Diagnosis of silent pheochromocytoma and paraganglioma

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1 Expert of Endocrinology & Metabolism ISSN: (Print) (Online) Journal homepage: Diagnosis of silent pheochromocytoma and paraganglioma Roderick Clifton-Bligh To cite this article: Roderick Clifton-Bligh (2013) Diagnosis of silent pheochromocytoma and paraganglioma, Expert of Endocrinology & Metabolism, 8:1, 47-57, DOI: / eem To link to this article: Published online: 10 Jan Submit your article to this journal Article views: 503 View related articles Citing articles: 2 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 22 November 2017, At: 09:24

2 CME For reprint orders, please contact Diagnosis of silent pheochromocytoma and paraganglioma Expert Rev. Endocrinol. Metab. 8(1), (2013) Roderick Clifton-Bligh University of Sydney, Sydney, NSW, Australia and Royal North Shore Hospital, St Leonards, NSW 2065, Australia Tel.: Fax: Pheochromocytomas or functioning paragangliomas can present in a dramatic manner with headache, palpitations and sometimes shock, but many occur with few symptoms despite at times markedly elevated catecholamine levels. Hypertension is not invariable, and may be paroxysmal. Increased diligence in the diagnosis of presymptomatic pheochromocytoma/paraganglioma is warranted from autopsy studies, suggesting that many of these tumors may be fatal at first presentation. Fortunately, an increasing number of pheochromocytomas/paragangliomas are now diagnosed before the advent of symptoms, either as an incidental finding on abdominal imaging or by targeted surveillance in subjects with known genetic susceptibility. The challenges and pitfalls associated with diagnosis of these silent pheochromocytoma/paragangliomas are reviewed in this article. Keywords: catecholamine multiple endocrine neoplasia neurofibromatosis paraganglioma phaeochromocytoma succinate dehydrogenase von Hippel Lindau Medscape: Continuing Medical Education Online This activity has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education through the joint sponsorship of Medscape, LLC and Expert s Ltd. Medscape, LLC is accredited by the ACCME to provide continuing medical education for physicians. Medscape, LLC designates this Journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit(s). Physicians should claim only the credit commensurate with the extent of their participation in the activity. All other clinicians completing this activity will be issued a certificate of participation. To participate in this journal CME activity: (1) review the learning objectives and author disclosures; (2) study the education content; (3) take the post-test with a 70% minimum passing score and complete the evaluation at (4) view/print certificate. Release date: 21 December 2012; Expiration date: 21 December 2013 Learning objectives Upon completion of this activity, participants will be able to: Analyze the biochemistry of pheochromocytoma Evaluate characteristics of imaging studies for pheochromocytoma Distinguish first-line testing in the genetic analysis of patients with pheochromocytoma Assess the use of genetic testing among patients with pheochromocytoma /EEM Expert s Ltd ISSN

3 Clifton-Bligh CME Financial & competing interests disclosure Editor Elisa Manzotti Publisher, Future Science Group, London, UK. Disclosure: Elisa Manzotti has disclosed no relevant financial relationships. CME Author Charles P Vega, MD, Health Sciences Clinical Professor; Residency Director, Department of Family Medicine, University of California, Irvine, CA, USA Disclosure: Charles P. Vega, MD, has disclosed no relevant financial relationships. Author and Credentials Roderick Clifton-Bligh, PhD, FRACP, Associate Professor in Medicine, University of Sydney, Sydney, NSW, Australia and Endocrinologist, Royal North Shore Hospital, St Leonards, NSW, Australia Disclosure: Roderick Clifton-Bligh, PhD, FRACP, has disclosed no relevant financial relationships. Tumors arising from the adrenal medulla ( pheochromocytoma [PC]) or from chromaffin tissue in the sympathetic chain (sympathetic paraganglioma [PGL]) can secrete excess catecholamines causing protean clinical manifestations including hypertension, tachycardia, hyperhidrosis, hypertensive crisis or shock [1]. Perhaps surprisingly, there is no consistent correlation between circulating levels of the catecholamines and the presence of symptoms and/or hypertension [2] although, in general, norepinephrine-secreting tumors are usually associated with sustained hypertension whereas tumors that secrete both epinephrine and norepinephrine are associated with episodic or paroxysmal hypertension. Conversely, 10 15% of PCs are associated with few symptoms at the time of diagnosis despite at times considerably elevated catecholamine levels, presumably due to individual sensitivity to catecholamine action and/or desensitization of adrenergic receptors in target tissues [2 5]. Nevertheless, these so-called silent PCs/PGLs may still be hazardous, as exemplified by autopsy studies suggesting that these tumors can present fatally and that up to 42% of all PCs are diagnosed postmortem [6 8]. The discovery of such tumors before the onset of clinical symptoms therefore is potentially lifesaving, and surgery is usually indicated for PCs/PGLs regardless of symptoms, after appropriate α-adrenergic blockade [8,9]. Silent tumors are typically diagnosed either as an incidental imaging finding, or by surveillance in a genetically susceptible individual. Both scenarios have become more common in the past two decades and both involve significant challenges and pitfalls. This article will present an approach to correctly diagnosing presymptomatic PCs/PGLs. Since demonstrating elevated circulating catecholamines is often a key diagnostic feature for these tumors, a brief review of biochemical testing is needed first. Biochemical testing for catecholamine-secreting