Comparing gene expression in deep infiltrating endometriosis with adenomyosis uteri: evidence for dysregulation of oncogene pathways

Abstract

Background: The pathogenesis of deep infiltrating endometriosis (DIE) is poorly understood. It is considered a benign disease but has histologic features of malignancy, such as local invasion or gene mutations. Moreover, it is not clear whether its invasive potential is comparable to that of adenomyosis uteri (FA), or whether it has a different biological background. Therefore, the aim of this study was to molecularly characterize the gene expression signatures of both diseases in order to gain insight into the common or different underlying pathomechanisms and to provide clues to pathomechanisms of tumor development based on these diseases.

Methods: In this study, we analyzed formalin-fixed and paraffin-embedded tissue samples from two independent cohorts. One cohort involved 7 female patients with histologically confirmed FA, the other cohort 19 female patients with histologically confirmed DIE. The epithelium of both entities was microdissected in a laser-guided fashion and RNA was extracted. We analyzed the expression of 770 genes using the nCounter expression assay human PanCancer (Nanostring Technology).

Results: In total, 162 genes were identified to be significantly down-regulated (n = 46) or up-regulated (n = 116) in DIE (for log2-fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05) compared to FA. Gene ontology and KEGG pathway analysis of increased gene expression in DIE compared to FA revealed significant overlap with genes upregulated in the PI3K pathway and focal adhesion signaling pathway as well as other solid cancer pathways. In FA, on the other hand, genes of the RAS pathway showed significant expression compared to DIE.

Conclusion: DIE and FA differ significantly at the RNA expression level: in DIE the most expressed genes were those belonging to the PI3K pathway, and in FA those belonging to the RAS pathway.

Keywords: Adenomyosis; Deep infiltrating endometriosis; Gene expression analysis; PI3K pathway; RAS pathway.

Fig. 1

Light microscopy of deep infiltrating endometriosis of the rectum (A) and of adenomyosis uteri (B) stained with cresylviolet (10x). In each case the second image in the row shows the marking of the tissue for laser dissection [2]. The third image shows the tissue after laser dissection [3]

Fig. 2

Volcano plot showing genes with most significant dysregulation in DIE vs. adenomyosis (adj. p-adjusted < 0.001 and log2 FC > 1.5). 15 genes were highly significantly upregulated in DIE, and one gene (FZD2) was downregulated (log2 FC < 0.66).

Fig. 3

Heatmap of supervised hierarchical clustering of differential genes (adj. p < 0.05, log2 fold change > 1.5 or < 0.66 and adjusted p-value < 0.05, n = 162) for the FA versus DIE group. This includes 116 upregulated genes in DIE and 46 genes with upregulation in adenomyosis. Gene expression with cases of adenomyosis (FA) is clearly distinct from cases with deep infiltration endometriosis (DIE).

Fig. 4

(a) KEGG enrichment analyses for differentially upregulated genes (p < 0.05, n = 116), 12 most significantly upregulated pathways are shown. This analysis revealed upregulating of signaling pathways in DIE, most significantly the PI3K pathway, but also pathways involved in virus infection, focal adhesion, endocrine resistance and malignancy. (b) Same KEGG analysis, for differentially downregulated genes (p < 0.05, n = 46), 4 most significant pathways (p < 0.0001) are shown. Here, RAS, PI3K-AKT, RAP1 and Calcium signaling pathways are significant