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Aims: Thymic tumours are rare in routine pathology practice. Although the World Health Organization (WHO) classification describes a number of well-defined categories, the classification remains challenging. The aim of this study was to investigate the reproducibility of the WHO classification among a large group of international pathologists with expertise in thymic pathology and by using whole slide imaging to facilitate rapid diagnostic turnover. Methods and results: Three hundred and five tumours, consisting of 90 biopsies and 215 resection specimens, were reviewed with a panel-based virtual microscopy approach by a group of 13 pathologists with expertise in thymic tumours over a period of 6 years. The specimens were classified according to the WHO 2015 classification. The data were subjected to statistical analysis, and interobserver concordance (Fleiss kappa) was calculated. All cases were diagnosed within a time frame of 2 weeks. The overall level of agreement was substantial (κ = 0.6762), and differed slightly between resection specimens (κ = 0.7281) and biopsies (κ = 0.5955). When analysis was limited to thymomas only, and they were grouped according to the European Society for Medical Oncology Clinical Practice Guidelines into B2, B3 versus A, AB, B1 and B3 versus A, AB, B1, B2, the level of agreement decreased slightly (κ = 0.5506 and κ = 0.4929, respectively). Difficulties arose in distinguishing thymoma from thymic carcinoma. Within the thymoma subgroup, difficulties in distinction were seen within the B group. Conclusions: Agreement in diagnosing thymic lesions is substantial when they are assessed by pathologists with experience of these rare tumours. Digital pathology decreases the turnaround time and facilitates access to what is essentially a multinational resource. This platform provides a template for dealing with rare tumours for which expertise is sparse.

Background and study aims In this study, we evaluated the performance of community hospitals involved in the Dutch quality in endosonography team regarding yield of endoscopic ultrasound (EUS)-guided tissue acquisition (TA) of solid pancreatic lesions using cumulative sum (CUSUM) learning curves. The aims were to assess trends in quality over time and explore potential benefits of CUSUM as a feedback-tool. Patients and methods All consecutive EUS-guided TA procedures for solid pancreatic lesions were registered in five community hospitals between 2015 and 2018. CUSUM learning curves were plotted for overall performance and for performance per center. The American Society of Gastrointestinal Endoscopy-defined key performance indicators, rate of adequate sample (RAS), and diagnostic yield of malignancy (DYM) were used for this purpose. Feedback regarding performance was provided on multiple occasions at regional interest group meetings during the study period. Results A total of 431 EUS-guided TA procedures in 403 patients were included in this study. The overall and per center CUSUM curves for RAS improved over time. CUSUM curves for DYM revealed gradual improvement, reaching the predefined performance target (70 %) overall, and in three of five contributing centers in 2018. Analysis of a sudden downslope development in the CUSUM curve of DYM in one center revealed temporary absence of a senior cytopathologist to have had a temporary negative impact on performance. Conclusions CUSUM-derived learning curves allow for assessment of best practices by comparison among peers in a multidisciplinary multicenter quality improvement initiative and proved to be a valuable and easy-to-interpret means to evaluate EUS performance over time.

Background For progressive metastatic medullary thyroid carcinoma (MTC), the available treatment options with tyrosine kinase inhibitors result in grade 3–4 adverse events in a large number of patients. Peptide Receptor Radionuclide Therapy (PRRT), which has also been suggested to be a useful treatment for MTC, is usually well tolerated, but evidence on its effectivity is very limited. Methods Retrospective evaluation of treatment effects of PRRT in a highly selected group of MTC patients, with progressive disease or refractory symptoms. In addition, a retrospective evaluation of uptake on historical 111In-DTPA-octreotide scans was performed in patients with detectable tumor size > 1 cm. Results Over the last 17 years, 10 MTC patients were treated with PRRT. Four out of 10 patients showed stable disease at first follow-up (8 months after start of therapy) whereas the other 6 were progressive. Patients with stable disease were characterized by a combination of both a high uptake on 111In-DTPA-octreotide scan (uptake grade ≥ 3) and a positive somatostatin receptor type 2a (SSTR2a) expression of the tumor by immunohistochemistry. Retrospective evaluation of historical 111In-DTPA-octreotide scans of 35 non-treated MTC patients revealed low uptake (uptake grade 1) in the vast majority of patients 31/35 (89%) with intermediate uptake (uptake grade 2) in the remaining 4/35 (11%). Conclusions PRRT using 177Lu-octreotate could be considered as a treatment in those patients with high uptake on 111In-DTPA-octreotide scan (uptake grade 3) and positive SSTR2a expression in tumor histology. Since this high uptake was present in a very limited number of patients, this treatment is only suitable in a selected group of MTC patients.

