Summary We report a comprehensive molecular characterization of pheochromocytomas and paragangliomas (PCC/PGLs), a rare tumor type. Multi-platform integration revealed that PCC/PGLs are driven by diverse alterations affecting multiple genes and pathways. Pathogenic germline mutations occurred in eight PCC/PGL susceptibility genes. We identified CSDE1 as a somatically-mutated driver gene, complementing four known drivers (HRAS, RET, EPAS1, NF1). We also discovered fusion genes in PCC/PGL, involving MAML3, BRAF, NGFR and NF1. Integrated analysis classified PCC/PGLs into four molecularly-defined groups: a kinase signaling subtype, a pseudohypoxia subtype, a Wnt-altered subtype, driven by MAML3 and CSDE1, and a cortical admixture subtype. Correlates of metastatic PCC/PGL included the MAML3 fusion gene. This integrated molecular characterization provides a comprehensive foundation for developing PCC/PGL precision medicine.

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Background Malignant pheochromocytoma and paraganglioma (MPP) are characterized by prognostic heterogeneity. Our objective was to look for prognostic parameters of overall survival in MPP patients. Patients and Methods Retrospective multicentric study of MPP characterized by a neckthoraco-abdomino-pelvic CT or MRI at the time of malignancy diagnosis in European centers between 1998 and 2010. Results We included 169 patients from 18 European centers. Main characteristics of MPP patients were: primary pheochromocytoma in 53% of patients, tumor or hormone-related symptoms in 57% or 58% of cases, positive plasma or urine hormones in 81% of patients, identification of a mutation in SDHB in 42 % of cases. Metastatic sites included the bone (64%), lymph node (40%), lung (29%) and liver (26%); mean time between initial and malignancy diagnosis was 43 months (0-614). Median follow-up was 68 months and median survival 6.7 years. Using univariate analysis, better survival was associated with head and neck paraganglioma, age <40 years, metanephrines <5-fold the upper limits of the normal range and low proliferative index. In multivariate analysis, hypersecretion (Hazard Ratio 3.02[1.65-5.55]; p:0.0004) was identified as independent significant prognostic factors of worst overall survival. Conclusions Our results do not confirm SDHB mutations as a major prognostic parameter in MPP and suggest additional key molecular events involved in MPP tumor progression. Aside from SDHB mutation, the biology of aggressive MPP remains to be understood.

Adrenal phaechromocytomas and extra-adrenal sympathetic paragangliomas (PPGLs) are rare neuroendocrine tumours, characterised by production of the catecholamines: noradrenaline, adrenaline and dopamine. Tumoural secretion of catecholamines determines their clinical presentation which is highly variable among patients. Up to 10-15% of patients present entirely asymptomatic and in 5% of all adrenal incidentalomas a PPGL is found. Therefore, prompt diagnosis of PPGL remains a challenge for every clinician. Early consideration of the presence of a PPGL is of utmost importance, because missing the diagnosis can be devastating due to potential lethal cardiovascular complications of disease. First step in diagnosis is proper biochemical analysis to confirm or refute the presence of excess production of catecholamines or their metabolites. Biochemical testing is not only indicated in symptomatic patients but also in asymptomatic patients with adrenal incidentalomas or identified genetic predispositions. Measurements of metanephrines in plasma or urine offer the best diagnostic performance and are the tests of first choice. Paying attention to sampling conditions, patient preparation and use of interfering medications is important, as these factors can largely influence test results. When initial test results are inconclusive, additional tests can be performed, such as the clonidine suppression test. Test results can also be used for estimation of tumour size or prediction of tumour location and underlying genotype. Furthermore, tumoural production of 3-methoxytyramine is associated with presence of an underlying SDHB mutation and may be a biomarker of malignancy.

Pheochromocytomas and paragangliomas (PPGLs) can be localized by 18 F-FDG PET. The uptake is particularly high in tumors with an underlying succinate dehydrogenase (SDH) mutation. SDHxrelated PPGLs are characterized by compromised oxidative phosphorylation and a pseudohypoxic response, which mediates an increase in aerobic glycolysis, also known as the Warburg effect. The aim of this study was to explore the hypothesis that increased uptake of 18 F-FDG in SDHx-related PPGLs is reflective of increased glycolytic activity and is correlated with expression of different proteins involved in glucose uptake and metabolism through the glycolytic pathway. Methods: Twenty-seven PPGLs collected from patients with hereditary mutations in SDHB (n 5 2), SDHD (n 5 3), RET (n 5 5), neurofibromatosis 1 (n 5 1), and myc-associated factor X (n 5 1) and sporadic patients (n 5 15) were investigated. Preoperative 18 F-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. Results: 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 PPGLs than in sporadic PPGLs (P 5 0.022 and 0.025, respectively). The expression of HK-2 and VEGF was significantly higher in SDHx-related PPGLs than in other hereditary PPGLs (P 5 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 PPGLs than in sporadic tumors (P 5 0.050 and 0.010, respectively). Mean SUVs significantly correlated with the expression of HK-2 (P 5 0.027), HK-3 (P 5 0.013), VEGF (P 5 0.049), and MCT-4 (P 5 0.020). Conclusion: The activation of aerobic glycolysis in SDHx-related PPGLs is associated with increased 18 F-FDG accumulation due to accelerated glucose phosphorylation by hexokinases rather than increased expression of glucose transporters.

