Distinguishing pancreatic neuroendocrine tumors (pNETs) from solid pseudopapillary neoplasms (SPNs) is challenging, primarily due to their overlapping pathological characteristics. To address this, our study aims to identify and validate novel biomarkers that effectively differentiate between these two conditions. We focus on the exploration of new immunohistochemical markers to enhance this distinction.In this study, we analyzed genetic variations in pNETs and SPNs using the GSE43795 dataset from the Gene Expression Omnibus (GEO) database. Our approach was to identify genes with higher expression in pNETs compared to SPNs and normal pancreatic tissues. We conducted enrichment analyses to understand the functions of these genes. Furthermore, protein-protein interaction (PPI) network analysis was utilized to identify key genes associated with pNETs. Our sample consisted of 163 pancreatic tumor specimens, comprising 78 pNETs and 85 SPNs. We also collected clinicopathological data and used immunohistochemistry to measure the expression levels of these key genes.The enrichment analysis revealed that genes overexpressed in pNETs were mainly involved in signal release, vesicle transport, and ion pathway activation, playing significant roles in endocrine processes like insulin secretion, dopamine synapses, and circadian rhythm regulation. The PPI analysis identified secretogranin II (SCG2), carboxypeptidase E (CPE), and chromogranin A (CgA, CHGA) as key markers for differentiating pNETs from SPNs. Immunohistochemical validation of these markers demonstrated high sensitivity (SCG2: 98.7%, CPE: 97.4%) and specificity (100%), indicating their superior discriminative power compared to traditional markers like CgA, β-catenin, lymphoid enhancer-binding factor 1 (LEF1), and vimentin.Our study indicates that SCG2 and CPE are effective, novel immunohistochemical biomarkers for differentiating pNETs from SPNs.2024 Translational Cancer Research. All rights reserved.