Gene expression patterns related to vascular invasion and aggressive features in endometrial cancer

Abstract

The presence of tumor cells entering vascular channels is a prognostic marker for many cancers, including endometrial carcinoma. Vascular invasion is considered to be an early step in the metastatic process and important for the progress of malignant tumors. Here, we investigated the gene expression patterns related to vascular involvement in 57 primary endometrial cancers, using DNA microarray and quantitative PCR techniques. A vascular invasion signature of 18 genes was significantly associated with patient survival and clinicopathological phenotype. Vascular involvement was also related to gene sets for epithelial-mesenchymal transition, wound response, endothelial cells, and vascular endothelial growth factor (VEGF) activity. With immunohistochemical validation, both collagen 8 and matrix metalloproteinase 3 (MMP3) were associated with vascular invasion, whereas ANGPTL4 and IL-8 were associated with patient survival. Our findings indicate that vascular involvement within primary tumors is associated with gene expression profiles related to angiogenesis and epithelial-mesenchymal transition. These data could contribute to an improved understanding of potential targets for metastatic spread and may provide clinically important information for better management of endometrial cancer.

Figure 1

Estimated survival probability based on absent or present vascular invasion. A: In a prospective test series, 57 tumors from endometrial cancer patients were used for gene expression studies. B: In a retrospective validation series, 286 patients were used. Survival curves were estimated using the Kaplan-Meier method and log-rank significance test.

Figure 2

A: Unsupervised hierarchical clustering was done on the vascular invasion signature with Pearson correlation and average linkage (WPGMA). Two main clusters, A and B, were formed. Distribution of different clinicopathological features between the clusters is shown in the panel below the heat map. MGC10848 is an alias for ITIH5, KIAA1872 is an alias for ATCAY, and FPRL1 is an alias for FPR2 (as in Table 2). B: A summary score for the vascular invasion signature was based on microarrays and qPCR normalized expression data. Good correlation is seen between the two methods. C: The vascular invasion signature was significantly associated with decreased patient survival (Kaplan-Meier method, log-rank test).

Figure 3

Clustering of tumors with (red) and without (pink) vascular invasion using the endothelial signature of 28 genes (A), the expanded endothelial signature of 468 genes (B), the wound response signature of 389 genes (C), the combined signature of these 834 unique genes (D), the TGF-β response signature of 121 genes (E), and the TGF-β pathway of 163 genes (F).

Figure 4

An average expression value was calculated for the 13-gene VEGF signature, and two subgroups were obtained by the median value. Patients with a high VEGF signature score exhibited a worse prognosis than those with a low signature score.

Figure 5

A: Western blot analysis supported specificity of ANGPTL4 antibody showing two splice variants. B: Estimated patient survival according to ANGPTL4 protein expression in tumor cells using our validation series (series II) (log-rank significance test). In representative micrographs, endometrial cancer exhibits positive (C) and negative (D) ANGPTL4 protein expression (samples from two different cases). Original magnification, ×400.