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. Biochemical aspects of PHEO/PGL Measurement of plasma/urinary catecholamine metabolitesespecially normetanephrine (NMN)currently represents a gold standard in biochemical detection of succinate dehydrogenase subunit B (SDHB) and D (SDHD)-related PHEO and PGL.
We aim ed to assess diagnostic utility of chromogranin A (CgA) alone or in combination with NMN in patients with PHEO and PGL related to mutations in SDHB and SDHD. CgA and NMN were measured in forty-one patients with SDHB mutation-related PHEO/PGL and 18 patients with either SDHD or SDHB mutation-related head and neck PGL (HNPGL) at the time of diagnosis at NIH. In the SDHB group, CgA showed a sensitivity of 73.2% and a specificity of 95.9%, while for NMN they were 68.3% and 98.6%, respectively. Elevations in CgA and NMN were complimentary in 92.7% of patients with proven tumors. Both tests performed well on receiver operating characteristic curve analysis. CgA levels were elevated in 76.9% of patients in the general SDHB cohort with proven disease and in 80% of patients with metastatic disease and normal NMN levels. CgA value in patients with HNPGL was significantly lower. We concluded that CgA is a valuable complimentary biomarker in work-up of SDHB-related PHEOs/PGLs. In combination with measurements of plasma NMN, CgA further enhances the detection of these tumors by 24.4%. Although non-specific for PHEO/PGL, CgA may well supplement plasma NMN to facilitate diagnostic evaluation of SDHB-related PHEOs/PGLs, especially where the measurement of plasma MNs could otherwise be delayed by decreased availability or cost restriction. Hereditary PHEO/PGL The occurrence of ≥two distinct types of tumors, one of them PGL, is unusual in an individual patient, except in hereditary cancer syndromes. Four unrelated patients were investigated, with thorough clinical evaluation. Plasma and tissue catecholamines and MNs were measured by high-performance liquid chromatography. Anatomic and functional imaging was performed for tumor visualization. Germline and tumor tissue DNA were analyzed for hypoxia-inducible factor 2 alpha (HIF2A) mutations. The prolyl hydroxylation and stability of the mutant HIF-2αprotein, transcriptional activity of mutant HIF2A, and expression of hypoxia-related genes were also investigated. Immunohistochemical staining for HIF-1/2αwas performed on formalin-fixed, paraffin-embedded tumor tissue. Patients were found to have polycythemia, multiple PGLs, and duodenal somatostatinomas by imaging or biochemistry with somatic gain-of-function HIF2A mutations. Each patient carried an identical unique mutation in both types of tumors but not in germline DNA. The HIF2A mutations in these patients were clustered adjacent to an oxygen-sensing proline residue, affecting HIF-2αinteraction with the prolyl hydroxylase domain 2-containing protein, decreasing the hydroxylation of HIF-2α, and reducing HIF-2αaffinity for the von Hippel-Lindau protein and its degradation. An increase in the half-life of HIF-2αwas associated with upregulation of the hypoxia-related genes EPO, VEGFA, GLUT1, and END1 in tumors. Our findings indicate the existence of a new syndrome with multiple PGLs and somatostatinomas associated with polycythemia. This new syndrome results from somatic gain-of-function HIF2A mutations, which cause an upregulation of hypoxia-related genes, including EPO and genes important in cancer biology. To identify mutation/location-related characteristics among pseudohypoxic PHEOs/PGLs, we used comprehensive microarray profiling (SDHB: n = 18, SDHD-abdominal/thoracic (AT): n = 6, SDHD-head/neck (HN): n = 8, VHL: n = 13). To avoid location-specific bias, typical adrenal medulla genes were derived from matched normal medullas and cortices (n = 8) for data normalization. Unsupervised analysis identified two dominant clusters, separating SDHB and SDHD-AT PHEOs/PGLs (cluster A) from VHL PHEOs and SDHD-HN PGLs (cluster B). Supervised analysis yielded 6937 highly predictive genes (misclassification error rate of 0.175). Enrichment analysis revealed that energy metabolism and inflammation/fibrosis-related genes were most pronouncedly changed in clusters A and B, respectively. A minimum subset of 40 classifiers was validated by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction vs. microarray: r = 0.87). Expression of several individual classifiers was identified as characteristic for VHL and SDHD-HN PHEOs and PGLs. In the present study, we show for the first time that SDHD-HN PGLs share more features with VHL PHEOs than with SDHD-AT PGLs. The presented data suggested novel subclassification of pseudohypoxic PHEOs/PGLs and implies cluster-specific pathogenic mechanisms and treatment strategies. Imaging and PHEO/PGL One hundred sixteen patients (39.7% had germline mutations in known disease susceptibility genes) were evaluated for a total of 195 PHEO/PGL foci. 18F-FDOPA PET correctly detected 179 lesions (91.8%) in 107 patients (92.2%). Lesion-based sensitivities for parasympathetic PGLs (head, neck, or anterior/middle thoracic ones), PHEOs, and extra-adrenal sympathetic (abdominal or posterior thoracic) PGLs were 98.2% 96.5% for Timone and 100% for NIH, 93.9% 93.8 and 93.9% and 70.3% 47.1 and 90%, respectively (p <0.001). Sympathetic (adrenal and extra-adrenal) SDHx-related PGLs were at a higher risk for negative 18F-FDOPA PET than non-SDHx-related PGLs (14/24 vs 0/62, respectively, p <0.001). In contrast, the risk of negative 18F-FDOPA PET was lower for parasympathetic PGLs regardless of the genetic background (1/90 in SDHx vs 1/19 in non-SDHx tumors, p = 0.32). 18F-FDOPA PET failed to detect two head and neck PGLs (HNPGL), likely due to their small size, whereas most missed sympathetic PGL were larger and may have exhibited a specific 18F-FDOPA-negative imaging phenotype. 18F-FDG PET detected all the missed sympathetic lesions. In summary, 18F-FDOPA PET appeared to be a very sensitive functional imaging tool for HNPGL regardless of the genetic status of the tumors. Patients with false-negative tumors on 18F-FDOPA PET should be tested for SDHx mutations. Treatment and PHEO/PGL Drug repurposing is an important part of drug discovery. We applied this paradigm in the screening of an approximately 3,800 compound library (including FDA-approved drugs and pharmacologically active compounds) employing a model of metastatic PHEO, the most common tumor of the adrenal medulla in children and adults. Analysis of the dose-response screening data facilitated the selection of 50 molecules with potential bioactivity in PHEO cells. We participated in the PHEO/PGL Guideline Task Force for US Endocrine Society.
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