subsequent H\u0026amp;E and IHC staining procedures were conducted on these slides. For H\u0026amp;E staining, microscopic characteristics were thoroughly examined in each slide, encompassing the proportion of hydropic change and hyalinization, as well as vascular densities in the tumor parenchyma. Furthermore, all IVL cases were classified based on cellular, vascular, and usual morphology. The extent of these morphological features was scored as minimal (\u0026lt;\u0026thinsp;5%), focal (5\u0026ndash;24%), or diffuse (\u0026gt;\u0026thinsp;25%) following a prior study \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In the case of IHC staining, the final protein expression score was derived by multiplying the percentage of positive cells (PPC) with the intensity of staining (IS). PPC categories included: 0\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;10% of positive cells, 1\u0026thinsp;=\u0026thinsp;10\u0026ndash;25%, 2\u0026thinsp;=\u0026thinsp;25\u0026ndash;50%, 3\u0026thinsp;=\u0026thinsp;50\u0026ndash;75%, and 4\u0026thinsp;=\u0026thinsp;\u0026ge;\u0026thinsp;75% of positive cells. IS was classified as 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). The cumulative score was then categorized as \u0026ldquo;low\u0026rdquo; for scores ranging from 0 to 3, \u0026ldquo;medium\u0026rdquo; for scores between 4 and 7, and \u0026ldquo;high\u0026rdquo; for scores from 8 to 12. Random fields under the 100x microscope were selectively chosen for vessel calculation.\u003c/p\u003e \u003cp\u003eThe specific protocol for this study is summarized in the Supplemental Methods.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIVL and UF Exhibit Profound Differences in Cellular Composition through scRNA-seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delve into the cellular heterogeneity between IVL and UF, scRNA-seq profiles were generated from a paired IVL and UF tissue sample. The results, illustrated in Figure 2A, unveiled the existence of 22 distinct clusters among high-quality cells. These 22 clusters were further categorized into eight subpopulations, encompassing fibroblasts, SMCs, endothelial cells, macrophages, T cells, monocytes, mast cells, and progenitor cells (Figure 2B).\u0026nbsp;Examining the distribution in Figure 2C and Figure 2D, fibroblasts dominated the cellular landscape of the IVL lesion, constituting 84.73% of the identified cells. SMCs and endothelial cells accounted for 4.38% and 2.37%, respectively. In contrast, the UF lesion exhibited a distinct composition, with endothelial cells comprising 56.64% and SMCs making up 37.02%, while fibroblasts constituted only 5.35%. These findings underscore a substantial cellular component disparity between IVL and UF, characterized by reduced levels of SMCs and a markedly increased prevalence of fibroblasts in IVL. Moreover, the observation of limited endothelial cells in IVL suggests a potential hypovascular characteristic. The heat map in Figure 2E summarizes the top five expressed genes in each cluster. In conclusion, the single-cell analysis underscores the profound differences in cellular components between IVL and UF, emphasizing the necessity for a comprehensive exploration of the pathological and molecular changes associated with IVL.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePathomorphological\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ealterations in IVL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, we scrutinized the pathomorphological distinctions between IVL and normal myometrium in a cohort of 23 IVL patients. The clinical attributes of these patients are succinctly outlined in Supplementary Table 1. Macroscopically, IVL exhibited a \u0026quot;worm-like\u0026quot; tissue appearance, softer in texture compared to both normal myometrium and UF (Figure 3A). Histologically, IVL was characterized as a benign smooth muscle tumor located within venous vascular spaces (Figure 3B). Pathomorphologically, numerous IVL tissues displayed hydropic changes and areas of hyalinization in proximity to the tumor parenchyma (Figure 3C/D). In rare instances, adipose tissue was observed in IVL, accompanied by focal hemorrhage and neutrophil infiltration (Figure 3E/F). Vascular morphology exhibited variability, with some instances featuring large vessels characterized by thick vascular walls (Figure 3G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the 23 IVL cases in our study, the prevailing morphology was vascular (16/23, 69.6%), followed by the cellular (2/23, 8.7%) and usual (2/23, 8.7%) types. Two patients exhibited a combination of cellular and vascular types, while one patient presented with both vascular and usual types.