Context: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine, usually benign, tumors. Currently, the only reliable criterion of malignancy is the presence of metastases. Objective: The aim of this study was to identify genes associated with malignancy in PPGLs. Design: Transcriptomic profiling was performed on 40 benign and 11 malignant PPGLs. Genes showing a significantly different expression between benign and malignant PPGLs with a ratio $4 were confirmed and tested in an independent series by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed for the validated genes on 109 benign and 32 malignant PPGLs. Functional assays were performed with hPheo1 cells. Setting: This study was conducted at the Department of Pathology of the Erasmus MC University Medical Center Rotterdam Human Molecular Genetics laboratory of the de Duve Institute, University of Louvain. Patients: PPGL samples from 179 patients, diagnosed between 1972 and 2015, were included. Main outcome measures: Associations between gene expression and malignancy were tested using supervised clustering approaches. Results: Ten differentially expressed genes were selected based on messenger RNA (mRNA) expression array data. Contactin 4 (CNTN4) was overexpressed in malignant vs benign tumors [4.62-fold; false discovery rate (FDR), 0.001]. Overexpression at the mRNA level was confirmed using qRTPCR (2.90-fold, P=0.02; validation set: 4.26-fold, P=0.005). Consistent findings were obtained in The Cancer Genome Atlas cohort (2.7-fold; FDR, 0.02). CNTN4 protein was more frequently expressed in malignant than in benign PPGLs by immunohistochemistry (58% vs 17%; P=0.002). Survival after 7 days of culture under starvation conditions was significantly enhanced in hPheo1 cells transfected with CNTN4 complementary DNA. Conclusion: CNTN4 expression is consistently associated with malignant behavior in PPGLs.

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ObjectiveAlthough the succinate dehydrogenase (SDH)-related tumor spectrum has been recently expanded, there are only rare reports of non-pheochromocytoma/paraganglioma tumors in SDHx-mutated patients. Therefore, questions still remain unresolved concerning the aforementioned tumors with regard to their pathogenesis, clinicopathological phenotype, and even causal relatedness to SDHx mutations. Absence of SDHB expression in tumors derived from tissues susceptible to SDH deficiency is not fully elucidated.Design and methodsThree unrelated SDHD patients, two with pituitary adenoma (PA) and one with papillary thyroid carcinoma (PTC), and three SDHB patients affected by renal cell carcinomas (RCCs) were identified from four European centers. SDHA/SDHB immunohistochemistry (IHC), SDHx mutation analysis, and loss of heterozygosity analysis of the involved SDHx gene were performed on all tumors. A cohort of 348 tumors of unknown SDHx mutational status, including renal tumors, PTCs, PAs, neuroblastic tumors, seminomas, and adenomatoid tumors, was investigated by SDHB IHC.ResultsOf the six index patients, all RCCs and one PA displayed SDHB immunonegativity in contrast to the other PA and PTC. All immunonegative tumors demonstrated loss of the WT allele, indicating bi-allelic inactivation of the germline mutated gene. Of 348 tumors, one clear cell RCC exhibited partial loss of SDHB expression.ConclusionsThese findings strengthen the etiological association of SDHx genes with pituitary neoplasia and provide evidence against a link between PTC and SDHx mutations. Somatic deletions seem to constitute the second hit in SDHB-related renal neoplasia, while SDHx alterations do not appear to be primary drivers in sporadic tumorigenesis from tissues affected by SDH deficiency.

