Mirko Peitzsch

Whole-Exome Sequencing Identifies MDH2 as a New Familial Paraganglioma Gene

Disruption of the Krebs cycle is a hallmark of cancer. IDH1 and IDH2 mutations are found in many neoplasms, and germline alterations in SDH genes and FH predispose to pheochromocytoma/paraganglioma and other cancers. We describe a paraganglioma family carrying a germline mutation in MDH2, which encodes a Krebs cycle enzyme. Whole-exome sequencing was applied to tumor DNA obtained from a man age 55 years diagnosed with multiple malignant paragangliomas. Data were analyzed with the two-sided Students t and Mann-Whitney U tests with Bonferroni correction for multiple comparisons. Between six- and 14-fold lower levels of MDH2 expression were observed in MDH2-mutated tumors compared with control patients. Knockdown (KD) of MDH2 in HeLa cells by shRNA triggered the accumulation of both malate (mean ± SD: wild-type [WT] = 1±0.18; KD = 2.24±0.17, P = .043) and fumarate (WT = 1±0.06; KD = 2.6±0.25, P = .033), which was reversed by transient introduction of WT MDH2 cDNA. Segregation of the mutation with disease and absence of MDH2 in mutated tumors revealed MDH2 as a novel pheochromocytoma/paraganglioma susceptibility gene.

Imaging Work-Up for Screening of Paraganglioma and Pheochromocytoma in SDHx Mutation Carriers: A Multicenter Prospective Study from the PGL.EVA Investigators

CONTEXT Recommendations have not been established concerning imaging to screen SDHx mutation carriers for paraganglioma and pheochromocytoma. OBJECTIVE Our objective was to compare the performance of gadolinium-enhanced magnetic resonance angiography, contrast-enhanced computed tomography, and [(123)I]metaiodo-benzylguanidine and somatostatin receptor scintigraphies for detecting head and neck and thoracic-abdominal-pelvic paragangliomas in SDHx mutation carriers. DESIGN AND SETTING We conducted a prospective, multicenter study from June 2005 to December 2009 at 23 French medical centers. PATIENTS A total of 238 index cases or relatives carrying mutations in SDHD, SDHB, or SDHC genes were included. INTERVENTION Images obtained by each technique were analyzed blind, without knowledge of results from other tests, first in each local center and then centrally. MAIN OUTCOME MEASURES We evaluated sensitivity, specificity, and likelihood ratios for individual and combinations of tests, the gold standard being the consensus of an expert committee. RESULTS Two hundred two tumors were diagnosed in 96 subjects. At local assessment, the sensitivity of anatomical imaging for detecting all tumors was higher (85.7%) than that of both scintigraphic techniques (42.7% for [(123)I]metaiodo-benzylguanidine and 69.5% for somatostatin receptor scintigraphy), except for thoracic localizations where somatostatin receptor scintigraphy was more sensitive (61.5 vs. 46.2% for anatomical imaging and 30.8% for [(123)I]metaiodo-benzylguanidine scintigraphy). The best diagnostic performance during local assessment was obtained by combining anatomical imaging tests and somatostatin receptor scintigraphy (sensitivity 91.7%). Central assessment significantly increased the sensitivity (98.6%) of tests in combination. CONCLUSIONS In routine practice, the imaging work-up for screening SDHx mutation carriers should include thoraco-abdomino-pelvic computed tomography, head and neck magnetic angiography, and somatostatin receptor scintigraphy. Expert centralized image assessment is recommended.

