Graeme Eisenhofer

Background: Testing for SDHB mutations is recommended in all patients presenting with metastatic pheochromocytomas or paragangliomas (PPGLs). However, PPGLs among patients with SDHB mutations do not produce adrenaline (Adr), suggesting testing for SDHB mutations may not be required when metastatic disease is accompanied by Adr production. Methods: Presence of SDHB mutations or deletions was tested for by Sanger sequencing and multiplex ligand probe amplification in 186 patients (101 males) aged 41 ± 16 yr at diagnosis of metastatic PPGLs (age range, 9 to 86 yr). Plasma concentrations of normetanephrine (NMN) and metanephrine (MN) were measured in all patients at presentation of metastatic disease. Adr-producing tumors were defined by increased plasma concentrations of MN above the upper cut-offs (88 pg/mL) and with increases larger than 5% of the combined increases of both plasma NMN and MN. Results: Twenty-two of the 186 patients (12%) with metastatic PPGLs had disease characterized by production of Adr, none of which had SDHB mutations. Of the other 165 patients with no tumoral Adr production, a much higher (P < 0.0001) proportion of 90 patients (55%) had SDHB mutations. Among all patients, 140 (75%) had metastases in bone, 74 (40%) in liver and 66 (35%) in lungs. Bone metastases were more prevalent among patients with than without SDHB mutations (85% vs. 66%, p < 0.005), and liver metastases were more prevalent among patients with adrenal than extra-adrenal primary tumors (57% vs. 31%, p = 0.0045). Conclusions: Lack of findings of SDHB mutations in patients with Adr-producing metastatic PPGLs indicates that it is unnecessary to test for SDHB mutations among such patients. In contrast, the high prevalence of SDHB mutations in other patients with metastatic PPGLs supports recommendations that these patients should all receive SDHB mutation testing. Our data also indicate associations of patterns of metastatic spread with primary tumor location and presence of SDHB mutations.

Mutations in succinate dehydrogenase (SDH) genes are responsible for cancer formation in certain tumors, including in pheochromocytomas and paragangliomas (PPGLs). Especially mutations in SDH subunit B result in tumors with immature phenotypic features of low catecholamine contents, significant production of dopamine, and high prevalence to metastases. The present study evaluates the succinate: fumarate ratio (SFR) as a new biomarker for identification of SDHx mutations in PPGLs and also assesses changes in the metabolome more broadly. Tumor tissue metabolites (including most Krebs cycle intermediates, pyruvate and lactate) of 234 PPGLs were measured by LC-MS/MS. Among our patient cohort, 45 had confirmed germline mutations in SDHB, SDHC or SDHD. A subset of samples (50) formed a training set and the majority (184) was analysed blind to the mutational status in a validation series. Predictions about the presence of SDHx mutations were provided based on the SFR. Median SFRs in tumors from patients with SDHx germline mutations were dramatically increased by 454-fold in the training and 94-fold in the validation set above tumors without SDHx mutations. Succinate was elevated 25-fold in tumors of patients with SDHx germline mutations, whereas fumarate, citrate, cis-aconitate and isocitrate were respectively decreased by 80%, 60%, 82%, and 80%. The diagnostic performance of the SFR as a sole predictor for SDHx mutations was estimated at 97% specificity and 93% sensitivity. With exclusion of head and neck paragangliomas sensitivity reached 100%. Among patients with SDHx mutations, higher SFRs were measured in tumors due to SDHB mutations or associated with metastatic disease than in those due to SDHC or SDHD mutations or without metastatic disease. The SFR of PPGLs predicts the presence of SDHx mutations with high diagnostic performance, and may also be useful for assessing metabolic factors responsible for malignant risk and responses to various treatment options.

All of more than 30 neuroimaging studies and at least 4 postmortem pathology studies have agreed that Parkinson disease entails a loss of myocardial noradrenergic innervation, implying that in addition to being a movement disorder from loss of nigrostriatal dopaminergic neurons, Parkinson disease is also a dysautonomia from loss of postganglionic noradrenergic neurons. Cardiac sympathetic denervation has been reported even in the earliest stages of the disease; however, whether the denervation can actually precede the movement disorder has been unknown. As part of an evaluation of hemodynamic instability suggesting pheochromocytoma, a 52-year-old man underwent 6-[18F]fluorodopamine scanning and other autonomic function tests. Four years later, over the course of 6-12 months, he developed slow movement, limb rigidity, masked face, and small handwriting and was diagnosed with mild Parkinson disease. Review of the earlier 6-[18F]fluorodopamine scan revealed that the patient had had markedly decreased 6-[18F]fluorodopamine-derived radioactivity throughout the left ventricular myocardium, confirmed by follow-up scanning. He also had had evidence of low baroreflex-cardiovagal gain and excessive adrenomedullary secretion, also confirmed at follow-up. In the interval, baroreflex-sympathoneural function declined, as indicated by development of abnormal beat-to-beat blood pressure responses to the Valsalva maneuver; however, the patient did not have orthostatic hypotension. The combination of denervation supersensitivity, baroreflex failure, and increased adrenomedullary secretion may have contributed to the patient's complaints related to neurocirculatory instability. The results lead to the proposition that cardiac sympathetic denervation may be a biomarker of presymptomatic Parkinson disease.