In this nationwide study of disease-affected and unaffected mutation carriers, we observed a lower rate of metastatic disease and a relatively high number of head and neck paragangliomas compared with previously reported referral-based cohorts.

Germline mutations in succinate dehydrogenase B (SDHB) predispose to hereditary paraganglioma (PGL) syndrome type 4. The risk of developing PGL or pheochromocytoma (PHEO) in SDHB mutation carriers is subject of recent debate. In the present nationwide cohort study of SDHB mutation carriers identified by the clinical genetics centers of the Netherlands, we have calculated the penetrance of SDHB associated tumors using a novel maximum likelihood estimator. This estimator addresses ascertainment bias and missing data on pedigree size and structure. A total of 195 SDHB mutation carriers were included, carrying 27 different SDHB mutations. The 2 most prevalent SDHB mutations were Dutch founder mutations: a deletion in exon 3 (31% of mutation carriers) and the c.423+1G>A mutation (24% of mutation carriers).One hundred and twelve carriers (57%) displayed no physical, radiological or biochemical evidence of PGL or PHEO. Fifty-four patients had a head and neck PGL (28%), 4 patients had a PHEO (2%), 26 patients an extra-adrenal PGL (13%). The overall penetrance of SDHB mutations is estimated to be 21% at age 50 and 42% at age 70 when adequately corrected for ascertainment. These estimates are lower than previously reported penetrance estimates of SDHB-linked cohorts. Similar disease risks are found for different SDHB germline mutations as well as for male and female SDHB mutation carriers. K E Y W O R D Sparaganglioma, penetrance, pheochromocytoma, SDHB

Static single-time-frame 18 F-FDG PET/CT is useful for the localization and functional characterization of pheochromocytomas and paragangliomas (PPGLs). 18 F-FDG uptake varies between PPGLs with different genotypes, and the highest SUVs are observed in cases of succinate dehydrogenase (SDH) mutations, possibly related to enhanced aerobic glycolysis in tumor cells. The exact determinants of 18 F-FDG accumulation in PPGLs are unknown. We performed dynamic PET/CT scanning to assess whether in vivo 18 F-FDG pharmacokinetics has added value over static PET to distinguish different genotypes. Methods: Dynamic 18 F-FDG PET/CT was performed on 13 sporadic PPGLs and 13 PPGLs from 11 patients with mutations in SDH complex subunits B and D, von Hippel-Lindau (VHL), RET, and neurofibromin 1 (NF1). Pharmacokinetic analysis was performed using a 2-tissue-compartment tracer kinetic model. The derived transfer rate-constants for transmembranous glucose flux (K 1 [in], k 2 [out]) and intracellular phosphorylation (k 3), along with the vascular blood fraction (V b), were analyzed using nonlinear regression analysis. Glucose metabolic rate (MR glc) was calculated using Patlak linear regression analysis. The SUV max of the lesions was determined on additional static PET/CT images. Results: Both MR glc and SUV max were significantly higher for hereditary cluster 1 (SDHx, VHL) tumors than for hereditary cluster 2 (RET, NF1) and sporadic tumors (P , 0.01 and P , 0.05, respectively). Median k 3 was significantly higher for cluster 1 than for sporadic tumors (P , 0.01). Median V b was significantly higher for cluster 1 than for cluster 2 tumors (P , 0.01). No statistically significant differences in K 1 and k 2 were found between the groups. Cutoffs for k 3 to distinguish between cluster 1 and other tumors were established at 0.015 min −1 (100% sensitivity, 15.8% specificity) and 0.636 min −1 (100% specificity, 85.7% sensitivity). MR glc significantly correlated with SUV max (P 5 0.001) and k 3 (P 5 0.002). Conclusion: In vivo metabolic tumor profiling in patients with PPGL can be achieved by assessing 18 F-FDG pharmacokinetics using dynamic PET/CT scanning. Cluster 1 PPGLs can be reliably identified by a high 18 F-FDG phosphorylation rate.