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUtilizing these morphological characteristics, we initially compared the hydropic change, hyalinization, and vascular density in tumor parenchyma between IVL and normal myometrium. In IVL, 8 (34.8%), 10 (43.5%), and 5 (21.7%) patients exhibited hydropic changes as minimal, focal, and diffused, respectively, whereas only 4 patients (3 minimal and 1 focal) showed hydropic changes in normal myometrium. Hyalinization manifested as minimal, focal, and diffused in 7 (30.4%), 11 (47.8%), and 5 (21.7%) patients in IVL, respectively, while only 2 patients (1 minimal and 1 focal) displayed hydropic changes in normal myometrium. The findings imply that the proportions of hydropic change and hyalinization were significantly higher in IVL (Figure 3C/D). The median blood vessel counts in IVL and normal myometrium tissues were 10 (range 3 to 25) and 20 (range 11 to 30), respectively, suggesting that blood vessel density in IVL was lower than that in normal myometrium (P \u0026lt; 0.001) (Figure 3H). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further investigate whether these morphological characteristics differed between IVL and UF, 14 paired normal myometrium, UF, and IVL tissues were selected. The results also indicated that hydropic change and hyalinization were more frequently observed in IVL tissue than in both normal myometrium and uterine fibroids. Conversely, the vascular density was decreased in IVL tissues compared to both normal myometrium and uterine fibroids (Figure 3I). In summary, when compared with normal myometrium and uterine fibroids, the proportions of hydropic change and hyalinization areas were significantly increased, and the vascular density was decreased in IVL tissues.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomic alterations between IVL and paired normal myometrium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delve deeper into the molecular changes within IVL, we conducted TMT-based proteomic analysis and WES on eight paired IVL and normal myometrium tissues. The clinical characteristics of these eight patients are summarized in Supplementary Table 2. A total of 263 DEPs were identified, with 133 proteins (50.6%) being upregulated and 130 downregulated (Figure 4A). The volcano plot and heat map illustrate the distribution of these DEPs (Figure 4B/C and Supplementary Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunctional annotation analysis using GO was performed to gain a better understanding of the biological functions of the DEPs. As depicted in Figure 4D, the most downregulated GO terms included cell-matrix adhesion, focal adhesion, and actin cytoskeleton, while the most upregulated GO terms included complement activation and extracellular exosome. Furthermore, KEGG enrichment analysis indicated that the DEPs are primarily involved in the regulation of actin cytoskeleton, extracellular matrix (ECM)-receptor interaction, and complement and coagulation cascades (Figure 4E and Supplementary Tables 4/5). The downregulated proteins in the protein-protein interaction (PPI) network included vinculin (VCL), ACTIN1, and ACTA2 (\u0026alpha;-SMA), while the upregulated proteins included C3, C4B, APOA1, APOB, and FN1 (Figure 4F). Vinculin, a cytoskeletal protein associated with cell-cell and cell-matrix junctions, plays a crucial role in cell morphology and locomotion. To further validate the results of the proteomic analysis, IHC staining of Vinculin was performed on the 23 paired IVL and normal myometrium paraffin-embedded tissues. As shown in Figure 4G, the expression of Vinculin was significantly lower in IVL than in normal myometrium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic variants between IVL and paired normal myometrium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWES was conducted to investigate the genetic variants between IVL and the corresponding normal myometrium. As illustrated in Figure 5A, a total of 381 mutations were identified, with nonsynonymous mutations constituting the most frequent type (61.4%), followed by synonymous mutations (24.4%). Excluding synonymous mutations, a total of 239 potential driver mutations were identified, with a median of 25 mutations in each sample. The prominently mutated genes included PKD1, CAPN 15, ZNF90, SNAPC4, and MUC4 (Figure 5B), with a mutation frequency of 25.0% (2/8). Details of each mutated gene mentioned above are summarized in Supplementary Table 6.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubsequently, we analyzed CNVs between the two groups. The cytobands of somatic CNVs for each IVL sample are depicted in Figure 5C. As indicated, the most frequent deletions in chromosomes were concentrated in 10q22.2, 10q24.32, 13q14, and 13q21-31, with a frequency of 37.5%. The top CNV cytobands of gain were observed in 8q24, 11q13, 12q24, 19q13, and 20q13 (Figure 5D).