BACKGROUND: The purpose of this study was to give an overview on hereditary syndromes associated with head and neck paragangliomas (HNPGs). METHODS: Our methods were the review and discussion of the pertinent literature. RESULTS: About one third of all patients with HNPGs are carriers of germline mutations. Hereditary HNPGs have been described in association with mutations of 10 different genes. Mutations of the genes succinate dehydrogenase subunit D (SDHD), succinate dehydrogenase complex assembly factor 2 gene (SDHAF2), succinate dehydrogenase subunit C (SDHC), and succinate dehydrogenase subunit B (SDHB) are the cause of paraganglioma syndromes (PGLs) 1, 2, 3, and 4. Succinate dehydrogenase subunit A (SDHA), von Hippel-Lindau (VHL), and transmembrane protein 127 (TMEM127) gene mutations also harbor the risk for HNPG development. HNPGs in patients with rearranged during transfection (RET), neurofibromatosis type 1 (NF1), and MYC-associated factor X (MAX) gene mutations have been described very infrequently. CONCLUSION: All patients with HNPGs should be offered a molecular genetic screening. This screening may usually be restricted to mutations of the genes SDHD, SDHB, and SDHC. Certain clinical parameters can help to set up the order in which those genes should be tested. (c) 2013 Wiley Periodicals, Inc. Head Neck 36: 907-916, 2014.

Functional zonation of the adrenal cortex is a consequence of the zone-specific expression of P450c17 ( CYP17A1) and its cofactors. Activin and inhibin peptides are differentially produced within the zones of the adrenal cortex and have been implicated in steroidogenic control. In this study, we investigated whether activin and inhibin can function as intermediates in functional zonation of the human adrenal cortex. Activin A suppressed CYP17A1 expression and P450c17 function in adrenocortical cell lines as well as in primary adrenal cell cultures. Inhibin βA-subunit mRNA and activin A protein levels were found to be increased up to 1,900-fold and 49-fold, respectively, after protein kinase C (PKC) stimulation through PMA or angiotensin II in H295R adrenocortical carcinoma cells. This was confirmed in HAC15 cells and for PMA in primary adrenal cell cultures. Both PMA and Ang II decreased CYP17A1 expression in the adrenocortical cell lines, whereas PMA concurrently suppressed CYP17A1 levels in the primary cultures. Inhibition of activin signaling during PKC stimulation through silencing of the inhibin βA-subunit or blocking of the activin type I receptor opposed the PMA-induced downregulation of CYP17A1 expression and P450c17 function. In contrast, PKA stimulation through adrenocorticotrophin or forskolin increased expression of the inhibin α-subunit and betaglycan, both of which are antagonists of activin action. These data indicate that activin A acts as a PKC-induced paracrine factor involved in the suppression of CYP17A1 in the zona glomerulosa and can thereby contribute to functional adrenocortical zonation.

Most gastrointestinal stromal tumors (GISTs) harbor oncogenic mutations in KIT or platelet-derived growth factor receptor-α. However, a small subset of GISTs lacks such mutations and is termed 'wild-type GISTs'. Germline mutation in any of the subunits of succinate dehydrogenase (SDH) predisposes individuals to hereditary paragangliomas and pheochromocytomas. However, germline mutations of the genes encoding SDH subunits A, B, C or D (SDHA, SDHB, SDHC or SDHD; collectively SDHx) are also identified in GISTs. SDHA and SDHB immunohistochemistry are reliable techniques to identify pheochromocytomas and paragangliomas with mutations in SDHA, SDHB, SDHC and SDHD. In this study, we investigated if SDHA immunohistochemistry could also identify SDHA-mutated GISTs. Twenty-four adult wild-type GISTs and nine pediatric/adolescent wild-type GISTs were analyzed with SDHB, and where this was negative, then with SDHA immunohistochemistry. If SDHA immunohistochemistry was negative, sequencing analysis of the entire SDHA coding sequence was performed. All nine pediatric/adolescent GISTs and seven adult wild-type GISTs were negative for SDHB immunohistochemistry. One pediatric GIST and three SDHB-immunonegative adult wild-type GISTs were negative for SDHA immunohistochemistry. In all four SDHA-negative GISTs, a germline SDHA c.91C>T transition was found leading to a nonsense p.Arg31X mutation. Our results demonstrate that SDHA immunohistochemistry on GISTs can identify the presence of an SDHA germline mutation. Identifying GISTs with deficient SDH activity warrants additional genetic testing, evaluation and follow-up for inherited disorders and paragangliomas.