tumors PCs are typically functional: approximately half secrete only norepinephrine whereas half secrete a mixture of epinephrine and norepinephrine [10]. Rarely, PC secretes only dopamine (which is either clinically silent or associated with hypotension) [11], and truly nonfunctional tumors are rarer still [12]. Sympathetic PGLs occurring in extra-adrenal abdominal or thoracic locations are functional in about 67 80% of cases and secrete norepinephrine [13]. Although epinephrine and norepinephrine are the biologically active hormones secreted by PCs and PGLs, the secretory pattern may be variable and occur in spells. In contrast, metanephrine and/or normetanephrine are produced continuously within chromaffin tumors (by catechol-o-methyltransferase enzymatic activity) and measurement of these circulating metabolites has become the gold standard for diagnosis of PC or functioning PGL [14 20]. There remains some controversy over whether these measurements are best performed in plasma or urine; several studies attest to better sensitivity of plasma total metanephrines but with lower specificity compared with 24 h urinary total metanephrine measurements. For instance, in a key study from the NIH, sensitivity and specificity for plasma metanephrines was 97 and 85%, respectively, whereas for urinary metanephrines these were 90 and 98%, respectively [16]. To avoid the hazard of missed diagnoses, current expert consensus favors first-line testing with plasma metanephrine and normetanephrine [8,9], ideally using liquid chromatography/tandem MS (but alternatively with HPLC and electrochemical detection) [21]. Plasma metanephrines are best measured from a fasting, recumbent subject and the clinician should be aware that certain drugs can cause biological or analytical interference with the assay [22]. When collected properly, fourfold or more elevations are highly likely to be diagnostic of PC/PGL whereas less elevated measurements may represent falsepositive results and require further evaluation [23], such as with repeat testing (e.g., by 24-h urinary fractionated metanephrines), measurement of serum chromogranin A or by functional imaging (discussed later). One study has reported utility of serum chromogranin A in minimizing false-positive diagnoses when metanephrine measurements are borderline, but this test is also subject to suboptimal specificity being elevated by hepatic or renal impairment, use of proton pump inhibitors or by the presence of other neuroendocrine tumors [23]. Incidental discovery on imaging The increasing use of abdominal ultrasound, CT and MRI has led to a predictable rise in the discovery of incidental adrenal lesions, some of which will be silent PCs. Two questions therefore arise: 48 Expert Rev. Endocrinol. Metab. 8(1), (2013)

4 CME Diagnosis of silent pheochromocytoma & paraganglioma how many adrenal incidentalomas are PCs? And how many PCs are now discovered incidentally? The prevalence of PCs in adrenal incidentalomas Estimates of PC prevalence from now more than 20 studies range from 0 to 18.6% [24 42]. Meta-analyses in expert reviews also arrive at different estimates, ranging between 3.1 and 5.6% [43 48]. Heterogenous inclusion criteria for incidentalomas explains some of this variance [47], but perhaps more obviously the prevalence of PCs among incidentally discovered adrenal lesions will depend on their size range (discussed below). Nevertheless, a low overall prevalence creates an important clinical challenge: namely that biochemical testing has lower positive-predictive value because of suboptimal specificity. Using a Bayesian approach, if PC prevalence is 5%, then biochemical testing with either plasma or urinary fractionated metanephrines is likely to find two or more false-positive results for every true PC [47]. Accuracy of biochemical testing is improved by selecting adrenal lesions with higher a priori probability of being PCs, and this can be achieved by considering the importance of size and (in the case of CT) density as follows. Adrenal size Most benign, nonfunctioning adrenal incidentalomas are 3 cm in size, whereas most PCs are >3 cm at diagnosis (Figures 1 & 2A). For instance, considering data from the Swedish Research Group ( ), the prevalence of PCs was approximately 2% in adrenal incidentalomas 3 cm, but 8.3% in lesions >3 cm [25]. This distinction is clinically relevant since biochemical testing becomes more accurate in these larger lesions such that Bayesian analysis would predict one or fewer false-positive result for every true PC case. Conversely, however, PC is more difficult to diagnose when 3 cm, particularly when silent. Compounding this difficulty is that circulating metanephrine levels are somewhat proportional to tumor size [49]; 0.3 therefore, small PCs may be associated with only borderline elevation in metanephrines. Nevertheless, small PCs are not infrequent 0.2 (in one recent series, 32% of PCs were 3 cm [50]) and their accurate diagnosis may spare the devastating consequences of hypertensive crises. A diagnostic approach 0.1 that considers CT density may overcome this problem. CT density It has been known for some time that PCs are usually associated with higher Hounsfield densities [51]. A recent study directly assessed the utility of considering CT density in diagnosing PCs among adrenal incidentalomas [52]. PC was diagnosed in nine out of 59 patients (15.2%) having Cumulative frequency 0 lesions of density greater than or equal to 10 Hounsfield units, including two subjects with adrenal lesions <3 cm and borderline urinary fractionated metanephrine levels. In contrast, urinary metanephrines were not elevated in any of 115 patients having lesions less than 10 Hounsfield units. Whether such low density adrenal lesions might avoid testing for PCs altogether requires further study; it is important to note that larger PCs may be hetergenous with areas of low density (Figure 