Genotype-Specific Steroid Profiles Associated With Aldosterone-Producing Adenomas

Primary aldosteronism comprises 2 main subtypes: unilateral aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia. Somatic KCNJ5 mutations are found in APA at a prevalence of around 40% that drive and sustain aldosterone excess. Somatic APA mutations have been described in other genes (CACNA1D, ATP1A1, and ATP2B3) albeit at a lower frequency. Our objective was to identify genotype-specific steroid profiles in adrenal venous (AV) and peripheral venous (PV) plasma in patients with APAs. We measured the concentrations of 15 steroids in AV and PV plasma samples by liquid chromatography–tandem mass spectrometry from 79 patients with confirmed unilateral primary aldosteronism. AV sampling lateralization ratios of steroids normalized either to cortisol or to DHEA+androstenedione were also calculated. The hybrid steroid 18-oxocortisol exhibited 18- and 16-fold higher concentrations in lateralized AV and PV plasma, respectively, from APA with KCNJ5 mutations compared with all other APA combined together (P<0.001). Lateralization ratios for the KCNJ5 group were also generally higher. Strikingly, we demonstrate that a distinct steroid signature can differentiate APA genotype in AV and PV plasma. Notably, a 7-steroid fingerprint in PV plasma correctly classified 92% of the APA according to genotype. Prospective studies are necessary to translate these findings into clinical practice and determine if steroid fingerprinting could be of value to select patients with primary aldosteronism who are particularly suitable candidates for adrenal venous sampling because of a high probability of having an APA.

Biochemical diagnosis of phaeochromocytoma using plasma-free normetanephrine, metanephrine and methoxytyramine: importance of supine sampling under fasting conditions.

To document the influences of blood sampling under supine fasting versus seated nonfasting conditions on diagnosis of phaeochromocytomas and paragangliomas (PPGL) using plasma concentrations of normetanephrine, metanephrine and methoxytyramine.

Mass Spectrometry–Based Adrenal and Peripheral Venous Steroid Profiling for Subtyping Primary Aldosteronism

BACKGROUND Differentiating patients with primary aldosteronism caused by aldosterone-producing adenomas (APAs) from those with bilateral adrenal hyperplasia (BAH), which is essential for choice of therapeutic intervention, relies on adrenal venous sampling (AVS)-based measurements of aldosterone and cortisol. We assessed the utility of LC-MS/MS-based steroid profiling to stratify patients with primary aldosteronism. METHODS Fifteen adrenal steroids were measured by LC-MS/MS in peripheral and adrenal venous plasma from AVS studies for 216 patients with primary aldosteronism at 3 tertiary referral centers. Ninety patients were diagnosed with BAH and 126 with APAs on the basis of immunoassay-derived adrenal venous aldosterone lateralization ratios. RESULTS Among 119 patients confirmed to have APAs at follow-up, LC-MS/MS-derived lateralization ratios of aldosterone normalized to cortisol, dehydroepiandrosterone, and androstenedione were all higher (P < 0.0001) than immunoassay-derived ratios. The hybrid steroids, 18-oxocortisol and 18-hydroxycortisol, also showed lateralized secretion in 76% and 35% of patients with APAs. Adrenal venous concentrations of glucocorticoids and androgens were bilaterally higher in patients with BAH than in those with APAs. Consequently, peripheral plasma concentrations of 18-oxocortisol were 8.5-fold higher, whereas concentrations of cortisol, corticosterone, and dehydroepiandrosterone were lower in patients with APAs than in those with BAH. Correct classification of 80% of cases of APAs vs BAH was thereby possible by use of a combination of steroids in peripheral plasma. CONCLUSIONS LC-MS/MS-based steroid profiling during AVS achieves higher aldosterone lateralization ratios in patients with APAs than immunoassay. LC-MS/MS also enables multiple measures for discriminating unilateral from bilateral aldosterone excess, with potential use of peripheral plasma for subtype classification.

