Cost-minimization analysis of sequential genetic testing versus targeted next-generation sequencing gene panels in patients with pheochromocytoma and paraganglioma.

INTRODUCTION: Pheochromocytomas and paragangliomas (PPGLs) are highly heritable tumours, with up to 40% of cases carrying germline variants. Current guidelines recommend genetic testing for all patients with PPGLs. Next-generation sequencing (NGS) enables accurate, fast, and inexpensive genetic testing. This study aimed to compare the costs related to PPGL genetic testing between the sequential testing using the decisional algorithm proposed in the 2014 Endocrine Society guidelines and targeted NGS gene panels. METHODS: Patients with proven PPGLs were enrolled. A gene list covering 17 susceptibility genes related to hereditary PPGLs was developed for targeted sequencing. Validation was carried out by Sanger sequencing. We simulated the diagnostic workflow to examine the anticipated costs based on each strategy for genetic testing. RESULTS: Twenty-nine patients were included, among whom a germline variant was identified in 34.5%. A total of 22.7% with apparently sporadic PPGL carried a variant. Five genes were involved (RET, n = 3; SDHB, n = 3; SDHD, n = 2; EGLN1, n = 1; and NF1, n = 1). According to the diagnostic workflow, the average cost of the targeted NGS (534.7 US dollars per patient) is lower than that of the sequential testing (734.5 US dollars per patient). The targeted NGS can also reduce the number of hospital visits from 4.1 to 1 per person. The cost can be further reduced to 496.24 US dollars per person (32% reduction) if we apply a new syndromic-driven diagnostic algorithm to establish priorities for specific genetic testing for syndromic and selected cases, and targeted NGS for non-syndromic patients. CONCLUSIONS: Targeted NGS can reduce both the cost of PPGL genetic testing and the number of hospital visits, compared with the conventional approach. Our proposed algorithm is the preferred approach due to its significant reduction of the cost of genetic testing.Key messagePheochromocytomas and paragangliomas are highly heritable neoplasms.The targeted next-generation sequencing (NGS) gene panels have proven to be fast, accurate, and inexpensive for the genetic analysis.According to this cost analysis, it is economically reasonable to use targeted NGS gene panels for genetic screening.

Algorithmic assessment of missense mutation severity in the Von-Hippel Lindau protein.

Von Hippel-Lindau disease (VHL) is an autosomal dominant rare disease that causes the formation of angiogenic tumors. When functional, pVHL acts as an E3 ubiquitin ligase that negatively regulates hypoxia inducible factor (HIF). Genetic mutations that perturb the structure of pVHL result in dysregulation of HIF, causing a wide array of tumor pathologies including retinal angioma, pheochromocytoma, central nervous system hemangioblastoma, and clear cell renal carcinoma. These VHL-related cancers occur throughout the lifetime of the patient, requiring frequent intervention procedures, such as surgery, to remove the tumors. Although VHL is classified as a rare disease (1 in 39,000 to 1 in 91,000 affected) there is a large heterogeneity in genetic mutations listed for observed pathologies. Understanding how these specific mutations correlate with the myriad of observed pathologies for VHL could provide clinicians insight into the potential severity and onset of disease. Using a select set of 285 ClinVar mutations in VHL, we developed a multiparametric scoring algorithm to evaluate the overall clinical severity of missense mutations in pVHL. The mutations were assessed according to eight weighted parameters as a comprehensive evaluation of protein misfolding and malfunction. Higher mutation scores were strongly associated with pathogenicity. Our approach establishes a novel in silico method by which VHL-specific mutations can be assessed for their severity and effect on the biophysical functions of the VHL protein.

Exosomal double-stranded DNA as a biomarker for the diagnosis and preoperative assessment of pheochromocytoma and paraganglioma.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are the most heritable endocrine tumors. Genetic testing for 12 driver susceptibility genes is recommended in all PCC and PGL cases. However, detection of somatic mutations in PCC and PGL remains unrealizable for genetic diagnosis and preoperative assessment. We compared the serum exosomal DNA and tumor tissue DNA from patients or mice with PCC or PGL and found double-stranded DNA (dsDNA) fragments in the circulating exosomes of patients with PCC or PGL. Exosomal dsDNA shared the same mutations in the susceptibility genes with that of the parent tumor cells. Moreover, our research showed that serum-derived exosomal dsDNA in PCC and PGL was highly consistent with the paired tumor genome. Our findings provide the first definitive evidence of the presence of exosomal dsDNA that can be used as a noninvasive genetic marker in one of the most effective somatic mutation screens for the diagnosis and preoperative assessment of PCCs and PGLs.