Objective: Phaeochromocytomas in patients with multiple endocrine neoplasia type 2 (MEN 2) produce adrenaline, whereas those with von Hippel-Lindau (VHL) syndrome do not. This study assessed whether these distinctions relate to differences in expression of the transporters responsible for uptake and storage of catecholamines – the noradrenaline transporter and the vesicular monoamine transporters (VMAT 1 and VMAT 2). Methods: Tumour tissue and plasma samples were obtained from 31 patients with hereditary phaeochromocytoma − 18 with VHL syndrome and 13 with MEN 2. We used quantitative PCR, Western blotting, electron microscopy, immunohistochemistry and measurements of plasma and tumour catecholamines to assess differences in expression of the transporters in noradrenaline-producing vs adrenaline-producing hereditary tumours. These differences were compared with those in a further group of 26 patients with non-syndromic phaeochromocytoma. Results: Adrenaline-producing phaeochromocytomas in MEN 2 patients expressed more noradrenaline transporter mRNA and protein than noradrenaline-producing tumours in VHL patients. In contrast, there was greater expression of VMAT 1 in VHL than MEN 2 tumours, while expression of VMAT 2 did not differ significantly. These differences were associated with larger numbers of storage vesicles and higher tissue contents of catecholamines in MEN 2 than in VHL tumours. Differences in expression of the noradrenaline transporter were weaker, and those of VMAT 1 and VMAT 2 stronger, in noradrenaline and adrenaline-producing non-syndromic than in hereditary tumours. Conclusions: The findings show that, in addition to differences in catecholamine biosynthesis, phaeochromocytomas in MEN 2 and VHL syndrome also differ in expression of the transporters responsible for uptake and vesicular storage of catecholamines. Abstract Objective: Phaeochromocytomas in patients with multiple endocrine neoplasia type 2 (MEN 2) produce adrenaline, whereas those with von Hippel-Lindau (VHL) syndrome do not. This study assessed whether these distinctions relate to differences in expression of the transporters responsible for uptake and storage of catecholamines – the noradrenaline transporter and the vesicular monoamine transporters (VMAT 1 and VMAT 2). Methods: Tumour tissue and plasma samples were obtained from 31 patients with hereditary phaeochromocytoma − 18 with VHL syndrome and 13 with MEN 2. We used quantitative PCR, Western blotting, electron microscopy, immunohistochemistry and measurements of plasma and tumour catecholamines to assess differences in expression of the transporters in noradrenaline-producing vs adrenaline-producing hereditary tumours. These differences were compared with those in a further group of 26 patients with non-syndromic phaeochromocytoma. Results: Adrenaline-producing phaeochromocytomas in MEN 2 patients expressed more noradrenaline transporter mRNA and protein than noradrenaline-producing tumours in VHL patients. In contrast, there was greater expression of VMAT 1 in VHL than MEN 2 tumours, while expression of VMAT 2 did not differ significantly. These differences were associated with larger numbers of storage vesicles and higher tissue contents of catecholamines in MEN 2 than in VHL tumours. Differences in expression of the noradrenaline transporter were weaker, and those of VMAT 1 and VMAT 2 stronger, in noradrenaline and adrenaline-producing non-syndromic than in hereditary tumours. Conclusions: The findings show that, in addition to differences in catecholamine biosynthesis, phaeochromocytomas in MEN 2 and VHL syndrome also differ in expression of the transporters responsible for uptake and vesicular storage of catecholamines.

