Despite advances in endoscopic techniques, most NFPA patients are at increased risk of postoperative recurrence and lack of therapeutic response due to their invasive growth in surrounding normal tissues at the time of diagnosis[9, 29]. Therefore, it is indispensable to explore the pathogenesis of NFPAs and its invasiveness, which will help us understand the behavior of NFPAs invasion and recurrence and provide systematic treatment methods. TME (tumor microenvironment) is implicated in the initiation and development of various tumours[14, 30], but research on the NFPAs is still scarce. In the present study, we devoted to screen out the TME-related genes that act on NFPAs RFS and invasiveness in our database.
First, the immune scores and stromal scores was obtained by using ESTIMATE algorithm. Although the relation between the immune/stromal score and pathological type, Knosp classification, and Hardy classification was not significant in our results, the low immune scores were significantly correlated with better recurrence-free survival, suggesting that the immune components in the TME may have deep effect in tumor progression. This is consisted with previous studies that tumor-associated macrophages (TAMs), T regulatory cells (Tregs) and mast cells are associated with poor prognosis in many type of cancer[14, 30–32]. Although our analysis showed the relationship between stromal scores and prognosis was not significant, this does not mean that stromal-specific genes have nothing to do with prognosis.
Second, we further analyzed 497 intersection DEGs (478 overexpressed and 19 underexpressed) which obtained from high vs. low immune/stromal scores groups, since the intersection genes are the most highly conserved. Among the 497 intersection genes analyzed by GO terms, we found that the most of them enrichened in the immune/inflammatory response (BP), cytokine activity (MF) and extracellular matrix (CC). KEGG pathway enrichment analysis shows that these IDEGs mainly clustered in PI3K-Akt signaling pathway, cell adhesion molecules (CAMs), Cytokine-cytokine receptor interaction. The PI3K-Akt signaling pathway is a signal transduction casacde involved in cell growth and metabolism[33]. Long, R. et al. study revelas that COL6A6 could block PI3K-Akt-pathway to supress the growth and metastasis of pituitary adenoma[34]. Moreover, we were able to construct a protein-protein interaction module based on these 497 intersection genes. Similarity, these hub genes are related with the B cell activation, T cell activation, angiogenesis and so on, which indicated that these genes were related to immune/inflammation and stromal response by function enrichment analysis.
Third, we analyzed 497 interaction genes and identified 23 genes that were significantly differentially expressed between invasion group and non-invasive group. Several identified genes in our results have previously been reported to play important roles in several types of cancer. Wang, Y. et al. study found that the expression of ABC transporter gene (ABCA8) was significantly down-regulated in prostate cancer as compared to noncancerous prostate tissues[35]. This was consistent with our result that the expression level of ABCA8 was significantly decresed in invasive group vs. non-invasive group. Although several studies demonstrated that Angiopoietin-like 4 (ANGPTL4) was overexpressed in colorectal cancer, breast cancer, prostate cancer and gastric cancer[36–39], downregulated by promoter methylation in gastric cancer and mammary carcinoma[40, 41]. Similarly, Angiopoietin-like 4 (ANGPTL4) was downregulated in invasive group based on our data. Therefore, the gene of ANGPTL4 could play an important role in tumor microenvironmental, either as a tumor suppressor or oncogene. In addition, PDLIM4 (PDZ and LIM domain 4) was found significantly underexpressed in invasive group compared with non-invasive group. It also has been validated downregulation of PDLIM4 is associated with aggressive tumor features and poor prognosis in several cancer such as prostate cancer[42] and ovarian cancer[43]. These genes may be potential biomarkers for early diagnosis and related to the prognosis of NFPAs. However, the function of these identified genes is still needed to investigate.
Finally, 5 TME-related genes were screen out which was significant related with the RFS. Among them, the upregulated four genes ADGRG6, CD52, GPR183 and NNMT are correlate with unfavorable outcomes of NFPAs. ADGRG6 is a member of the adhesion G protein-coupled receptor family and the depletion of ADGRG6 expression in urothelial bladder carcinomas cells compromised their abilities to recruit endothelial cell and induce tube formation[44]. The upregulated CD52 is correlated with poor survival in patient with Lung adenocarcinoma by analysis[45]. CD52 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on the surface of normal T and B lymphocytes and also known as CAMPATH-1 antigen[46]. In addition, NNMT(Nicotinamide N-methyltransferase) was found to be overexpressed in gastric carcinoma tissue compared with adjacent tissues and is related with the poor prognosis[47], which is a phase II metabolizing enzyme, mainly catalyzes the methylation of nicotinamide and other pyridines into pyridinium ions[48]. Moreover, Wang, Y. et al. found that the overexpressed NNMT is correlated with poor survival and chemotherapy response in breast cancer patients who received chemotherapy[49]. As explained above, a serious of genes we identified through bioinformatics methods are related to tumor microenvironment, and these genes are related to patients’ prognosis. Besides, although the function and role of some of these genes have been reported in multiple types of tumor, further validation with biological experiments in our patient’s samples are still needed.