Assessment of the potential activity of major dietary compounds as selective estrogen receptor modulators in two distinct cell models for proliferation and differentiation.

Estrogen receptors (ERs) α and ß are distributed in most tissues of women and men. ERs are bound by estradiol (E2), a natural hormone, and mediate the pleiotropic and tissue-specific effects of E2, such as proliferation of breast epithelial cells or protection and differentiation of neuronal cells. Numerous environmental molecules, called endocrine disrupting compounds, also interact with ERs. Phytoestrogens belong to this large family and are considered potent therapeutic molecules that act through their selective estrogen receptor modulator (SERM) activity. Using breast cancer cell lines as a model of estrogen-dependent proliferation and a stably ER-expressing PC12 cell line as a model of neuronal differentiating cells, we studied the SERM activity of major dietary compounds, such as apigenin, liquiritigenin, daidzein, genistein, coumestrol, resveratrol and zearalenone. The ability of these compounds to induce ER-transactivation and breast cancer cell proliferation and enhance Nerve Growth Factor (NGF) -induced neuritogenesis was assessed. Surprisingly, although all compounds were able to activate the ER through an estrogen responsive element reporter gene, they showed differential activity toward proliferation or differentiation. Apigenin and resveratrol showed a partial or no proliferative effect on breast cancer cells but fully contributed to the neuritogenesis effect of NGF. However, daidzein and zearalenone showed full effects on cellular proliferation but did not induce cellular differentiation. In summary, our results suggest that the therapeutic potential of phytoestrogens can diverge depending on the molecule and the phenotype considered. Hence, apigenin and resveratrol might be used in the development of therapeutics for breast cancer and brain diseases.

Risk assessment of maternally inherited SDHD paraganglioma and phaeochromocytoma.

BACKGROUND: Germline mutations in the SDHD tumour suppressor gene (11q23.1) predispose to phaeochromocytomas and paragangliomas (PPGL) mainly on a paternal transmission. However, PPGL have been recently reported in three carriers of a maternally inherited SDHD mutation. OBJECTIVE: To assess the risk of PPGL occurrence on maternal transmission of SDHD mutation. METHODS: Pedigrees of 80 SDHD-related families have been reviewed. 35 asymptomatic subjects carrying a maternally transmitted SDHD mutation were identified. 20 of them accepted to benefit from a PPGL imaging screening. RESULTS: A unique histologically proven biochemically negative phaeochromocytoma has been diagnosed in a 35-year-old woman. Molecular investigations carried out on tumour tissue revealed that the loss of heterozygosity encompassed the paternally derived q arm and the maternally derived p arm of chromosome 11. CONCLUSIONS: This study demonstrates that the risk of developing PPGL for a subject carrying a germline SDHD mutation on the maternal allele remains a rare scenario but does exist. Our data suggest an adjustment of current genetic counselling and clinical care recommendations for at-risk subjects. A targeted familial genetic test should be proposed from the age of 18 years to every subject having a mother carrying a germline SDHD mutation and a first medical workup, including imaging, should be recommended to SDHD-positive mutation carriers.

Functional and in silico assessment of MAX variants of unknown significance.

UNLABELLED: The presence of germline mutations affecting the MYC-associated protein X (MAX) gene has recently been identified as one of the now 11 major genetic predisposition factors for the development of hereditary pheochromocytoma and/or paraganglioma. Little is known regarding how missense variants of unknown significance (VUS) in MAX affect its pivotal role in the regulation of the MYC/MAX/MXD axis. In the present study, we propose a consensus computational prediction based on five "state-of-the-art" algorithms. We also describe a PC12-based functional assay to assess the effects that 12 MAX VUS may have on MYC's E-box transcriptional activation. For all but two of these 12 VUS, the functional assay and the consensus computational prediction gave consistent results; we classified seven variants as pathogenic and three as nonpathogenic. The introduction of wild-type MAX cDNA into PC12 cells significantly decreased MYC's ability to bind to canonical E-boxes, while pathogenic MAX proteins were not able to fully repress MYC activity. Further clinical and molecular evaluation of variant carriers corroborated the results obtained with our functional assessment. In the absence of clear heritability, clinical information, and molecular data, consensus computational predictions and functional models are able to correctly classify VUS affecting MAX. KEY MESSAGES: A functional assay assesses the effects of MAX VUS over MYC transcriptional activity. A consensus computational prediction and the functional assay show high concordance. Variant carriers' clinical and molecular data support the functional assessment.