2A). Clearly, however, the positive predictive value of elevated metanephrines is improved by the presence of higher CT density. Adrenal MRI PC was classically thought to have a distinct appearance on adrenal MRI: isointense with respect to the liver on T1-weighted images and hyperintense on T2-weighted images [53]. A recent study has highlighted a wider spectrum of MRI findings in PC, such that only 11% of PCs had a classical homogenous T2-hyperintense appearance and more than half demonstrated heterogenous T2 signal [53]. Therefore, although favorable for its lack of ionizing radiation, MRI does not appear to have superior diagnostic utility for PCs in comparison to adrenal CT. Functional imaging Nuclear medicine imaging with MIBG or PET may be useful for diagnosis of PCs in adrenal lesions with equivocal biochemical testing. Two recent studies examining 123 I-MIBG found strikingly similar sensitivity for diagnosis of (adrenal) PC at 85 88%, and a similarly lower sensitivity for diagnosis of functioning PGL (58 and 67%, respectively) [55,56]. Importantly, however, MIBG is less sensitive in smaller adrenal lesions such that tumors <2.5 cm are likely to be negative [55] >10 Size (cm) Figure 1. Frequency distribution of adrenal incidentalomas by size (cm); incidentally discovered pheochromocytomas are shown in hatched area. The dotted line at 3 cm illustrates that at 3 cm the prevalence of pheochromocytoma is only 2%, whereas at >3cm the prevalence is 8.3%. Data taken from [25]. 49

5 Clifton-Bligh CME loci were neurofibromatosis 1 (NF1), Von Hippel Lindau (VHL) and rearranged A B during transfection (RET ), but mutations in these genes explain only about twothirds of PC/PGL heritability [62]. Eight additional loci have since been discovered, encoding: the four subunits of succinate dehydrogenase, namely SDHB (familial PC/PGL syndrome 4, OMIM [63] ), SDHC (PGL3, OMIM [64]), SDHD (PGL1, OMIM [65]) and SDHA (PGL5, OMIM [66]); Figure 2. Computed tomography imaging of pheochromocytoma. (A) Left SDH5 (PGL2, OMIM [67]), a propheochromocytoma. Abdominal CT scan from an asymptomatic man aged 65 years tein necessary for flavination of SDHA; showing a heterogenous left adrenal mass, 6 cm (arrow). (B) Bilateral TMEM125, a transmembrane protein that pheochromocytoma. Abdominal CT scan from a man aged 35 years with known MEN2, showing a left adrenal mass, 2 cm with density of >25 Hounsfield units (thick arrow), regulates mtor activity [68] ; MAX, a tranand thickened medial limb of the right adrenal gland (thin arrow). scriptional coregulator of cmyc function [69] ; and KIF1B β [70]. The mechanism(s) by Standard 18F-FDG-PET imaging was reported to have 88% which mutations in such diverse genes can produce tumors that sensitivity in the diagnosis of nonmetastatic PCs/PGLs [57], are otherwise histologically indistinguishable remains a mystery. although this series included a relatively large number of tumors An important clue is that two broad groups of PCs/PGLs can be containing SDHB mutations (which are more likely to be posi- defined on the basis of gene expression: one with hypoxic-response tive due to altered glucose transport). Other PET tracers such as gene expression (associated with mutations in VHL, and the SDH 18 F-DOPA [58] and 18F-FDA [59] have shown utility in PC/PGL subunit genes); and the other with growth factor-kinase signaling diagnosis and these may be more useful than MIBG in assessment expression (associated with RET, NF1, MAX and TMEM127) [71]. of smaller adrenal lesions. Several recent excellent reviews have summarized the overall prevalence of these mutations among PCs and PGLs, and the curthe proportion of PCs that are incidentally discovered rent knowledge regarding penetrance of PCs/PGLs in individuals Four large surgical series of PCs have examined temporal trends carrying these mutations [62,72,73]. A summary of the different over the past 35 years and found that incidental discovery has genetic diagnoses is presented in Table 1, together with associated been increasing as the mode of diagnosis (Figure 3) [2 5]. Over this syndromic features. The clinician needs answers to three main time frame, the propotion of PCs detected as an incidental imag- questions: who should be referred for testing, what should haping finding has increased from about 10% up to now 25 30% pen if a positive test is returned and what should happen if the of all cases [3 5]. Compared with patients diagnosed on the basis tests are negative. of symptomatic presentation, those with incidentally discovered tumors were older and were slightly less likely to be hypertensive, Who should be referred for genetic testing? although there was no difference in tumor size [3,4]. These findings Expert consensus had been that all patients younger than 50 years argue that a substantial proportion of previously missed (and of age with PC/PGL tumors should be offered genetic testing for potentially fatal) PCs are now being appropriately diagnosed by these susceptibility loci, after appropriate genetic counseling [9]. In judicious triage of incidental imaging findings. fact, as the costs associated with genetic testing decrease year by year, there seems less rationale for excluding older subjects from Genetic susceptibility to PC/PGL testing; although uncommon, there are reports of large kindreds The other major area for which potential exists for diagnosis of being first recognized through diagnosis of an older proband [74]. PCs before symptoms develop is via the increasing recognition of The laboratory approach to genetic testing has gained considerits genetic susceptibility. Determining heritable PC/PGL suscep- able momentum over the past decade and important stimuli for tibility provides the opportunity for presymptomatic diagnosis of this have included, first, identification of particular phenotypes these tumors not only in the proband but also in affected family