The Section is conducting patient-oriented research about the etiology, pathophysiology, genetics, diagnosis, and treatment of pheochromocytoma (PHEO) and paraganglioma (PGL). Projects include not only translational research-applying basic science knowledge to clinical diagnosis, pathophysiology, and treatment-but also reverse translation research where appreciation of clinical findings leads to new concepts that basic researchers can pursue in the laboratory. In order to achieve our goals, the strategy of the Section is based on the multidisciplinary collaborations among NIH investigators and outside medical centers. Our Section links together a patient-oriented component with two bench-level components. The patient-oriented component (Medical Neuroendocrinology) is currently the main driving force for our hypotheses and discoveries. The two bench-level components (Tumor Pathogenesis and Chemistry & Biomarkers) emphasize first, technologies of basic research tailored for pathway and target discovery and second, the development of the discoveries into clinical applications. Metabolomic aspects of PHEO/PGL Mutations of SDHx genes increase susceptibility to development of PHEOs/PGLs, with particularly high rates of malignancy associated with SDHB mutations. We assessed whether altered succinate dehydrogenase product-precursor relationships, manifested by differences in tumor ratios of succinate to fumarate or other metabolites, might aid in identifying and stratifying patients with SDHx mutations. PHEO/PGL tumor specimens from 233 patients, including 45 with SDHx mutations, were provided from eight tertiary referral centers for mass spectrometric analyses of Krebs cycle metabolites. Diagnostic performance of the succinate:fumarate ratio was used for the identification of pathogenic SDHx mutations. SDH-deficient PHEOs/PGLs were characterized by 25-fold higher succinate and 80% lower fumarate, cis-aconitate, and isocitrate tissue levels than PHEOs/PGLs without SDHx mutations. Receiver-operating characteristic curves for the use of succinate to fumarate or to cis-aconitate and isocitrate ratios to identify SDHx mutations indicated areas under the curves of 0.94 to 0.96; an optimal cut-off of 97.7 for the succinate:fumarate ratio provided a diagnostic sensitivity of 93% at a specificity of 97% to identify SDHx-mutated PHEOs/PGLs. Succinate:fumarate ratios were higher in both SDHB-mutated and metastatic tumors than in those due to SDHD/C mutations or without metastases. Mass spectrometric-based measurements of ratios of succinate:fumarate and other metabolites in PHEOs/PGLs offer a useful method to identify patients for testing of SDHx mutations, with additional utility to quantitatively assess functionality of mutations and metabolic factors responsible for malignant risk. The present study investigated the impact of genetic alterations on metabolic networks in PGLs. 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 1H-NMR spectroscopy at 500 MHz for untargeted and HPLC tandem mass spectrometry for targeted metabolite profiling. 1H-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<0.0001) and low ATP/ADP/AMP content (p<0.001) in SDH related PGLs compared to sporadics and PGLs of other genotypes. Extending these results, low levels of N-acetylaspartic acid (NAA, p<0.05) in SDH tumors and creatine (p<0.05) in VHL tumors were observed compared to sporadics and other genotypes. A positive correlation was observed between NAA and ATP/ADP/AMP content (p<0.001) and NAA and complex II activity (p<0.0001) of PGLs. Targeted purine analysis in PHEOs/PGLs showed low adenine in cluster 1 compared to cluster 2 tumors (SDH p<0.0001; VHL p<0.05) while lower levels (p<0.05) of guanosine and hypoxanthine were observed in RET tumors compared to SDH tumors. Principal component analysis (PCA) of metabolites could distinguish PGLs of different genotypes. The present study gives a comprehensive picture of alterations in energy metabolism in SDH and VHL related PGLs and establishes the interrelationship between energy metabolism and amino acid and purine metabolism in PGLs. Hereditary PHEO/PGL We have investigated the genetic/pathogenetic factors associated with a new clinical entity in patients presenting with PHEO/PGL and polycythemia. Two patients without hypoxia-inducible factor 2α (HIF2A) mutations, who presented with similar clinical manifestations, were analyzed for other gene mutations, including prolyl hydroxylase (PHD) mutations. We have found for the first time a germ-line mutation in PHD1 in one patient and a novel germ-line PHD2 mutation in a second patient. Both mutants exhibited reduced protein stability with substantial quantitative protein loss and thus compromised catalytic activities. Due to the unique association of patients' polycythemia with borderline or mildly elevated erythropoietin (EPO) levels, we also performed an in vitro sensitivity assay of erythroid progenitors to EPO and for EPO receptor (EPOR) expression. The results show inappropriate hypersensitivity of erythroid progenitors to EPO in these patients, indicating increased EPOR expression/activity. In addition, the present study indicates that HIF dysregulation due to PHD mutations plays an important role in the pathogenesis of these tumors and associated polycythemia. The PHD1 mutation appears to be a new member contributing to the genetic landscape of this novel clinical entity. Our results support the existence of a specific PHD1- and PHD2-associated PHEO/PGL-polycythemia disorder. Imaging and PHEO/PGL The aim of this study was to explore the hypothesis that increased uptake of 18F-FDG in SDHx-related PHEOs/PGLs is reflective of increased glycolytic activity and is correlated with expression of different proteins involved in glucose uptake and metabolism through the glycolytic pathway. Twenty-seven PHEOs/PGLs collected from patients with hereditary mutations in SDHB (n = 2), SDHD (n = 3), RET (n = 5), neurofibromatosis 1 (n = 1), and myc-associated factor X (n = 1) as well as sporadic patients (n = 15) were investigated. Preoperative 18F-FDG PET/CT studies were analyzed; mean and maximum standardized uptake values (SUVs) in manually drawn regions of interest were calculated. The expression of proteins involved in glucose uptake (glucose transporters types 1 and 3 GLUT-1 and -3, respectively), phosphorylation (hexokinases 1, 2, and 3 HK-1, -2, and -3, respectively), glycolysis (monocarboxylate transporter type 4 MCT-4), and angiogenesis (vascular endothelial growth factor VEGF, CD34) were examined in paraffin-embedded tumor tissues using immunohistochemical staining with peroxidase-catalyzed polymerization of diaminobenzidine as a read-out. The expression was correlated with corresponding SUVs. Both maximum and mean SUVs for SDHx-related tumors were significantly higher than those for sporadic and other hereditary tumors (P < 0.01). The expression of HK-2 and HK-3 was significantly higher in SDHx-related PHEOs/PGLs than in sporadic PHEOs/PGLs (P = 0.022 and 0.025, respectively). The expression of HK-2 and VEGF was significantly higher in SDHx-related PHEOs/PGLs than in other hereditary PHEOs/PGLs (P = 0.039 and 0.008, respectively). No statistical differences in the expression were observed for GLUT-1, GLUT-3, and MCT-4. The percentage anti-CD 34 staining and mean vessel perimeter were significantly higher in SDHx-related PHEOs/PGLs than in sporadic tumors (P = 0.050 and 0.010, respectively). Mean SUVs significantly correlated with the expression of HK-2 (P = 0.027), HK-3 (P = 0.013), VEGF (P = 0.049), and MCT-4 (P = 0.020).
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