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation analysis of TMT-based proteomics and WES\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo consolidate the findings from both proteomic and WES analyses, we conducted Pearson correlation analysis. The P-value was \u0026lt; 0.05 for 87 CNVs-DEPs, among which 47 exhibited a correlation value of \u0026gt; 0 (Figure 6A). GO analyses were subsequently employed to unravel the potential functions of these 47 CNVs-DEPs. As depicted in Figure 6B, ECM organization, cell-matrix adhesion, focal adhesion, and integrin binding were the primary enrichments. Subsequently, we delved into the cytoband distributions corresponding to these 47 altered CNVs-DEPs. Figure 6C reveals that the most frequent cytoband was located on chromosome 10q, with a frequency of 21.3% (10/47).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUpon comparing protein quantitation between IVL and the paired normal myometrium, we identified significant differences in three out of the 10 proteins. The expression levels of GLUD1, PDLIM1, and SLK were notably decreased in IVL (Figure 6D and Figure 6E). Significantly, both PDLIM1 and SLK play roles in the regulation of focal adhesion. Intriguingly, in the TMT-based proteomic analysis, we observed that the gene for vinculin, a cytoskeletal protein enriched in focal adhesions, is also located on chromosome 10q. These findings suggest that a chromosome 10q deletion in IVL may lead to the downregulation of several proteins involved in the regulation of the actin cytoskeleton and focal adhesion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntegrated analysis of clinical and pathological characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore potential correlations between clinical characteristics and pathological changes, we scrutinized data from 23 IVL patients, stratified by the extent of tumor disease (as illustrated in Table 1). Among the 15 aggressive IVL patients (with extra-pelvic disease), 26.7% exhibited diffuse hyalinization, with an equal percentage showing hydropic changes. These manifestations were observed in 12.5% of patients with intra-pelvic disease. In the subgroup of eight patients with intra-pelvic disease, Vinculin high expression was identified in 37.5% of cases, while the proportion of Vinculin high expression was 13.3% (2/15) in aggressive IVL cases. Despite the small sample size limiting our ability to draw definitive conclusions about the relationship between IVL tumor extent and vinculin IHC staining score, the results suggest that lower vinculin protein expression levels may be positively correlated with a more advanced tumor stage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Clinical characteristics and pathological changes in 23 IVL patients\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"88%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003eCase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eTumor extension\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003eAge (y)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003ehydropic change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHyalinization\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"top\"\u003e\n \u003cp\u003eVinculin expression\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"top\"\u003e\n \u003cp\u003eMorphology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eIntrapelvic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003emiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003ecellular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eIntrapelvic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eIntrapelvic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003eLow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003eusual\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eIntrapelvic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003emiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003ecellular, vascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eIntrapelvic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eIntrapelvic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003ecellular, vascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eIntrapelvic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eIntrapelvic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eInferior Vena Cava\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eInferior Vena Cava\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003ecellular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eInferior Vena