associated with individual susceptibility genes; and second, the members. Increasing numbers of patients with PCs/PGLs are now recognition that immunohistochemistry of tumors could be used being recognized and treated on the basis of surveillance following to triage the order in which these multiple genes are tested. a positive genetic test result [60,61]. PCs and PGLs represent the most heritable of all tumor types. Characteristic phenotypes associated with PC/PGL The inherited predisposition to PC/PGLs was first recognized susceptibility genes in 1993 and initially genetic mutations were thought to cause These are summarized in Table 1, but some additional points only 10% of all PCs/PGLs. Germline mutations have now been should be emphasized. First, past medical history is imporfound in at least 20 30% of cases [60]. The first recognized tant. PC/PGLs occurring in multiple endocrine neoplasia type 50 Expert Rev. Endocrinol. Metab. 8(1), (2013)

6 CME Diagnosis of silent pheochromocytoma & paraganglioma 2 (MEN2; associated with RET mutations) (Figure 2B) and Von Recklinghausen s disease (associated with NF1 mutations) are rarely the index event in those syndromes [75,76]; conversely, if PC is the presenting feature of a patient later diagnosed with a RET mutation, then thyroidectomy is additionally indicated since medullary thyroid cancer (MTC) is likely to be present [72]. The diagnosis of VHL syndrome will be already known in about 50 70% of patients with PC/PGLs associated with VHL mutations [77]. Although diagnosis of familial PGL syndromes is currently less likely to be known at the time of index presentation, this is expected to change as genetic testing in PC/PGLs becomes more widespread. Second, location matters [60 70,78 83]. PCs are associated with mutations in RET, VHL, NF1 and sometimes SDHB, SDHD, SDHA, MAX and TMEM127. Tumors of sympathetic paraganglia are more likely to be associated with mutations in SDHB, sometimes in SDHD, and less commonly in SDHC, SDHA, VHL or MAX [60 62,78 81]. Tumors of parasympathetic paraganglia (in the head and neck) are more likely to be associated with mutations in SDHD, sometimes in SDHC or SDHB, and rarely in SDHA or TMEM127. Third, an adrenegic pattern of catecholamine secretion by PCs is more likely to be associated with mutation in RET, NF1, TMEM127 or MAX [75,76,82 84], whereas purely noradrenergic secretion is more likely to be associated with VHL, SDHB or SDHD [60,61,84]. Finally, malignant PCs/PGLs are more likely to be associated with mutations in SDHB and less commonly in VHL [85]. Conversely, a diagnosis of SDHB mutation should alert the clinician to the possibility of malignant PGLs. SDHB immunohistochemistry is a surrogate marker for mitochondrial complex II dysfunction Recently, several groups reported the intriguing finding that negative immunohistochemical (IHC) staining for SDHB is highly specific for PCs and PGLs associated with mutations in any of the SDHA, SDHB, SDHC or SDHD subunit genes [86,87]. Tumors associated with mutations in VHL, RET or NF1 on the other hand show positive granular SDHB cytoplasmic staining (consistent with normal mitochondrial location of SDH) [86,87]. More recently, IHC for SDHA has also been used to identify tumors associated with germline mutations in that gene [88,89]. Some groups have therefore proposed that IHC should be used to triage the order in which these genes are tested, in order to reduce time and cost of genetic testing [86,87]. At this time, however, expert consensus favors that genetic testing should be guided by the clinical scenario as described above [9]. An additional advantage of IHC is that it can be applied to other tumors associated with these syndromic diagnoses in a manner that can facilitate appropriate SDH Incidentally discovered PC (%) Shen et al. [5] Amar et al. [3] ( ) Kopetschke et al. [4] genetic testing even when PC or PGL is not the presenting tumor. Around 14% of patients with mutations in SDHB and 8% with SDHD mutations will develop renal cell cancer (RCC) [88] and these RCCs are also negative for SDHB immunostaining (in contrast to other RCCs which retain SDHB staining) [89]. It is important to note that some (~10%) of PC/PGLs are SDHB negative by IHC but are not associated with identifiable SDHB, SDHC or SDHD mutations, raising the possibility that other mechanisms of mitochondrial complex 2 instability exist which lead to tumorigenesis. As a case in point, the PC/PGLs of the Carney triad (syndromic but not hereditary association of PC/PGL, gastrointestinal stromal tumor [GIST] and pulmonary chondroma) show negative staining for SDHB [87]. Similarly, GISTs that are negative for SDHB staining are unlikely to be associated with underlying germline SDH mutations, even though this IHC phenotype defines a clinically useful subgroup ( Carney pediatric wild-type ) characterized by young age of onset, multifocality and resistance to imatinib [90]. It therefore appears that negative staining for SDHB is a marker for impaired function of mitochondrial complex II, regardless of the mechanism by which this is disrupted. Massive parallel sequencing is likely to dramatically improve cost & time of genetic testing for PC/PGL susceptibility genes The advent of new sequencing technologies that permit rapid analysis of multiple genes in parallel offers significant advantages for disorders associated with multiple susceptibility loci such as PCs. In essence, these technologies sequence millions of relatively short reads of DNA which are then aligned to the human reference genome [91]. Two general approaches are in current use: amplicon sequencing, where multiple individual genes are included on a test panel that can be used to test many DNA samples in a single run; or whole-exome sequencing, in which essentially all genes are sequenced simultaneously in a subject s DNA sample. The very rapid reduction in sequencing costs over the past few years has brought the reality of whol-exome sequencing as a cost-effective ( ) ( ) Figure 3. Three surgical series showing temporal change in the proportion of pheochromocytomas that had been diagnosed on the basis of incidental imaging finding. Data taken from [3] (closed bars) [4], (open bars) and [5] (hatched bars). Amar et al. [3] had expressed temporal trend by chronological quartiles and these have been approximately re-expressed as decades as shown parenthetically. 51

