Intravenous leiomyomatosis is inclined to a solid entity different from uterine leiomyoma based on RNA-seq analysis with RT-qPCR validation

Abstract

Introduction: Intravenous leiomyomatosis (IVL) is currently regarded as a special variant of the common uterine leiomyoma (LM). Though IVL shows a similar histological morphology to LM, IVL is characterized by unique intravenous growth patterns and low-grade malignant potential, which are quite different from LM. There are currently few studies underlying the molecular alterations of IVL, though this information is important for understanding the pathogenesis of the disease, and for identifying potential biomarkers.

Method: We carried out a high-throughput whole transcriptome sequencing of tumor and normal tissue samples from five IVL patients and five LM patients and compared the differentially expressed genes (DEGs) between IVL and leiomyoma. We performed multiple different enrichment and target analyses, and the expression of selected DEGs was validated using RT-qPCR in formalin-fixed samples.

Results: Our study identified substantial different genes and pathways between IVL and LM, and functional enrichment analyses found several important pathways, such as angiogenesis and antiapoptosis pathways, as well as important related genes, including SH2D2A, VASH2, ADAM8, GATA2, TNF, and the lncRNA GATA6-AS1, as being significantly different between IVL and LM (P = .0024, P = .0195, P = .0212, P = .0435, P = .0401, and P = .0246, respectively). CXCL8, LIF, CDKN2A, BCL2A1, COL2A1, IGF1, and HMGA2 were also differently expressed between IVL and LM groups, but showed no statistical difference (P = .2409, P = .1773, P = .0596, P = .2737, P = .1553, P = .1045, and P = .1847, respectively) due to the large differences among individuals. Furthermore, RT-qPCR results for five selected DEGs in IVL tissues and adjacent nontumor tissues were mainly consistent with our sequencing results.

Conclusion: Our results indicated that IVL may be a solid entity that is unique and different from LM, proving consistent with previous studies. Furthermore, we identified DEGs, particularly within angiogenesis and antiapoptosis pathway-related genes that may play crucial roles in the development and pathogenesis of IVL and may be potential specific biomarkers.

Keywords: angiogenesis; differentially expressed genes; high-throughput whole transcriptome resequencing; intravenous leiomyomatosis.

FIGURE 1

IVL clinical image. (A) Curved planar reformation (CPR) CT shows IVL in inferior vena cava (IVC) and pelvic cavity. (B) Volume rendering (VR) three‐dimensional reconstruction CT shows the 3D form of IVL before operation. (C) Specimen of IVL after operation. The red and blue circles indicate the location from where tumor and tumor‐adjacent normal tissue were collected, respectively

FIGURE 2

Venn diagram for DEGs in term A (LMT vs LMN), term B (IVLT vs IVLN) and term C (IVLT vs LMT). IVLT represents IVL tumor tissues; IVLN represents IVL‐adjacent normal tissues; LMT represents leiomyoma tumor tissues; and LMN represents leiomyoma‐adjacent normal tissues. The number in the circle represents the number of identified DEGs

FIGURE 3

Venn diagram for up/downregulated DEGs in term A (LMT vs LMN), term B (IVLT vs IVLN) and term C (IVLT vs LMT). (A) Venn diagram for DEGs in terms A, B, and C where IT represents IVL tumor tissues; IN represents IVL‐adjacent normal tissues; LT represents leiomyoma tumor tissues; LN represents leiomyoma‐adjacent normal tissues; U represents upregulated DEGs; and D represents downregulated DEGs. The number in the triangle represents the number of DEGs. B: Quantification of Venn diagram for up/downregulated DEGs

FIGURE 4

Angiogenesis factors enriched in IVL tumors. FPKM values for angiogenesis factors enriched in IVL tumors, including VASH2, GATA2, CXCL8, LIF, SH2D2A, and ADAM8. Student's t‐test was used to analyze the differences between the two groups and to estimate the level of significance. Log‐transformation was performed to approximate normal distribution of data before implementation of parametric tests

FIGURE 5

Apoptosis factors enriched in IVL tumors. FPKM values for apoptosis factors enriched in IVL tumors, including CDKN2A, IGF1, TNF, COL2A1, and BCL2A1. Tukey's multiple comparison test was used to analyze the differences among 10 samples from two groups and to estimate the level of significance

FIGURE 6

Comparison of expression pattern of lncRNAs GATA6‐AS1 and HGMA2 in IVLT and LMT samples. Comparison of expression pattern of lncRNAs GATA6‐AS1 and HGMA2 in IVL and LM tumor samples. Tukey's multiple comparison test was used to analyze the differences among 10 samples from two groups and to estimate the level of significance

FIGURE 7

RT‐qPCR validation of selected DEGs in Chinese IVL Patients. RT‐qPCR validation of selected DEGs in IVL patient samples, including CXCL8, LIF, SH2D2A, VASH2, and GATA2. Tukey's multiple comparison test was used to analyze the differences from two groups and to estimate the level of significance. Log‐transformation was performed to approximate normal distribution of data before implementation of parametric tests