Cava\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003ehigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eInferior Vena Cava\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003emiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular, usual\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003emiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003eusual\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003eminimal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eHeart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003emiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003ediffuse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003elow\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"5.208333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\" valign=\"top\"\u003e\n \u003cp\u003eLung\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"bottom\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.625%\"\u003e\n \u003cp\u003efocal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.708333333333332%\" valign=\"bottom\"\u003e\n \u003cp\u003emiddle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.791666666666668%\" valign=\"bottom\"\u003e\n \u003cp\u003evascular\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong eight non-aggressive IVL patients, cellular, vascular, and usual morphology were observed in 25%, 50%, and 12.5% of patients, respectively. Two patients exhibited both cellular and vascular features. In the group of 15 aggressive IVL patients, a total of 80% were categorized as having vascular morphology, with one patient displaying both vascular and usual features. These findings imply that vascular morphology constitutes a substantial proportion in aggressive IVL cases.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eIVL exhibits a higher degree of fibrosis and hyalinization compared to normal myometrium and UF\u003c/h2\u003e \u003cp\u003eIn a recent study involving scRNA-seq analysis of five unpaired UF and normal myometrium samples, the cellular composition showed no significant differences between the two groups \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In our study, utilizing UF tissue as a control, we observed a striking difference in cellular composition between one paired IVL and UF tissue through scRNA-seq.\u0026nbsp;Specifically, there was a notable decrease in SMCs and an increase in fibroblasts in IVL. These findings were corroborated by our proteomic analysis, demonstrating a significant decrease in α-SMA (SMCs marker) and an increase in FN1 (fibroblasts marker) in IVL tissue. It's worth noting that although we initially dissected three paired UF and IVL fresh tissues for scRNA-seq, only one was successfully tested due to the low percentage of tumor cells in IVL tissue. The other two attempts failed because of inadequate cellular numbers, potentially linked to the increased fibrotic and hyalinized nature of IVL tissue.\u003c/p\u003e \u003cp\u003eHE staining revealed a significant increase in the proportions of hydropic change and hyalinization areas in IVL tissues compared to normal myometrium. While hydropic change and hyalinization are features also found in UF, we conducted a comparative analysis to discern differences between IVL and UF in terms of these histological features. The results further indicated a higher prevalence of hydropic change and hyalinization in IVL compared to both normal myometrium and UF. Given that hyalinization involves the transformation of a hard, cellular tumor into softer, acellular material, our findings provide insights into the clinical observation that IVL is noticeably softer than both normal myometrium and UF.\u003c/p\u003e \u003cp\u003eOne of the primary functions of fibroblasts is the synthesis of collagen proteins\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Collagen, a significant component of the extracellular matrix, contributes to the formation of a three-dimensional meshwork, providing physical support to cells and maintaining structural integrity in tissues\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Beyond its role in structural support, collagen plays a crucial role in various cellular processes, including cell migration, adhesion, angiogenesis, tissue development, and repair. Studies by Sahai E have highlighted that during migration, cancer cells utilize collagen fibers as tracks to leave the primary tumor, emphasizing the modulatory role of collagen in cell behavior\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, collagen has been identified as a novel driver of tumor invasion, promoting local invasion and metastasis\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, fibroblasts were predominantly identified in IVL. Upon comparing IVL with normal myometrium tissues and UF, we observed higher proportions of hyalinization areas. These areas, characterized by the replacement of smooth muscle cells with collagen, exhibited a similar transformation in blood vessels within regions of hyaline necrosis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Proteomic analysis further revealed an enrichment of upregulated proteins in the collagen-containing extracellular matrix. Consequently, we deduced that fibroblasts in IVL contribute to the increased production of collagen tissue, potentially correlating with the invasiveness of the tumor.