7 Clifton-Bligh CME approach for disorders associated with multiple different genes. Nevertheless, there remain many ethical challenges in adopting whole-exome sequencing for routine pathology use. In particular, whole-exome sequencing has a (probably small) risk of finding deleterious mutations in genes unrelated to the phenotypic presentation; a commonly quoted example is the possibility of discovering genetic susceptibility for an untreatable neurodegenerative disorder [92]. Regulatory and legal frameworks are still evolving to deal with this new genetic testing paradigm, which, notwithstanding, has become widely available. What should happen if a positive test is returned? Assuming that the reporting laboratory has included appropriate standards to avoid a false-positive result (sequencing both DNA strands on duplicate DNA samples [93]) or otherwise reporting a Table 1. Genes and clinical features (penetrance) of pheochromocytoma/paraganglioma syndromes. Syndrome Gene PC PGL Syndromic features MEN2 RET PC (20 50%) Bilateral 50% Malignant 3% NF1 NF1 PC (5%) Bilateral 10% Malignant 3% VHL VHL PC (10 30%) Bilateral 40% Malignant 5% Malignant 5% PGL1 SDHD PC (24%) Malignant 4% Multifocal 56% TAPGL (0.5%) TAPGL (3%) HNPGL (3%) HNPGL (85%) TAPGL (22%) PGL2 SDHAF2 HNPGL PGL3 SDHC Malignant 0% Multifocal 17% PGL4 SDHB PC (20%) Malignant 30% Multifocal 21% HNPGL (?%) TAPGL (?%) TAPGL (50%) HNPGL (22%) SDHA PC (?%) TAPGL (?%) HNPGL (?%) MAX PC (?%) Bilateral 21% Malignant 11% TMEM127 PC (?%) Bilateral 31% Malignant 4% TAPGL (few) TAPGL (few) HNPGL (few) known polymorphism, there are the following three important steps in follow-up of patients with positive genetic diagnoses: Family members should receive genetic counseling toward considering genetic testing and, if positive, then appropriate biochemical and imaging surveillance for tumors as described below; The proband (and other carriers in the family) should have appropriate surveillance for developing recurrent PC or PGL, with annual biochemical testing and targeted imaging if elevated metanephrines develop; for syndromes associated with PGL, imaging every 2 3 years is also required since many tumors (in particular of the head and neck) will be biochemically silent; The proband should have appropriate surveillance for other syndromic features, as detailed in Table 1. Medullary thyroid cancer (>95%) Hyperparathyroidism (10%) Neurofibromas Café au lait patches Lisch nodules Optic gliomas Bone dysplasia Peripheral nerve sheath tumors Cerebellar hemangioblastoma (60%) Spinal hemangioblastoma (15%) Retinal angioma (40 60%) Renal cell cancer (25%) Renal cell cancer (8%) Pituitary adenoma (?%) Gastrointestinal stromal cell tumor (?%) Renal cell cancer (14%) Gastrointestinal stromal cell tumor (2%) Gastrointestinal stromal cell tumor (?%) Paternal inheritance. HNPGL: Head and neck PGL; PC: Pheochromocytoma; PGL: Paraganglioma; TAPGL: Thoracoabdominal PGL; VHL: Von Hippel Lindau. Data taken from [60 72,78 83]. There remain two key deficiencies at this time in reporting positive genetic results: first, a lack of clear genotype phenotype correlation for most PC/PGL genes and, second, uncertainty regarding pathogenicity of new mutations. Reasonable genotype phenotype correlation exists only for RET mutations [94 96]: highest risk mutations are associated with the development of MTC in infancy, and development of PCs around 8 10 years later; conversely, lowest risk RET mutations are associated with MTC development typically after 8 years of age and PC after age 20 years. This information has been used to develop guidelines on timing of thyroidectomy according to specific RET mutation [94], and the age at which biochemical screening for PC should begin [95]. Unfortunately, genotype phenotype correlations are not as strong for other PC/PGL syndromes to guide how frequently carriers of these gene mutations should be screened for tumor development or whether indeed some carriers might be safely reassured. Some broad features exist: large-scale deletions involving VHL are more likely to be associated with type 1 VHL syndrome (in which PC does not occur), and conversely type 2 VHL (in which PC does occur) is mainly associated with missense or nonsense VHL mutations [78 81]; SDHD and SDH5 are imprinted, so that tumors almost exclusively develop only in carriers inheriting mutations on the paternal allele [63,67] and SDHD mutations are highly penetrant, with tumors (mainly head or neck PGLs) occurring in 80 90% of carriers (paternally inherited) by age 50 years [61,97]. For the most part, however, diagnosis of a pathogenic mutation 52 Expert Rev. Endocrinol. Metab. 8(1), (2013)

