Proteomic profiling of formalin-fixed paraffin-embedded specimens reveals candidate intraoperative diagnostic biomarkers for Hirschsprung disease

Background Rare diseases such as Hirschsprung disease (HSCR) pose challenges for prospective pediatric surgical research because of limited patient numbers. Archived formalin-fixed paraffin-embedded (FFPE) tissue samples provide a valuable resource for molecular analyses without requiring additional invasive procedures. In this study, we performed a comprehensive proteomic analysis of FFPE samples from patients with HSCR to identify potential novel biomarkers for intraoperative diagnosis and further explore HSCR pathophysiology. Methods Proteomic analysis was performed on 45 FFPE samples from 15 patients (mean age: 6.5 ± 3.3 months, male/female = 11:4), including samples from the recto-sigmoid or long-type aganglionosis regions. The study included the aganglionic segment (AS), the transitional zone (TZ), and the normal intestine (NI) samples from each patient. Statistical significance was determined using adjusted p-values (< 0.05). Functional analysis was performed using gene set enrichment analysis. Results A total of 10,541 proteins were identified, of which 13 were significantly upregulated in the NI compared with the AS and TZ. Immunohistochemical validation demonstrated that catenin alpha-2 (CTNNA2), mammalian ependymin-related protein 1 (EPDR1), NAC-alpha domain-containing protein 1 (NACAD), and neural cell adhesion molecule 2 (NCAM2), previously unassociated with HSCR or gastrointestinal development, exhibited staining restricted to ganglionic regions of the large intestine. Conclusions These findings indicate that CTNNA2, EPDR1, NACAD, and NCAM2 may be promising candidate biomarkers for intraoperative differentiation of the NI from the TZ and AS, demonstrating the feasibility of FFPE-based proteomics for HSCR research.