Biochemical characterisation and clinical correlation of neuropeptides in neuroblastoma with emphasis on neuropeptide Y

Abstract: Neuropeptides influence cellular events involved in tumour growth and differentiation. Neuroblastoma, a malignant childhood tumour of neural crest origin, synthesises and releases monoamines and neuropeptides. The concentrations of some of these neuropeptides in plasma are correlated to clinical stage and outcome. The neuropeptides exist in various molecular forms in plasma and tumour tissue but their biochemical structure in vivo are poorly investigated. The aim of the present work was to combine radioimmunoassays (RIA), different forms of liquid chromatography, amino acid sequence analysis and mass spectrometry to identify and characterise molecular forms of neuropeptides present in neuroblastoma tumour tissue, and to identify correlations to clinical parameters. The hypothesis tested was that the processing of neuropeptide Y (NPY) and other peptides in neuroblastoma generates molecular forms that may have clinical significance. Vasoactive intestinal peptide-like immunoreactivity (VIP-LI) and somatostatin-like immunoreactivity (SOM-LI) in neuroblastoma and benign ganglioneuroma tumour tissue were found in the form of the intact, biologically active peptides VIP-28, SOM-14 and SOM-28. However, the results for SOM-LI may be influenced by presence of the recently identified somatostatin homologoue cortistatin, that was shown to have similar affinity as somatostatin for the antiserum used. Pancreastatin-like immunoreactivity (PST-LI) showed molecular heterogeneity in neuroblastoma. The concentration of PST-LI in tumour tissue and plasma was higher in favourable neuroblastomas whereas low concentrations in both plasma and tumour tissue were correlated to poor outcome. NPY-like immunoreactivity (NPY-LI) in both neuroblastoma and ganglioneuroma tumour tissue showed considerable heterogeneity in general, as demonstrated by using two antisera specific for different epitopes of the NPY molecule. However, no clinical correlation was identified related to this heterogeneity. By gel- permeation chromatography the degree of proNPY processing could be measured. A low degree of proNPY processing was seen only in primary tumour tissue from neuroblastomas with regional or metastatic spread and was correlated to poor outcome. Using a combination of "micro-preparative" RP-HPLC and mass spectrometry, intact NPY(1-36) and the metabolic fragments NPY(3-36) and NPY(31-36) were isolated and identified in neuroblastoma tumour tissue. The identification of these fragments shows that NPY processing in vivo generates NPY-metabolites known to have different receptor selectivity than intact NPY. C-flanking peptide of NPY (CPON)-like immunorectivity was found in similar concentrations as NPY-LI. The fragment CPON(1-26) was identified eluting with NPY after several chromatographic steps, indicating that CPON and NPY may interact by non-covalent forces. Taken together, "micropreparative" chromatography in combination with tandem mass spectrometry were useful tools for biochemical characterisation of neuropeptides and their metabolites in biological samples. The results provide evidence that the processing of proNPY and NPY in neuroblastoma have clinical implications.