8 CME Diagnosis of silent pheochromocytoma & paraganglioma in a PC/PGL susceptibility gene implies at present a requirement for lifelong surveillance for possible tumor development. There are now large databases of mutations that have been associated with PC/PGL development [101,102]. However, finding a novel mutation (rare variant) within any of these genes requires a cautious approach before determining that it is pathogenic. It is generally reasonable to conclude pathogenecity if the mutation is present in multiple family members affected by tumor development, providing that it is not otherwise a common polymorphism. Subsequently, finding the same mutation in an unrelated subject with PC/PGL more certainly fulfils the genetic equivalent of Koch s postulates. However in the absence of such clinical correlation, there are now several in silico programs used to predict severity of mutation on gene function [103]. These have not been widely tested yet against clinical databases, and more study is required before their common use in this field. What should happen if the tests are negative? Negative results from genetic testing in a patient presenting with PC/PGL essentially support diagnosis of a sporadic tumor. There are some caveats however: as with any clinical test, there is likely to be a small false-negative error rate; it is likely that not all PC/PGL susceptibility genes have been identified yet and of course the tumor may still have malignant potential and particularly, if large, should be accompanied by careful clinical follow-up [1]. False-negative genetic test results might arise from preanalytical or analytical errors; in general, most laboratories have good-quality controls for minimizing false-positive errors (by sequencing both DNA strands and testing duplicate samples), but false-negative errors are more difficult to control for. Missense, nonsense and small insertion/deletion (INDEL) mutations are routinely tested for by Sanger sequencing in most laboratories, but large-scale deletions require additional methodology and may not be part of the routine test panel. Both the referring clinician and the genetic pathology laboratory need to keep an open mind regarding negative test results, particularly in younger patients and/or those presenting with bilateral PCs. Even with stringent pathology testing, so-called sporadic PCs have a risk of contralateral recurrence of approximately 6% [72]. Key issues Expert commentary & five-year view Recognition of silent PC and PGL tumors requires clinical finesse but is potentially life-saving since these tumors can be fatal before the advent of symptoms. A practical approach for diagnosing PC among adrenal incidentalomas is to biochemically test those patients with lesions >3 cm and/or 10 Hounsfield units; elevated plasma fractionated metanephrines in these cases should prompt preparation for surgery. Functional imaging with MIBG or PET can be considered where needed to resolve doubt, remembering that MIBG is often negative for small tumors or those in extraadrenal locations. All patients with PCs or PGLs should be offered genetic testing after appropriate genetic counseling. Gene testing may be triaged by SDHB immunohistochemistry on the tumor; positive IHC directs first-line testing toward VHL, RET, MAX or TMEM127; negative IHC directs testing towards SDHB, SDHC, SDHD or SDHA. Neurofibromatosis is accurately diagnosed on phenotypic grounds alone. Most laboratories currently use Sanger sequencing for diagnosis of mutations, most of which are missense, nonsense or small insertions/deletions. Genotype phenotype correlations are only robust for RET mutations in MEN2. Mutations in other PC/PGL genes usually prompt periodic but lifelong surveillance for tumor development. Family members of probands with PC/PGL gene mutations should receive genetic counseling. In the next 5 years, several changes can be confidently predicted in this area: sequencing will be done by massive parallel technologies, allowing rapid testing of all PC/PGL genes in tandem for every patient; cost of sequencing will be reduced, but bioinformatic processing required to generate a result may counterbalance this; more genes will be identified to be associated with PC/PGL; genetic and/or environmental modifiers for tumor development will be identified and better algorithms will then be developed to direct appropriate screening for tumor development in carriers of PC/PGL gene mutations, or indeed to avoid screening in very low-risk situations. Acknowledgements The author gratefully acknowledges Dr Diana Benn for critical review of this manuscript % of subjects with incidentally discovered PCs are completely asymptomatic and up to 50% are normotensive. Phaeochromocytomas (PCs; or functioning paragangliomas [PGLs]) should be treated surgically regardless of symptoms, after appropriate preoperative adrenergic blockade. PC is diagnosed in 3 5% of adrenal incidentalomas, but is more likely in lesions >3 cm or if 10 HU. Biochemical testing is essential, and expert consensus favors measuring plasma-free fractionated metanephrines; the positive predictive value is enhanced using imaging criteria above. The proportion of PCs that have been discovered incidentally/presymptomatically has been increasing: recent series, 25 30% of PCs have been discovered incidentally on computed tomography. And an additional 10% due to screening genetically susceptible individuals, but this is likely to increase in an era of routine genetic testing. Genetic testing should be offered to most patients with PC/PGL after appropriate genetic counseling. Germline mutations in 11 loci have now been associated with these tumors; the order of testing these multiple genes can be triaged by clinical risk factors and/or by SDHB immunohistochemistry of tumor. Massive parallel sequencing assays are likely to replace traditional Sanger sequencing in the near future, which will result in faster and less expensive gene tests but with greater requirement for bioinformatic curating of data. 53