Krebs Cycle Metabolite Profiling for Identification and Stratification of Pheochromocytomas/ Paragangliomas due to Succinate Dehydrogenase Deficiency

CONTEXT Mutations of succinate dehydrogenase A/B/C/D genes (SDHx) increase susceptibility to development of pheochromocytomas and paragangliomas (PPGLs), with particularly high rates of malignancy associated with SDHB mutations. OBJECTIVE 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. DESIGN, SETTING, AND PATIENTS PPGL 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. MAIN OUTCOME MEASURE Diagnostic performance of the succinate:fumarate ratio for identification of pathogenic SDHx mutations. RESULTS SDH-deficient PPGLs were characterized by 25-fold higher succinate and 80% lower fumarate, cis-aconitate, and isocitrate tissue levels than PPGLs without SDHx mutations. Receiver-operating characteristic curves for use of ratios of succinate to fumarate or to cis-aconitate and isocitrate to identify SDHx mutations indicated areas under 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 PPGLs. Succinate:fumarate ratios were higher in both SDHB-mutated and metastatic tumors than in those due to SDHD/C mutations or without metastases. CONCLUSIONS Mass spectrometric-based measurements of ratios of succinate:fumarate and other metabolites in PPGLs 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.

Opposing effects of HIF1α and HIF2α on chromaffin cell phenotypic features and tumor cell proliferation: Insights from MYC-associated factor X

Pheochromocytomas and paragangliomas (PPGLs) are catecholamine‐producing chromaffin cell tumors with diverse phenotypic features reflecting mutations in numerous genes, including MYC‐associated factor X (MAX). To explore whether phenotypic differences among PPGLs reflect a MAX‐mediated mechanism and opposing influences of hypoxia‐inducible factor (HIF)s HIF2α and HIF1α, we combined observational investigations in PPGLs and gene‐manipulation studies in two pheochromocytoma cell lines. Among PPGLs from 140 patients, tumors due to MAX mutations were characterized by gene expression profiles and intermediate phenotypic features that distinguished these tumors from other PPGLs, all of which fell into two expression clusters: one cluster with low expression of HIF2α and mature phenotypic features and the other with high expression of HIF2α and immature phenotypic features due to mutations stabilizing HIFs. Max‐mutated tumors distributed to a distinct subcluster of the former group. In cell lines lacking Max, re‐expression of the gene resulted in maturation of phenotypic features and decreased cell cycle progression. In cell lines lacking Hif2α, overexpression of the gene led to immature phenotypic features, failure of dexamethasone to induce differentiation and increased proliferation. HIF1α had opposing actions to HIF2α in both cell lines, supporting evolving evidence of their differential actions on tumorigenic processes via a MYC/MAX‐related pathway. Requirement of a fully functional MYC/MAX complex to facilitate differentiation explains the intermediate phenotypic features in tumors due to MAX mutations. Overexpression of HIF2α in chromaffin cell tumors due to mutations affecting HIF stabilization explains their proliferative features and why the tumors fail to differentiate even when exposed locally to adrenal steroids.

Plasma metanephrine for assessing the selectivity of adrenal venous sampling

Adrenal vein sampling is used to establish the origins of excess production of adrenal hormones in primary aldosteronism. Correct catheter positioning is confirmed using adrenal vein measurements of cortisol, but this parameter is not always reliable. Plasma metanephrine represents an alternative parameter. The objective of our study was to determine the use of plasma metanephrine concentrations to establish correct catheter positioning during adrenal vein sampling with and without cosyntropin stimulation. We included 52 cosyntropin-stimulated and 34 nonstimulated sequential procedures. Plasma cortisol and metanephrine concentrations were measured in adrenal and peripheral venous samples. Success rates of sampling, using an adrenal to peripheral cortisol selectivity index of 3.0, were compared with success rates of metanephrine using a selectivity index determined by receiver operating characteristic curve analysis. Among procedures assessed as selective using cortisol, the adrenal to peripheral vein ratio of metanephrine was 6-fold higher than that of cortisol (94.0 versus 15.5; P<0.0001). There were significant positive relationships between adrenal to peripheral vein ratios of cortisol and metanephrine for cosyntropin-stimulated samplings but not for nonstimulated samplings. Receiver operating characteristic curve analysis indicated a plasma metanephrine selectivity index cutoff of 12. Using this cutoff, concordance in sampling success rates determined by cortisol and metanephrine was substantially higher in cosyntropin-stimulated than in nonstimulated samplings (98% versus 59%). For the latter procedures, sampling success rates determined by metanephrine were higher (P<0.01) than those determined by cortisol (91% versus 56%). In conclusion, metanephrine provides a superior analyte compared with cortisol in assessing the selectivity of adrenal vein sampling during procedures without cosyntropin stimulation.