Usefulness of Succinate dehydrogenase B (SDHB) immunohistochemistry in guiding mutational screening among patients with pheochromocytoma-paraganglioma syndromes.

Genetic testing of pheochromocytomas (PCC) and paragangliomas (PGL), although expensive, is gradually becoming a part of the routine laboratory investigation for patients with PCC-PGL syndrome. Recently, Succinate dehydrogenase B (SDHB) immunochemistry has been shown to be an excellent indicator of germline mutations in the SDH genes and could help significantly reduce cost. This study assesses the utility of SDHB immunohistochemical analysis when used to guide genetic analysis, with emphasis on cost benefits it could provide in a resource-limited setting. Forty-four cases of PCC/PGL characterized by genetic analysis were included to determine their SDHB expression pattern by immunohistochemistry. SDHB antibody expression was negative among three cases each, with SDHB and SDHD mutations. Immunohistochemistry results were positive for all three cases of RET, a single case of neurofibromatosis and for two cases with Von Hippel-Lindau (VHL) mutations while the remaining two cases with VHL mutations showed a diffuse 'cytoplasmic blush'. Thirty of the remaining 31 samples demonstrated positive staining and were negative for mutations, while a lone sample that was negative for staining and mutation was not included in the final analysis as the internal control for the sample was not adequately stained. Cost analysis in our settings showed that triaging with SDHB immunohistochemistry could potentially reduce costs by USD 320-500 per patient. SDHB immunohistochemistry, when used as a guide to genetic testing, can significantly reduce the effort, time and costs of testing among patients with PCC-PGL, a huge benefit in resource limited settings.

Familial pheochromocytomas and paragangliomas.

Pheochromocytomas and paragangliomas are neural crest cell tumors of the adrenal medulla and parasympathetic/sympathetic ganglia, respectively, that are often associated with catecholamine production. Genetic research over the years has led to our current understanding of the association 13 susceptibility genes with the development of these tumors. Most of the susceptibility genes are now associated with specific clinical presentations, biochemical makeup, tumor location, and associated neoplasms. Recent scientific advances have highlighted the role of somatic mutations in the development of pheochromocytoma/paraganglioma as well as the usefulness of immunohistochemistry in triaging genetic testing. We can now approach genetic testing in pheochromocytoma/paraganglioma patients in a very organized scientific way allowing for the reduction of both the financial and emotional burden on the patient. The discovery of genetic predispositions to the development of pheochromocytoma/paraganglioma not only facilitates better understanding of these tumors but will also lead to improved diagnosis and treatment of this disease.

Usefulness of negative and weak-diffuse pattern of SDHB immunostaining in assessment of SDH mutations in paragangliomas and pheochromocytomas.

This is a confirmatory study about usefulness of SDHB and SDHA immunostaining in assessment of SDH mutations in paragangliomas and pheochromocytomas. Paraganglioma/pheochromocytoma syndrome (PGL/PCC syndrome) consists of different entities, associated with germline mutations in five different genes: SDHD, SDHAF2, SDHC, SDHA and SDHB. It has been suggested that negative immunostaining of SDHB can be taken as an indicator of the presence of a mutation in one of the five SDH genes. We have performed SDHB and SDHA immunohistochemical staining in a series of paragangliomas and pheochromocytomas from 64 patients. The patients had been previously checked for mutations in SDHD, SDHC and SDHB, but also for mutation in RET and VHL. All 14 patients with SDH mutations (9 with SDHB and 5 with SDHD mutations) exhibited negative or weak-diffuse SDHB staining pattern in tumour tissue, whereas cells of the 23 RET mutated and 8 VHL mutated tumours showed a positive SDHB immunostaining. Sixteen of the patients that did not exhibit a mutation in any gene showed positive SDHB immunostaining in tumour tissue, while only three of the patients without mutation exhibited negative staining. All patients exhibited positive pattern of SDHA immunostaining. The results confirm the value of SDHB immunohistochemical status in assessment of germline mutations in PGL/PCC syndrome.

