The Fibrosis-Targeted Collagen/Integrins Gene Profile Predicts Risk of Metastasis in Pulmonary Neuroendocrine Neoplasms

Abstract

Recently, collagen/integrin genes have shown promise as predictors of metastasis mainly in non-small cell lung cancer and breast cancer. However, it is unknown if these gene expression profiling differ in metastatic potential of pulmonary neuroendocrine neoplasms (PNENs). In this study, we sought to identify differentially expressed collagen/integrin genes in PNENs in order to understand the molecular mechanisms underlying the development of stroma-associated fibrosis for invasion and metastasis. We compared collagen/integrin gene expression profiling between PNE tumors (PNETs) and PNE carcinomas (PNECs) using a two-stage design. First, we used PCR Array System for 84 ECM-related genes, and among them, we found COL1A2, COL3A1, COL5A2, ITGA5, ITGAV, and ITGB1 functionally involved in the formation of the stroma-associated fibrosis among PNENs histological subtypes. Second, we examined the clinical association between the six collagen/integrin genes in tumor tissues from 24 patients with surgically excised PNENs. However, the pathological exam of their resected tissues demonstrated that 10 developed lymph node metastasis and 7 distant metastasis. We demonstrated and validated up regulation of the six fibrogenic genes in PNECs and down regulation in PNETs that were significantly associated with metastasis-free and overall survival (P<0.05). Our study implicates up regulation of fibrogenic genes as a critical molecular event leading to lymph node and distant metastasis in PNENs.

Keywords: collagens; extracellular matrix; integrins; metastasis; pulmonary neuroendocrine neoplasms; surgical resection.

Figure 1

Box plots of differentially expressed COL and ITG gene expressions among PNENs histological subtypes in a log scale. The top and bottom of the box plot represents the 25th and 75th percentile range. The line across the box shows the median of gene expression and the top and bottom bars show the maximum and minimum values, outliers were showed. The association between COL and ITG gene expressions and PNENs histological types was calculated by non-parametric Kruskal-Wallis test. (A) Difference of COL1A2 mRNA expression level among PNENs histological subtypes (P=0.004; Adjusted P-value [AC-SCLC]=0.007); (B) Difference of COL3A1 mRNA expression level among PNENs histological subtypes (P=0.004; Adjusted P-value [AC-SCLC]=0.004); (C) Difference of COL5A2 mRNA expression level among PNENs histological subtypes (P=0.003; Adjusted P-value [AC-SCLC]=0.010; Adjusted P-value [TC-SCLC]=0.019); (D) Difference of ITGA5 mRNA expression level among PNENs histological subtypes (P=0.009; Adjusted P-value [AC-SCLC]=0.008); (E) Difference of ITGAV mRNA expression level among PNENs histological subtypes (P=0.028; Adjusted P-value [AC-SCLC]=0.018); (F) Difference of ITGB1 mRNA expression level among PNENs histological subtypes (P=0.057). The horizontal bars represents the statistical difference between groups, the pairwise comparison was adjusted by Bonferroni’s correction for multiple tests (Adjusted P-value). (*) was used when P > 0.05, and (**) was used when P ≥ 0.01. SCLC, small cell lung carcinoma; LCNEC, large cell neuroendocrine carcinoma; AC, atypical carcinoid; TC, typical carcinoid.

Figure 2

Kaplan-Meier plots for overall survival for PNEN patients according to the difference in COL and ITG gene expressions. (A) In the Kaplan-Meier survival curve of COL5A2, patients who had high expression (top of the curve) presented lower survival rate than those who had lower expression (bottom of the curve), 112.78 vs 16.61 months, (P=0.018); (B) In the Kaplan-Meier survival curve of ITGA5, patients who had high expression (top of the curve) presented lower survival rate than those who had lower expression (bottom of the curve), 126.38 vs 13.50 months, (P=0.001); (C) In the Kaplan-Meier survival curve of ITGB1, patients who had high expression (top of the curve) presented lower survival rate than those who had lower expression (bottom of the curve), 123.54 vs 15.15 months, (P=0.006).