Levodopa therapy in Parkinson’s disease: influence on liquid chromatographic tandem mass spectrometric-based measurements of plasma and urinary normetanephrine, metanephrine and methoxytyramine

Background Medication-related interferences with measurements of catecholamines and their metabolites represent important causes of false-positive results during diagnosis of phaeochromocytomas and paragangliomas (PPGLs). Such interferences are less troublesome with measurements by liquid chromatography with tandem mass-spectrometry (LC-MS/MS) than by other methods, but can still present problems for some drugs. Levodopa, the precursor for dopamine used in the treatment of Parkinson’s disease, represents one potentially interfering medication. Methods Plasma and urine samples, obtained from 20 Parkinsonian patients receiving levodopa, were analysed for concentrations of catecholamines and their O-methylated metabolites by LC-MS/MS. Results were compared with those from a group of 120 age-matched subjects and 18 patients with PPGLs. Results Plasma and urinary free and deconjugated (free + conjugated) methoxytyramine, as well as urinary dopamine, showed 22- to 148-fold higher (P < 0.0001) concentrations in patients receiving levodopa than in the reference group. In contrast, plasma normetanephrine, urinary noradrenaline and urinary free and deconjugated normetanephrine concentrations were unaffected. Plasma free metanephrine, urinary adrenaline and urinary free and deconjugated metanephrine all showed higher (P < 0.05) concentrations in Parkinsonian patients than the reference group, but this was only a problem for adrenaline. Similar to normetanephrine, plasma and urinary metanephrine remained below the 97.5 percentiles of the reference group in almost all Parkinsonian patients. Conclusions These data establish that although levodopa treatment confounds identification of PPGLs that produce dopamine, the therapy is not a problem for use of LC-MS/MS measurements of plasma and urinary normetanephrine and metanephrine to diagnose more commonly encountered PPGLs that produce noradrenaline or adrenaline.

Characteristics of Pediatric vs Adult Pheochromocytomas and Paragangliomas

Context Pheochromocytomas and paragangliomas (PPGLs) in children are often hereditary and may present with different characteristics compared with adults. Hereditary PPGLs can be separated into cluster 1 and cluster 2 tumors due to mutations impacting hypoxia and kinase receptor signaling pathways, respectively. Objective To identify differences in presentation of PPGLs between children and adults. Design A retrospective cross-sectional clinical study. Setting Seven tertiary medical centers. Patients The study included 748 patients with PPGLs, including 95 with a first presentation during childhood. Genetic testing was available in 611 patients. Other data included locations of primary tumors, presence of recurrent or metastatic disease, and plasma concentrations of metanephrines and 3-methoxytyramine. Results Children showed higher (P < 0.0001) prevalence than adults of hereditary (80.4% vs 52.6%), extra-adrenal (66.3% vs 35.1%), multifocal (32.6% vs 13.5%), metastatic (49.5% vs 29.1%), and recurrent (29.5% vs 14.2%) PPGLs. Tumors due to cluster 1 mutations were more prevalent among children than adults (76.1% vs 39.3%; P < 0.0001), and this paralleled a higher prevalence of noradrenergic tumors, characterized by relative lack of increased plasma metanephrine, in children than in adults (93.2% vs 57.3%; P < 0.0001). Conclusions The higher prevalence of hereditary, extra-adrenal, multifocal, and metastatic PPGLs in children than adults represents interrelated features that, in part, reflect the lower age of disease presentation of noradrenergic cluster 1 than adrenergic cluster 2 tumors. The differences in disease presentation are important to consider in children at risk for PPGLs due to a known mutation or previous history of tumor.