A comprehensive next generation sequencing-based genetic testing strategy to improve diagnosis of inherited pheochromocytoma and paraganglioma.

CONTEXT: Pheochromocytomas and paragangliomas are notable for a high frequency of inherited cases, many of which present as apparently sporadic tumors. OBJECTIVE: The objective of this study was to establish a comprehensive next generation sequencing (NGS)-based strategy for the diagnosis of patients with pheochromocytoma and paraganglioma by testing simultaneously for mutations in MAX, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL. DESIGN: After the methodology for the assay was designed and established, it was validated on DNA samples with known genotype and then patients were studied prospectively. SETTING: The study was performed in a diagnostic genetics laboratory. PATIENTS: DNA samples from 205 individuals affected with adrenal or extraadrenal pheochromocytoma/head and neck paraganglioma (PPGL/HNPGL) were analyzed. A proof-of-principle study was performed using 85 samples known to contain a variant in 1 or more of the genes to be tested, followed by prospective analysis of an additional 120 samples. MAIN OUTCOME MEASURES: We assessed the ability to use an NGS-based method to perform comprehensive analysis of genes implicated in inherited PPGL/HNPGL. RESULTS: The proof-of-principle study showed that the NGS assay and analysis gave a sensitivity of 98.7%. A pathogenic mutation was identified in 16.6% of the prospective analysis cohort of 120 patients. CONCLUSIONS: A comprehensive NGS-based strategy for the analysis of genes associated with predisposition to PPGL and HNPGL was established, validated, and introduced into diagnostic service. The new assay provides simultaneous analysis of 9 genes and allows more rapid and cost-effective mutation detection than the previously used conventional Sanger sequencing-based methodology.

Evaluation of SDHB, SDHD and VHL gene susceptibility testing in the assessment of individuals with non-syndromic phaeochromocytoma, paraganglioma and head and neck paraganglioma.

OBJECTIVES: Research studies have reported that about a third of individuals with phaeochromocytoma/paraganglioma (PPGL) have an inherited predisposition, although the frequency of specific mutations can vary between populations. We evaluated VHL, SDHB and SDHD mutation testing in cohorts of patients with non-syndromic PPGL and head and neck paraganglioma (HNPGL). DESIGN: Prospective, observational evaluation of NHS practice. PATIENTS: Individuals with PPGL/HNPGL referred to a supraregional genetics testing service over a 10-year period. MEASUREMENTS: Clinical (age, tumour site, malignancy, etc.), mutation frequencies and characteristics. RESULTS: A total of 501 probands with PPGL (n = 413) or HNPGL (n = 88) were studied. Thirty-one percent of patients with PPGL presented had a pathogenic mutation in SDHB, SDHD or VHL. Mutation detection rates were highest in those with a positive family history (62%), malignancy (53%), multiple tumours (33%) or PGL (44%). Twenty-eight percent of individuals with a single sporadic phaeochromocytoma had a mutation. Overall, 63% of patients with HNPGL had a mutation (92% of those with a family history, 89% of those with multicentric tumours and 34% of those with a single sporadic HNPGL). Penetrance was calculated in 121 SDHB mutation-positive probands and 187 of their mutation-positive relatives. Most relatives were asymptomatic and lifetime penetrance in non-proband SDHB mutation carriers was <50%. CONCLUSIONS: Practice-based evaluations of genetic testing in PPGL reveal high mutation detection rates. Although clinical criteria can be used to prioritize mutation testing, mutations were detected in 'low risk groups' indicating a need for comprehensive and inexpensive genetic testing strategies for PPGL and HNPGL.

Somatic mutation analysis of the SDHB, SDHC, SDHD, and RET genes in the clinical assessment of sporadic and hereditary pheochromocytoma.

