Genotype-specific differences in the tumor metabolite profile of pheochromocytoma and paraganglioma using untargeted and targeted metabolomics.

CONTEXT AND OBJECTIVE: Pheochromocytomas and paragangliomas (PGLs) are neuroendocrine tumors of sympathetic or parasympathetic paraganglia. Nearly 40% of PGLs are caused by germline mutations. The present study investigated the effect of genetic alterations on metabolic networks in PGLs. DESIGN: Homogenates of 32 sporadic PGLs and 48 PGLs from patients with mutations in SDHB, SDHD, SDHAF-2, VHL, RET, and NF-1 were subjected to proton ((1)H) nuclear magnetic resonance (NMR) spectroscopy at 500 MHz for untargeted and HPLC tandem mass spectrometry for targeted metabolite profiling. RESULTS: (1)H NMR spectroscopy identified 28 metabolites in PGLs of which 12 showed genotype-specific differences. Part of these results published earlier reported low complex II activity (P < .0001) and low ATP/ADP/AMP content (P < .001) in SDH-related PGLs compared with sporadics and PGLs of other genotypes. Extending these results, low levels of N-acetylaspartic acid (NAA; P < .05) in SDH tumors and creatine (P < .05) in VHL tumors were observed compared with sporadics and other genotypes. Positive correlation was observed between NAA and ATP/ADP/AMP content (P < .001) and NAA and complex II activity (P < .0001) of PGLs. Targeted purine analysis in PGLs showed low adenine in cluster 1 compared with cluster 2 tumors (SDH P < .0001; VHL P < .05) whereas lower levels (P < .05) of guanosine and hypoxanthine were observed in RET tumors compared with SDH tumors. Principal component analysis (PCA) of metabolites could distinguish PGLs of different genotypes. CONCLUSIONS: The present study gives a comprehensive picture of alterations in energy metabolism in SDH- and VHL-related PGLs and establishes the interrelationship of energy metabolism and amino acid and purine metabolism in PGLs.

Overexpression of the natural antisense hypoxia-inducible factor-1alpha transcript is associated with malignant pheochromocytoma/paraganglioma.

Paragangliomas (PGLs) have widely different metastastic potentials. Two different types of PGLs can be defined by expression profiling. Cluster 1 PGLs exhibit VHL and/or succinate dehydrogenase (SDH) mutations and a pseudohypoxic phenotype. RET and neurofibromatosis type 1 (NF1) mutations occur in cluster 2 tumors characterized by deregulation of the RAS/RAF/MAP kinase signaling cascade. Sporadic PGLs can exhibit either profile. During sustained hypoxia, a natural antisense transcript of hypoxia-inducible factor 1 (aHIF) is expressed. The role of aHIF in the metastatic potential of PGL has not yet been investigated. The aim was to test the hypothesis that genotype-specific overexpression of aHIF is associated with an increased metastatic potential. Tumor samples were collected from 87 patients with PGL. Quantitative PCR was performed for aHIF, vascular endothelial growth factor (VEGF), aquaporin 3, cytochrome b561, p57Kip2, slit homolog 3, and SDHC. Expression was related to mutation status, benign versus malignant tumors, and metastasis-free survival. We found that both aHIF and VEGF were overexpressed in cluster 1 PGLs and in metastatic tumors. In contrast, slit homolog 3, p57Kip2, cytochrome b561, and SDHC showed overexpression in non-metastatic tumors, whereas no such difference was observed for aquaporin 3. Patients with higher expression levels of aHIF and VEGF had a significantly decreased metastasis-free survival. Higher expression levels of SDHC are correlated with an increased metastasis-free survival. In conclusion, we not only demonstrate a higher expression of VEGF in cluster 1 PGL, fitting a profile of pseudohypoxia and angiogenesis, but also of aHIF. Moreover, overexpression of aHIF and VEGF marks a higher metastatic potential in PGL.