\u003c/p\u003e \u003cp\u003eIn summary, our findings lead us to conclude that IVL exhibits more fibrotic and hyalinization features than normal myometrium and UF. These characteristics may be associated with the tumor development observed in IVL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eIVL exhibits a poorer blood supply compared to normal myometrium and and UF\u003c/h2\u003e \u003cp\u003eOur scRNA-seq results indicated significantly lower proportions of endothelial cells in IVL compared to UF. Additionally, HE staining results confirmed a lower blood vessel density in IVL compared to normal myometrium and UF. These findings suggest that IVL experiences inadequate blood supply, potentially explaining the higher frequency of hyalinization in IVL. Insufficient blood supply within the myoma is recognized as a key factor contributing to hyalinization. Interestingly, our results diverge from a previous study suggesting that IVL tumors possess a strong ability to promote angiogenesis. The earlier study proposed that IVL tumors exhibit upregulated expression of proangiogenic factors, including GATA1, LIF, CXCL8, SH2D2A, et al \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo further investigate angiogenesis-associated pathway changes in IVL, we conducted a thorough analysis by identifying enriched angiogenesis-related terms in the GO analysis based on our proteomic data. DEPs associated with GO: 0016525 (negative regulation of angiogenesis) demonstrated significant upregulation in IVL. Notably, APOH (Apolipoprotein H; fold change\u0026thinsp;=\u0026thinsp;1.72), THBS1 (Thrombospondin 1; fold change\u0026thinsp;=\u0026thinsp;1.79), HRG (Histidine Rich Glycoprotein; fold change\u0026thinsp;=\u0026thinsp;1.60), THBS4 (Thrombospondin 4; fold change\u0026thinsp;=\u0026thinsp;2.19), and THBS2 (Thrombospondin 2; fold change\u0026thinsp;=\u0026thinsp;1.68) exhibited substantial increases in expression.\u003c/p\u003e \u003cp\u003eConversely, DEPs associated with GO: 0045766 (positive regulation of angiogenesis) displayed significant downregulation in IVL. Noteworthy examples include HSPB1 (Heat Shock Protein Family B Member 1; fold change\u0026thinsp;=\u0026thinsp;0.58), RRAS (RAS Related; fold change\u0026thinsp;=\u0026thinsp;0.59), and AQP1 (Aquaporin 1; fold change\u0026thinsp;=\u0026thinsp;0.61), all showing marked decreases in expression. Additionally, proteins involved in the general process of angiogenesis (GO: 0001525) underwent significant changes in IVL, with MYH9 (Myosin Heavy Chain 9; fold change\u0026thinsp;=\u0026thinsp;0.66), FN1 (Fibronectin 1; fold change\u0026thinsp;=\u0026thinsp;1.93), TGFBI (Transforming Growth Factor Beta Induced; fold change\u0026thinsp;=\u0026thinsp;1.72), and APOD (Apolipoprotein D; fold change\u0026thinsp;=\u0026thinsp;1.58) exhibiting altered expression levels.\u003c/p\u003e \u003cp\u003eCollectively, these results align with our earlier findings, supporting the notion that IVL experiences a poor blood supply in comparison to normal myometrium. However, to validate and further elucidate this conclusion, additional studies are warranted.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDysregulation of focal adhesion and the actin cytoskeleton appears to be closely associated with the development of IVL.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur TMT-based proteomic analysis revealed that the most significantly downregulated signaling pathway was related to the regulation of the actin cytoskeleton and focal adhesion. Notably, key actin cytoskeletal proteins such as vinculin, α-actinin 1, Filamin A, and others exhibited downregulation. Focal adhesions, crucial for ECM interactions, play a fundamental role in maintaining tissue homeostasis and have been implicated in cancer development and pathogenesis \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The disassembly of focal adhesions in cells has been linked to cytoskeletal transmission, promotion of epithelial-mesenchymal transition, and induction of cell migration \u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, all of which contribute to cancer formation and progression.