The fibrosis-targeted collagen/integrins gene profile predicts risk of metastasis in pulmonary neuroendocrine neoplasms

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. The neoplastic area was micro dissected during the frozen section procedure to ensure the inclusion of neoplastic tissue and distant non-neoplastic tissue as control. Total mRNA was extracted from 24 fresh-frozen tumor and normal tissues using the QIAsymphony miRNA CT 400 kit (Qiagen, CA, USA) according to the manufacturer’s instructions. RNA integrity and quality were determined using the Bioanalyzer 2100 (Agilent Technologies). Complementary DNA was synthesized using the c-DNA – RT² First Strand Kit (Qiagen Sample & Assay Technologies) according to the manufacturer’s protocol. The difference of expression in EMT genes was evaluated by the real-time PCR method. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using the RT² Profiler PCR Array System (PAHS-090Z; Qiagen, Dusseldorf, Germany) kit for the human epithelial-to-mesenchymal transition (EMT) pathway with 84 target genes.