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Classification of pheochromocytomas according to their type of secretion. Surgery 100(6), (1986). 12 Mannelli M, Pupilli C, Lanzillotti R et al. A nonsecreting pheochromocytoma presenting as an incidental adrenal mass. Report on a case. J. Endocrinol. Invest. 16(10), (1993). 13 Erickson D, Kudva YC, Ebersold MJ et al. Benign paragangliomas: clinical presentation and treatment outcomes in 236 patients. J. Clin. Endocrinol. Metab. 86(11), (2001). 14 Lenders JW, Pacak K, Walther MM et al. Biochemical diagnosis of pheochromocytoma: which test is best? JAMA 287(11), (2002). 15 Eisenhofer G, Goldstein DS, Walther MM et al. Biochemical diagnosis of pheochromocytoma: how to distinguish true- from false-positive test results. J. Clin. Endocrinol. 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The incidental indeterminate adrenal mass on CT (> 10 H) in patients without cancer: is further imaging necessary? Follow-up of 321 consecutive indeterminate adrenal masses. AJR. Am. J. Roentgenol. 189(5), (2007). 30 Bondanelli M, Campo M, Trasforini G et al. Evaluation of hormonal function in a series of incidentally discovered adrenal masses. Metab. Clin. Exp. 46(1), (1997). 31 Reincke M, Nieke J, Krestin GP, Saeger W, Allolio B, Winkelmann W. Preclinical Cushing s syndrome in adrenal incidentalomas : comparison with adrenal Cushing s syndrome. J. Clin. Endocrinol. Metab. 75(3), (1992). 32 Bastounis EA, Karayiannakis AJ, Anapliotou ML, Nakopoulou L, Makri GG, Papalambros EL. Incidentalomas of 54 Expert Rev. Endocrinol. Metab. 8(1), (2013)

10 CME Diagnosis of silent pheochromocytoma & paraganglioma the adrenal gland: diagnostic and therapeutic implications. Am. Surg. 63(4), (1997). 33 Tsvetov G, Shimon I, Benbassat C. Adrenal incidentaloma: clinical characteristics and comparison between patients with and without extraadrenal malignancy. J. Endocrinol. Invest. 30(8), (2007). 34 Bovio S, Cataldi A, Reimondo G et al. Prevalence of adrenal incidentaloma in a contemporary computerized tomography series. J. Endocrinol. Invest. 29(4), (2006). 35 Bencsik Z, Szabolcs I, Góth M et al. Incidentally detected adrenal tumours (incidentalomas): histological heterogeneity and differentiated therapeutic approach. J. Intern. Med. 237(6), (1995). 36 Linos DA, Stylopoulos N, Raptis SA. Adrenaloma: a call for more aggressive management. World J. Surg. 20(7), ; discussion 792 (1996). 37 Kasperlik-Zeluska AA, Roslonowska E, Slowinska-Srzednicka J et al. Incidentally discovered adrenal mass (incidentaloma): investigation and management of 208 patients. Clin. Endocrinol. (Oxf.) 46(1), (1997). 38 Proye C, Jafari Manjili M, Combemale F et al. Experience gained from operation of 103 adrenal incidentalomas. Langenbecks. Arch. Surg. 383(5), (1998). 39 Murai M, Baba S, Nakashima J, Tachibana M. Management of incidentally discovered adrenal masses. World J. Urol. 17(1), 9 14 (1999). 40 Rossi R, Tauchmanova L, Luciano A et al. Subclinical Cushing s syndrome in patients with adrenal incidentaloma: clinical and biochemical features. J. Clin. Endocrinol. Metab. 85(4), (2000). 41 Favia G, Lumachi F, Basso S, D Amico DF. Management of incidentally discovered adrenal masses and risk of malignancy. Surgery 128(6), (2000). 42 Barzon L, Fallo F, Sonino N, Boscaro M. Development of overt Cushing s syndrome in patients with adrenal incidentaloma. Eur. J. Endocrinol. 146(1), (2002). 43 Mantero F, Terzolo M, Arnaldi G et al. A survey on adrenal incidentaloma in Italy. Study Group on Adrenal Tumors of the Italian Society of Endocrinology. J. Clin. Endocrinol. Metab. 85(2), (2000). 44 Barzon L, Sonino N, Fallo F, Palu G, Boscaro M. Prevalence and natural history of adrenal incidentalomas. Eur. J. Endocrinol. 149(4), (2003). 45 Mansmann G, Lau J, Balk E, Rothberg M, Miyachi Y, Bornstein SR. The clinically inapparent adrenal mass: update in diagnosis and management. Endocr. Rev. 25(2), (2004). 46 Young WF Jr. Management approaches to adrenal incidentalomas. A view from Rochester, Minnesota. Endocrinol. Metab. Clin. North Am. 29(1), , x (2000). 47 Cawood TJ, Hunt PJ, O Shea D, Cole D, Soule S. Recommended evaluation of adrenal incidentalomas is costly, has high false-positive rates and confers a risk of fatal cancer that is similar to the risk of the adrenal lesion becoming malignant; time for a rethink? Eur. J. Endocrinol. 161(4), (2009). 48 Androulakis II, Kaltsas G, Piaditis G, Grossman AB. The clinical significance of adrenal incidentalomas. Eur. J. Clin. Invest. 41(5), (2011). 49 Eisenhofer G, Lenders JW, Goldstein DS et al. Pheochromocytoma catecholamine phenotypes and prediction of tumor size and location by use of plasma free metanephrines. Clin. Chem. 51(4), (2005). 50 Yu R, Pitts A, Wei M. Small pheochromocytomas: significance, diagnosis and outcome. J. Clin. Hypertens. 14, (2012). 51 Korobkin M, Brodeur FJ, Yutzy GG et al. Differentiation of adrenal adenomas from nonadenomas using CT attenuation values. AJR. Am. J. Roentgenol. 166(3), (1996). 52 Sane T, Schalin-Jäntti C, Raade M. Is biochemical screening for pheochromocytoma in adrenal incidentalomas expressing low unenhanced attenuation on computed tomography necessary? J. Clin. Endocrinol. Metab. 97(6), (2012). 53 Fink IJ, Reinig JW, Dwyer AJ, Doppman JL, Linehan WM, Keiser HR. MR imaging of pheochromocytomas. J. Comput. Assist. Tomogr. 9(3), (1985). 54 Jacques AE, Sahdev A, Sandrasagara M et al. Adrenal phaeochromocytoma: correlation of MRI appearances with histology and function. Eur. Radiol. 18(12), (2008). 55 Bhatia KSS, Ismail MM, Sahdev et al. 123 I-metaiodobenzylguanidine (MIBG) scintigraphy for the detection of adrenal and extra-adrenal phaeochromocytomas: CT and MRI correlation. Clin. Endocrinol. 69, (2008). 56 Wiseman GA, Pacak K, O Dorisio MS et al. Usefulness of 123 I-MIBG scintigraphy in the evaluation of patients with known or suspected primary or metastatic pheochromocytoma or paraganglioma: results from a prospective multicenter trial. J. Nucl. Med. 50(9), (2009). 57 Timmers HJ, Chen CC, Carrasquillo JA et al. Comparison of 18 F-fluoro-l-DOPA, 18 F-fluoro-deoxyglucose, and 18 F-fluorodopamine PET and 123 I-MIBG scintigraphy in the localization of pheochromocytoma and paraganglioma. J. Clin. Endocrinol. Metab. 94(12), (2009). 58 Hoegerle S, Nitzsche E, Altehoefer C et al. Pheochromocytomas: detection with 18 F DOPA whole body PET initial results. Radiology 222(2), (2002). 59 Pacak K, Eisenhofer G, Carrasquillo JA, Chen CC, Li ST, Goldstein DS. 6-[ 18 F] fluorodopamine positron emission tomographic (PET) scanning for diagnostic localization of pheochromocytoma. Hypertension 38(1), 6 8 (2001). 