Systemic analysis of somatic mutations of other susceptibility genes in syndromic tumors as well as apparently sporadic tumors in well-characterized specimens is lacking. Its clinical relevance has not been studied. Our objective was to determine the frequency of second allele inactivation in syndromic tumors and determine the frequency and potential clinical impact of somatic mutations and loss of heterozygosity (LOH) of the known susceptibility genes in syndromic and sporadic tumors. Nine tumor specimens from clinically characterized VHL mutation, five from SDHB mutation, four from SDHD mutation, two from RET mutation carriers, and eight from apparently sporadic cases were analyzed. Tumor DNA mutation screening of the SDHx, VHL, and RET genes and LOH analyses of the SDHx and VHL genes were performed. The Yates-corrected chi-squared test was used for comparison of the clinical data and the molecular-genetic results. Second allele inactivation in tumors was identified in 83% of VHL, 80% of SDHB, and 50% of SDHD specimen. High prevalence of VHL (6/6, p=0.024) and SDHB (7/7, p=0.018) somatic mutations has been identified in the sporadic group compared to all others. In the group of the VHL tumors the SDHB somatic events were significantly lower (2/6; p=0.045). In 18/19 (95%) of cases, we were able to demonstrate the presence of at least two concomitant affected susceptibility genes. We conclude that LOH is the most prevalent second allele-inactivating event. SDHB and VHL somatic mutation might play a role in the sporadic forms of tumor development. There is no clinical impact of mutation screening or LOH analysis of tumor specimens.

Clinical predictors and algorithm for the genetic diagnosis of pheochromocytoma patients.

PURPOSE: Six pheochromocytoma susceptibility genes causing distinct syndromes have been identified; approximately one of three of all pheochromocytoma patients carry a predisposing germline mutation. When four major genes (VHL, RET, SDHB, SDHD) are analyzed in a clinical laboratory, costs are approximately $3,400 per patient. The aim of the study is to systematically obtain a robust algorithm to identify who should be genetically tested, and to determine the order in which genes should be tested. EXPERIMENTAL DESIGN: DNA from 989 apparently nonsyndromic patients were scanned for germline mutations in the genes VHL, RET, SDHB, SDHC, and SDHD. Clinical parameters were analyzed as potential predictors for finding mutations by multiple logistic regression, validated by bootstrapping. Cost reduction was calculated between prioritized gene testing compared with that for all genes. RESULTS: Of 989 apparently nonsyndromic pheochromocytoma cases, 187 (19%) harbored germline mutations. Predictors for presence of mutation are age <45 years, multiple pheochromocytoma, extra-adrenal location, and previous head and neck paraganglioma. If we used the presence of any one predictor as indicative of proceeding with gene testing, then 342 (34.6%) patients would be excluded, and only 8 carriers (4.3%) would be missed. We were also able to statistically model the priority of genes to be tested given certain clinical features. E.g., for patients with prior head and neck paraganglioma, the priority would be SDHD>SDHB>RET>VHL. Using the clinical predictor algorithm to prioritize gene testing and order, a 44.7% cost reduction in diagnostic process can be achieved. CONCLUSIONS: Clinical parameters can predict for mutation carriers and help prioritize gene testing to reduce costs in nonsyndromic pheochromocytoma presentations.

Chemically induced pheochromocytomas in rats: mechanisms and relevance for human risk assessment.

Pheochromocytomas are tumors originating from chromaffin cells of the adrenal medulla, which have been observed in numerous carcinogenicity studies. The authors have evaluated pheochromocytoma concurrence with other effects and the possible mechanisms, in order to assess the relevance of such data for the classification of carcinogenic effects and their relevance to humans. The evaluation revealed that pheochromocytomas occur with relatively higher frequency in male rats, especially when the following conditions are involved: hypoxia, uncoupling of oxidative phosphorylation, disturbance in calcium homeostasis, and disturbance of the hypothalamic endocrine axis. The underlying biochemical mechanisms suggest that other substances that interfere with these biochemical endpoints also produce pheochromocytomas. Such endpoints include enzymes involved in catecholamine synthesis, receptor tyrosine kinase (RET), hypoxia-inducible factor (HIF), succinate dehydrogenase, fumarate hydratase, and pyruvate dehydrogenase. To date, there is no indication that the substances inducing pheochromocytomas in animal experiments also induce corresponding tumors in humans. Because the mechanisms of action identified in rats are to be expected in humans, pheochromocytomas may be induced after exposure conditions similar to those used in the animal studies. Whether hereditary mutations represent a risk factor in humans is not clear. Pheochromocytomas that occur in animal experiments currently appear to have little relevance for conditions at the work place. When sufficiently documented and evaluated, such secondary pheochromocytomas are not relevant for classification and human risk assessment.

Should genetic testing be performed in each patient with sporadic pheochromocytoma at presentation?

