Abstract
Although the postoperative recurrence rate for pancreatic neuroendocrine tumors (PNETs) is reported to be 13.5%‐30%, the paucity of valuable biomarkers to predict recurrence poses a problem for the early detection of relapse. Hence, this study aimed to identify new biomarkers to predict the recurrence of PNETs. We performed RNA sequencing (RNA‐Seq) on RNA isolated from frozen primary tumors sampled from all localized G1/G2 PNETs resected curatively from 1998 to 2015 in our institution. We calculated differentially expressed genes (DEGs) in tumor with and without recurrence (≥3 years) for the propensity‐matched cohort. Gene ontology analysis for the identified DEGs was also performed. Furthermore, we evaluated the expression levels of candidate genes as recurrence predictors via immunostaining. Comparison of transcriptional levels in tumors with and without recurrence identified 166 DEGs. Up‐ and downregulated genes with high significance in these tumors were mainly related to extracellular organization and cell adhesion, respectively. We observed the top three upregulated genes, C‐type lectin domain family 3 member A (CLEC3A), matrix metalloproteinase‐7 (MMP7), and lipocalin2 (LCN2) immunohistochemically and compared their levels in recurrent and nonrecurrent tumors. Significantly higher recurrence rate was shown in patients with positive expression of CLEC3A (P = 0.028), MMP7 (P = 0.003), and LCN2 (P = 0.040) than that with negative expression. We identified CLEC3A, MMP7, and LCN2 known to be associated with the phosphatidylinositol‐3‐kinase/Akt pathway, as potential novel markers to predict the postoperative recurrence of PNETs.
Highlights
The detection of pancreatic neuroendocrine tumors (PNETs) is increasing due to improved diagnostic performance, with the incidence is reported to have increased by 10%.1,2Surgical resection—the only treatment known to achieve a cure—is recommended for locoregional disease control and should still be considered for patients with advanced disease if it can be performed safely.[3]
The major finding in the present study is that the top three upregulated genes C‐type lectin domain family 3 member A (CLEC3A), LCN2, and matrix metalloproteinase‐7 (MMP7) showed significantly higher expression at the protein level in tumors with recurrence than in those without recurrence by IHC, which might be useful for predicting recurrence
Significant downregulated genes in tumor with recurrence were related to cell‐cell adhesion as mentioned in the result, the difference in gene expression between recurrence and nonrecurrence was too small to expect to be a marker predicting recurrences
Summary
The detection of pancreatic neuroendocrine tumors (PNETs) is increasing due to improved diagnostic performance, with the incidence is reported to have increased by 10%.1,2. To the best of our knowledge, no study has yet performed comprehensive gene expression analysis to compare tumors with and without recurrence after R0 resection. Within 2 hours of resection, tumor and nonneoplastic pancreatic tissues were chopped into 3‐4 mm cubes using a double‐edged razor placed into the shallow pockets of a freezing plate (Meiko Medical) with 2‐3 mL a small amount of Tissue‐Tek® OCTTM compound (Sakura Finetek) and frozen by liquid nitrogen for 20‐30 seconds until completely frozen as previously described.[20] We conducted gene expression analysis by performing RNA‐Seq on frozen primary tumor specimens and nonneoplastic pancreatic tissues from the patients. The libraries were sequenced on Illumina HiSeq 1500 sequencers (Illumina) and the reads were mapped to the GRCh38 human reference genome assembly using HISAT (v. 2.2.6).[23]
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