\u003c/p\u003e \u003cp\u003eA prior study conducted RNA-seq to explore differentially expressed genes between IVL and normal uterine muscle tissues. Consistent with our proteomic findings, the results from this study also suggested that IVL may disrupt the homeostasis of gene networks involved in ECM and cytoskeleton regulation \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These collective observations underscore the potential significance of focal adhesion and actin cytoskeletal dysregulation in the pathogenesis of IVL, emphasizing the need for further investigation into these molecular mechanisms.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosome 10q deletions and vascular morphology could be important markers predicting the aggressive behavior of IVL\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWES outcomes revealed an enrichment of chromosome deletions at specific loci, including 10q22.2, 10q24.32, 13q14, and 13q21-31, within IVL tissues. Correlation analyses unveiled that the deletion of chromosome 10q in IVL might lead to the downregulation of proteins crucial in the regulation of the actin cytoskeleton and focal adhesion, which could play a significant role in IVL tumorigenesis. The validity of these findings was substantiated through vinculin IHC staining. Additionally, our investigation unveiled a correlation between lower vinculin protein expression and the distinct vascular morphology with the aggressive behavior observed in IVL.\u003c/p\u003e \u003cp\u003eChromosome 10q deletions have been implicated in various diseases, including cutaneous T-cell lymphoma \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, low-grade gliomas \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, and prostate cancer \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. A prior study by Ordulu et al. further supported the association of a 10q deletion in IVL lesions \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In Ordulu's research, hierarchical clustering analysis identified three groups, with all IVL instances in group 3 (n\u0026thinsp;=\u0026thinsp;5) exhibiting a deletion on 10q. Notably, 80% of IVL cases in group 3 displayed aggressive behavior. In our study, the removal of all eight IVL tissues for TMT-based proteomic and WES from the inferior vena cava already signaled their aggressive nature. Furthermore, Ordulu's study highlighted that 80% of cases with a vascular morphology among the 5 IVL instances associated with recurrence, aligning with our results where 80% (12 out of 15) of aggressive IVL patients exhibited vascular morphology. Collectively, these findings strongly suggest that chromosome 10q deletions and vascular morphology serve as crucial markers for predicting the aggressive behavior of IVL.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study sought to investigate the pathological and molecular alterations in IVL patients through a comprehensive multi-omics approach. However, the study is subject to several limitations that warrant attention in future research. Firstly, the impact of our findings is constrained by the availability of only one paired IVL and UF sample for scRNA-seq.\u0026nbsp;Secondly, to prevent the overinterpretation of IVL origin, further validation of WES results, including genetic variations, is essential with a larger sample size. Lastly, additional basic research is imperative to enhance our understanding of the origin and development of IVL.\u003c/p\u003e \u003cp\u003eIn summary, this pioneering study conducted an integrated analysis, incorporating scRNA-seq, H\u0026amp;E, and IHC staining, TMT-based quantitative proteomics analysis, and WES of IVL. Our investigation unveiled both the pathomorphological and molecular characteristics in IVL, paving the way for new avenues in basic research and clinical treatment for IVL.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIVL Intravenous leiomyomatosis\u003c/p\u003e\n\u003cp\u003eIVC: Inferior vena cava\u003c/p\u003e\n\u003cp\u003eUF Uterine fibroid\u003c/p\u003e\n\u003cp\u003eSMCs Smooth muscle cells\u003c/p\u003e\n\u003cp\u003eMED12 Mediator complex subunit 12\u003c/p\u003e\n\u003cp\u003escRNA-seq Single-cell RNA sequencing\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E Hematoxylin and eosin\u003c/p\u003e\n\u003cp\u003eIHC Immunohistochemistry\u003c/p\u003e\n\u003cp\u003eTMT Tandem mass tag\u003c/p\u003e\n\u003cp\u003eWES Whole-Exome Sequencing\u003c/p\u003e\n\u003cp\u003etSNE t-distributed Stochastic Neighbor Embedding\u003c/p\u003e\n\u003cp\u003eGO Gene Ontology\u003c/p\u003e\n\u003cp\u003eKEGG Kyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\n\u003cp\u003eDEPs Differentially expressed proteins\u003c/p\u003e\n\u003cp\u003eCNV Copy number variation\u003c/p\u003e\n\u003cp\u003ePPC Percentage of positive cells\u003c/p\u003e\n\u003cp\u003eIS Intensity of staining\u003c/p\u003e\n\u003cp\u003eECM Extracellular matrix\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiarong Zhang, Yingyong Hou and Chunsheng Wang designed the study; Sheng Yin, Peipei Shi, Jing Han, Aimin Ren, Li Ma, Wenbin Tang, Hua Li, Wenxue Liu, Sihui Yu and Tingting Li collected the data; Sheng Yin, Peipei Shi and Jing Han analyzed the data; Sheng Yin and Peipei Shi wrote the original draft; Jiarong Zhang revised the manuscript. All authors approved the final paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Zhongshan Development Program (XK-066).