60 Neumann HP, Bausch B, McWhinney SR et al.; Freiburg Warsaw Columbus Pheochromocytoma Study Group. Germ-line mutations in nonsyndromic pheochromocytoma. N. Engl. J. Med. 346(19), (2002). 61 Benn DE, Gimenez-Roqueplo AP, Reilly JR et al. Clinical presentation and penetrance of pheochromocytoma/ paraganglioma syndromes. J. Clin. Endocrinol. Metab. 91(3), (2006). 62 Gimenez-Roqueplo AP, Dahia PL, Robledo M. An update on the genetics of paraganglioma, pheochromocytoma, and associated hereditary syndromes. Horm. Metab. Res. 44(5), (2012). 63 Baysal BE, Ferrell RE, Willett-Brozick JE et al. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287(5454), (2000). 64 Schiavi F, Boedeker CC, Bausch B et al. Predictors and prevalence of paraganglioma syndrome associated with mutations of the SDHC gene. J. Am. Med. Assoc. 294, (2005). 65 Astuti D, Latif F, Dallol A et al. Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. Am. J. Hum. Genet. 69(1), (2001). 66 Burnichon N, Briere JJ, Libe R et al. SDHA is a tumor suppressor gene causing paraganglioma. Hum. Mol. Genet. 19, (2010). 55

11 Clifton-Bligh CME 67 Hao HX, Khalimonchuk O, Schraders M et al. SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science 325(5944), (2009). 68 Qin Y, Yao L, King EE et al. Germline mutations in TMEM127 confer susceptibility to pheochromocytoma. Nat. Genet. 42(3), (2010). 69 Comino-Méndez I, Gracia-Aznárez FJ, Schiavi F et al. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nat. Genet. 43(7), (2011). 70 Yeh IT, Lenci RE, Qin Y et al. A germline mutation of the KIF1B beta gene on 1p36 in a family with neural and nonneural tumors. Hum. Genet. 124(3), (2008). 71 Dahia PLM, Ross KN, Wright ME et al. A Hif1alpha regulatory loop links hypoxia and mitochondrial signals in pheochromocytomas. PLoS Gen. 25(1), (2005). 72 Welander J, Söderkvist P, Gimm O. Genetics and clinical characteristics of hereditary pheochromocytomas and paragangliomas. Endocr. Relat. 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12 CME Diagnosis of silent pheochromocytoma & paraganglioma Diagnosis of silent pheochromocytoma and paraganglioma To obtain credit, you should first read the journal article. After reading the article, you should be able to answer the following, related, multiple-choice questions. To complete the questions (with a minimum 70% passing score) and earn continuing medical education (CME) credit, please go to journal/expertendo. Credit cannot be obtained for tests completed on paper, although you may use the worksheet below to keep a record of your answers. You must be a registered user on Medscape.org. If you are not registered on Medscape.org, please click on the New Users: Free Registration link on the left hand side of the website to register. Only one answer is correct for each question. Once you successfully answer all post-test questions you will be able to view and/or print your certificate. For questions regarding the content of this activity, contact the accredited provider, CME@medscape.net. For technical assistance, contact CME@webmd.net. American Medical Association s Physician s Recognition Award (AMA PRA) credits are accepted in the US as evidence of participation in CME activities. For further information on this award, please refer to org/ama/pub/category/2922.html. The AMA has determined that physicians not licensed in the US who participate in this CME activity are eligible for AMA PRA Category 1 Credits. Through agreements that the AMA has made with agencies in some countries, AMA PRA credit may be acceptable as evidence of participation in CME activities. If you are not licensed in the US, please complete the questions online, print the AMA PRA CME credit certificate and present it to your national medical association for review. Activity Evaluation Where 1 is strongly disagree and 5 is strongly agree 1. The activity supported the learning objectives. 2. The material was organized clearly for learning to occur. 3. The content learned from this activity will impact my practice. 4. The activity was presented objectively and free of commercial bias You are seeing a 59-year-old man with a left adrenal mass identified incidentally on a CT of the abdomen for suspected diverticulitis. You suspect that he may have a pheochromocytoma. What should you consider in general regarding this diagnosis? A Incident high circulating levels of catecholamines correlate well with symptoms B Incident high circulating levels of catecholamines correlate well with hypertension C Pheochromocytoma is always symptomatic by the time of diagnosis D Tumors that secrete noradrenaline alone are usually associated with sustained hypertension 2. What should you consider regarding biochemical and imaging studies for this patient? A Size on imaging studies does not correlate with the diagnosis of pheochromocytoma vs incidentaloma B Pheochromocytomas have higher Hounsfield densities compared with incidentalomas C Pheochromocytomas have a classical homogenous T2-hyperintense appearance in over 99% of tumors D The sensitivity of urinary metanephrines exceeds that of plasma metanephrines in the diagnosis of pheochromocytoma 3. The patient is diagnosed with pheochromocytoma. What is the best study to initiate the genetic workup for this case? A Direct testing for NF1 mutations B Immunohistochemical (IHC) staining for SDHB C Direct testing for RET mutations D Direct testing for MAX, SDHD, and VHL mutations 4. What else should you consider regarding genetic testing for this patient? A Genetic testing is not indicated for this patient based on his age alone B Positive staining for SDHB is associated with mutations in VHL, RET, or NF1 C Pheochromocytomas are usually the first manifestation of multiple endocrine neoplasia type 2 D Genotype phenotype correlation is strongest in cases of VHL mutation 57