Radioisotope dilution measurements of norepinephrine spillover (rate of entry of the transmitter into plasma) provide more accurate assessments of sympathoneural transmitter release than allowed by measurements of plasma catecholamine concentrations alone. Measurements of total body norepinephrine spillover, as an index of global sympathetic outflow, allow effects on plasma clearance to be distinguished from effects on release of catecholamines into plasma,while spillovers from specific tissues enable examination of regionalized sympathetic responses. However, spillovers of norepinephrine represent only a fraction of the transmitter that escapes neuronal and extraneuronal uptake after release by nerves. Numerous factors may influence this fraction and measures spillovers independently of transmitter release by nerves. Modified radioisotope dilution methods for assessment of rate processes operating within and between intracellular and extracellular compartments have further improved our understanding of the relationships of norepinephrine release, uptake, spillover, turnover, and metabolism. This article reviews the breadth of information about sympathetic nerve function attainable using catecholamine radioisotope dilution analyses against a backdrop of the relative advantages and methodological limitations associated with the methodology.

Measurements of plasma-free normetanephrine and metanephrine provide a sensitive test for diagnosis of pheochromocytoma but may fail to detect tumors that produce predominantly dopamine. Such tumors are extremely rare, usually found as extraadrenal paragangliomas. This report describes measurements of plasma concentrations of free methoxytyramine, the O-methylated metabolite of dopamine, in 120 patients with catecholamine-producing tumors, including nine with extraadrenal paragangliomas secreting predominantly dopamine. In seven of these nine patients, tumors were found incidentally or secondary to the space-occupying complications of the lesions. Plasma concentrations of free methoxytyramine and dopamine were increased in all nine patients, including two with normal plasma and urinary normetanephrine and metanephrine and normal urinary outputs of dopamine. Relative increases above normal for plasma methoxytyramine (104-fold) and dopamine (56-fold) were much greater (P < 0.001) than those for urinary dopamine (3-fold). Insensitivity of the latter for identification of dopamine-secreting tumors was due to dependence of the urinary amine on renal extraction and decarboxylation of circulating 3,4-dihydroxyphenylalanine. Measurements of plasma-free methoxytyramine, in addition to normetanephrine and metanephrine, are unlikely to improve diagnosis of pheochromocytomas in hypertensive patients with symptoms of catecholamine excess but may be useful in selected patients for identification of tumors that produce predominantly dopamine.

Background: Measurements of plasma free metanephrines (normetanephrine and metanephrine) provide a useful test for diagnosis of pheochromocytoma and may provide other information about the nature of these tumors.

PNMT (phenylethanolamine-N-methyl-transferase) is the enzyme that catalyzes the formation of epinephrine from norepinephrine. In transgenic mice over-expressing PNMT, observations revealed a very high level of aggression compared to their background strain, C57BL/6J. To evaluate the influence of PNMT on aggression and emotionality in this transgenic line, single-sex male and female groups were independently established that consisted of either four wild-type mice or four transgenic mice overexpressing PNMT. The members of each group were littermates. Mixed single-sex groups consisting of two transgenic mice and two wild-type mice were also established. Almost no fights were observed within the female groups. In males, the transgenic line showed a significantly higher level of fighting than controls (p = 0.007) and mixed male groups (p = 0.02). Housing mice from the transgenic line in mixed groups with wild-type mice seems to decrease the level of aggression in the transgenic line. In conclusion, this is the first study to demonstrate a clear, significant increase in aggression arising from PNMT overexpression. This suggests an important role for central epinephrine levels in aggressive behavior.

Pheochromocytomas are rare catecholamine-producing neuroendocrine tumors that are usually benign, but which may also present as or develop into a malignancy. Predicting such behavior is notoriously difficult and there are currently no curative treatments for malignant tumors. This report follows from a workshop at the Banbury Conference Center, Cold Spring Harbor, New York, on the 16th–18th November 2003, held to review the state of science and to facilitate future progress in the diagnosis and treatment of malignant pheochromocytoma. The rarity of the tumor and the resulting fragmented nature of studies, typically involving small numbers of patients, represent limiting factors to the development of effective treatments and diagnostic or prognostic markers for malignant disease. Such development is being facilitated by the availability of new genomics-based tools, but for such approaches to succeed ultimately requires comprehensive clinical studies involving large numbers of patients, stringently collected clinical data and tumor samples, and interdisciplinary collaborations among multiple specialist centers. Nevertheless, the well-characterized hereditary basis and the unique functional nature of these neuroendocrine tumors provide a useful framework that offers advantages for establishing the pathways of tumorigenesis and malignancy. Such findings may have relevance for understanding the basis of other more common malignancies where similar frameworks are not available. As the relevant pathways leading to pheochromocytoma are established it should be possible to take advantage of the new generation of drugs being developed to target specific pathways in other malignancies. Again the success of this will require well-designed and coordinated multicenter studies.