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary data of genetics and proteomics has been deposited to the GSA-human database (https://ngdc.cncb.ac.cn/gsa-human/; ID: PRJCA015471) and OMIX (https://ngdc.cncb.ac.cn/omix/; OMIX003497), which is available from the corresponding author on request.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approval by the ethical committee of Zhongshan Hospital, Fudan University (Ethics Committee document number: B2021-488R). Written informed consent was obtained from all participating patients.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNot applicable.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledge\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the patients enrolled in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCP HL. K. Intravenous leiomyomatosis with cardiac extension: tumor thrombectomy through an abdominal approach. 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Hum Pathol. 2014;45:1885\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Intravenous leiomyomatosis, multi-omics analysis, chromosome 10q deletion, vascular morphology, aggressive behavior","lastPublishedDoi":"10.21203/rs.3.rs-4210065/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4210065/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIntravenous leiomyomatosis (IVL) is a histologically well differentiated smooth muscle tumor with aggressive behavior, capable of extending throughout the venous system. Due to its rarity, systematic studies on IVL are limited. We conducted a comprehensive multi-omics study, collecting tissues from IVL, uterine fibroid, and normal myometrium. Single-cell RNA sequencing analysis revealed a significant difference in cell composition between IVL and uterine fibroid. H\u0026amp;E staining demonstrated more frequent hydropic change and hyalinization, with decreased vascular density in IVL tissues compared to both normal myometrium and uterine fibroid. Proteomics analysis in 8 paired IVL and normal myometrium fresh frozen tissue identified differentially expressed proteins mainly enriched in focal adhesions and regulation of the actin cytoskeleton. The most frequently involved chromosomes included deletions in 10q22.2, 10q24.32, 13q14, and 13q21-31. Correlation analyses highlighted chromosome 10q as the most frequent cytoband, with corresponding proteins involved in regulating focal adhesions and the cytoskeleton. Integrated analysis between pathological and clinical characteristics indicated that chromosome 10q deletion and vascular morphology in IVL could serve as important markers predicting aggressive behavior. Our study illuminates the pathological and molecular changes associated with IVL, offering insights that may contribute to establishing new directions for IVL treatment.\u003c/p\u003e","manuscriptTitle":"Pathological and molecular insights into intravenous leiomyomatosis: an integrative multi-omics study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-03 11:02:15","doi":"10.21203/rs.3.rs-4210065/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-05-05T17:29:04+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-28T18:29:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-04T04:21:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2024-04-03T01:23:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"96343762-6f45-4331-8aff-e5acbb565b8d","owner":[],"postedDate":"May 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-03T16:07:35+00:00","versionOfRecord":{"articleIdentity":"rs-4210065","link":"https://doi.org/10.1186/s12967-024-05919-9","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2025-02-26 15:57:45","publishedOnDateReadable":"February 26th, 2025"},"versionCreatedAt":"2024-05-03 11:02:15","video":"","vorDoi":"10.1186/s12967-024-05919-9","vorDoiUrl":"https://doi.org/10.1186/s12967-024-05919-9","workflowStages":[]},"version":"v1","identity":"rs-4210065","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4210065","identity":"rs-4210065","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}