BACKGROUND: According to previous studies, around 15% of patients with an apparently sporadic pheochromocytoma and a negative family history had a hereditary disease. This high frequency together with the financial support provided to reference laboratories of molecular genetics by the French government led to a nearly systematic screening in each patient with a pheochromocytoma. OBJECTIVE: To check the efficiency of systematic genetic screening in patients with apparently sporadic pheochromocytoma, by analysing the 6 years experience of a multidisciplinary team in this field. METHODS: One hundred patients with a pheochromocytoma-only phenotype and no family history were included. Patients with extra-adrenal tumours were excluded. Prevalence of hereditary forms was determined and analyzed according to age at onset, sex. Cost of the genetic analysis was calculated. RESULTS: A germline mutation in one of the five susceptibility genes (VHL, RET, SDHD, SDHC, SDHB) was identified in eight patients (8%) with an age of onset between 13 and 57 years. Among them, six had a bilateral pheochromocytoma and only two had a unilateral tumour. If the guidelines for genetic screening were age of onset less than 50 or bilateral pheochromocytoma, no patients with a hereditary tumour would be missed and a 24% cost reduction would be achieved. CONCLUSIONS: According to these data, a genetic predisposition test for hereditary pheochromocytoma seems not recommended in patients with a unilateral adrenal tumour diagnosed after 50 in the absence of familial, clinical, biological or imaging features for a familial disease.

Detailed assessment of chromosome 22 aberrations in sporadic pheochromocytoma using array-CGH.

Pheochromocytoma is a predominantly sporadic neuroendocrine tumor derived from the adrenal medulla. Previous low resolution LOH and metaphase-CGH studies reported the loss of chromosomes 1p, 3q, 17p and 22q at various frequencies. However, the molecular mechanism(s) behind development of sporadic pheochromocytoma remains largely unknown. We have applied high-resolution tiling-path microarray-CGH with the primary aim to characterize copy number imbalances affecting chromosome 22 in 66 sporadic pheochromocytomas. We detected copy number alterations on 22q at a frequency of 44%. The predominant finding was monosomy 22 (30%), followed by terminal deletions in 8 samples (12%) and a single interstitial deletion. We further applied a chromosome 1 tiling-path array in 7 tumors with terminal deletions of 22q and found deletions of 1p in all cases. Our overall results suggest that at least 2 distinct regions on both 22q and 1p are important in the tumorigenesis of sporadic pheochromocytoma. A large proportion of pheochromocytomas also displayed indications of cellular heterogeneity. Our study is to our knowledge the first array-CGH study of sporadic pheochromocytoma. Future analysis of this tumor type should preferably be performed in the context of the entire human genome using genome-wide array-CGH, which is a superior methodological approach. Supplemental material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

[Diagnostic guidelines for adrenal gland nodules: typing, assessment of biological potential and prognosis, molecular pathogenesis]. / Diagnostische Leitlinien bei Knoten in der Nebenniere: Typisierung, Dignitäts- und Prognoseerfassung, molekulare Pathogenese.

The aim of morphological tumour diagnosis is to answer clinical questions on type, biological potential, prognosis and aetiology of individual neoplasms. The limitations and perspectives of different methods used in the diagnosis of adrenal tumours, ranging from histology to molecular genetic DNA analyses, are described. When surgical specimens from adrenal neoplasms cannot be typed on the basis of histology and/or with clinica data (e.g. endocrine symptoms and history) as adrenocortical tumours, phaeochromocytomas or metastases to the adrenal, immunohistological investigations with a panel of different antibodies are necessary. After identification of the tissue derivation of an individual adrenal tumour, its biological potential must be assessed. Among adrenocortical neoplasms, adenomas and carcinomas can be distinguished by evaluation of various histological parameters (including structural features and signs of invasion) according to defined algorithms. In addition, conventional histology (by estimation of mitotic activity) allows the discrimination of tumours with especially high malignant potential from other adrenocortical carcinomas. In contrast, among adrenomedullary tumours even the combined use of histological, immunohistological and DNA cytophotometric techniques only allows the definition of risk groups (benign versus suggestive of malignancy), while reliable recognition of an individual malignant phaeochromocytoma is so far impossible. The question as to whether a particular phaeochromocytoma represents a sporadic tumour or a neoplasm inherited as one feature of a defined syndrome cannot be answered with the above methods, but only by the application of molecular genetic techniques.