Background: Despite widely heterogenous histological appearance and genetic markers, high-risk neuroblastoma share a commonly aggressive disease course and poor outcome. While the genome and transcriptome of these tumors have been studied in detail, little is known about its proteome. Using a proteomic analysis, this study aimed to identify potential biomarkers associated with this clinico-pathological risk group that may account for their consistently aggressive behavior.
Methods: Primary NB tumors obtained from untreated high-risk patients were analyzed using two-dimensional gel electrophoresis (2DE) and mass spectrometry. Proteins extracted were pooled to construct a reference proteome map. A total of 602 protein spots were excised from 2DE gels for protein identification. Identified proteins were classified by gene ontology analysis and searched in the literature for biological significance. Immunohistochemical staining of selected proteins was scored on a neuroblastoma tumor microarray (TMA), and correlated with survival outcomes derived from the Singapore Childhood Cancer Registry using Log-rank analysis.
Results: Biopsy specimens were obtained from 6 high-risk treatment naïve patients of different demographics, disease site, stage, and biology to represent the diverse heterogeneity seen in this clinico-pathological group. We established the first known proteome map of high-risk neuroblastoma and identified 382 unique proteins with a minimum of 2 unique peptides matched and an additional 42 proteins were identified based on one peptide match. They included putative tumor suppressor proteins and oncoproteins not described in neuroblastoma before. Among them was nuclear distribution protein C (NudC), a nuclear movement protein that regulates microtubule organization. NudC staining correlated with degree of tumor differentiation, and was associated with INPC classification (p=0.023). In 86 TMA samples, 3+ NudC staining was associated with poorer overall survival (p=0.03) in a subgroup of low- and intermediate-risk patients.
Conclusion: We identified novel biomarkers through proteomic profiling of high-risk neuroblastoma that may be associated with tumor biology and behavior. NudC expression correlated with established pathological and clinical prognostic indicators, and identifies a subgroup of non-high risk patients with poorer outcomes.