ACTH stimulates adrenocortical steroid production through the melanocortin 2 receptor (MC2R). MC2R trafficking and signaling are dependent on the MC2R accessory protein (MRAP). The MRAP homolog MRAP2 also transports the MC2R to the cell surface but might prevent activation.The objective of the investigation was to study the regulatory pathways of MRAP and MRAP2 and their contributions to ACTH responsiveness in human adrenal tissues.MRAP, MRAP2, and MC2R expression levels were studied in 32 human adrenocortical samples. Regulation of these mRNAs was investigated in 43 primary adrenal cultures, stimulated with ACTH, forskolin, angiotensin II (AngII), phorbol-12-myristate-13-acetate (PMA), or dexamethasone. The induction of cortisol, cAMP, and ACTH-responsive genes after treatment with ACTH was related to MRAP, MRAP2, and MC2R expression levels.MRAP and MRAP2 levels were lower in adrenocortical carcinomas (ACC) than in other adrenal tissues (P0.001). Patient ACTH and cortisol levels were associated with adrenal levels of MRAP and MC2R in adrenal hyperplasia samples (P0.05) but not in tumors. ACTH induced the expression of MRAP 11 ± 2.1-fold and MC2R 20 ± 3.8-fold in all adrenal tissue types (mean ± SEM, both P0.0001), whereas AngII augmented these mRNAs 4.0 ± 1.2-fold and 12.6 ± 3.2-fold (P0.0001) in all but the ACC. MRAP2 expression was suppressed by forskolin (-24 ± 15%, P = 0.013) and PMA (-22 ± 7%, P = 0.0007). MRAP, MRAP2, or MC2R levels were not associated with the induction of cortisol, cAMP, or gene expression by ACTH in vitro.MRAP and MC2R expression is induced by ACTH and AngII, which would facilitate cell surface receptor availability. Physiological expression levels of MRAP, MRAP2, and MC2R were not limiting for ACTH sensitivity.

Despite the established role of Ki67 labeling index in prognostic stratification of adrenocortical carcinomas and its recent integration into treatment flow charts, the reproducibility of the assessment method has not been determined. The aim of this study was to investigate interobserver variability among endocrine pathologists using a web-based virtual microscopy approach. Ki67-stained slides of 76 adrenocortical carcinomas were analyzed independently by 14 observers, each according to their method of preference including eyeballing, formal manual counting, and digital image analysis. The interobserver variation was statistically significant (P

Context: Familial pheochromocytoma (PCC) has been associated with germline mutations in 16 genes. Here we investigated three siblings presenting with bilateral pheochromocytomas. In addition, the index patient also exhibited renal oncocytoma and erythrocytosis, whereas the second sibling presented with a lymph node metastasis. Design: First, single-nucleotide polymorphism array and exome sequencing were performed on germline and PCC-derived DNA to identify genomic alterations in the index patient. Second, alterations were confirmed and validated by Sanger sequencing, analyzed by (multiplexed) PCR to determine the loss of the wild-type allele, and investigated by immunohistochemistry in the tumors of the three siblings. Results: The index patient's germline DNA revealed a large complex genomic alteration encompassing the intragenic and promoter regions of Myc-associated factor X (MAX) and alpha-(1,6)fucosyltransferase (FUT8). In all three siblings the MAX alteration was confirmed, and the loss of the wild-type MAX and FUT8 alleles was demonstrated in all tumors. Uniparental disomy of chromosome 14q, previously demonstrated as a hallmark for MAX-related PCC, was shown in the index patient's PCC by single-nucleotide polymorphism array. Loss of MAX and FUT8 protein expression was demonstrated by immunohistochemistry in the tumors from the three siblings. Conclusions: Our results indicate that large genomic deletions of MAX should be considered in familial and bilateral PCC with prior negative testing for gene mutations. In addition, our results confirm that MAX is a tumor suppressor gene for renal oncocytomas.

Mutations in the tumor suppressor genes SDHB, SDHC, and SDHD (or collectively SDHx) cause the inherited paraganglioma syndromes, characterized by pheochromocytomas and paragangliomas. However, other tumors have been associated with SDHx mutations, such as gastrointestinal stromal tumors (GISTs) specifically in the context of Carney-Stratakis syndrome. Previously, we have shown that SDHB immunohistochemistry is a reliable technique for the identification of pheochromocytomas and paragangliomas caused by SDHx mutations. We hypothesized that GISTs in patients with SDHx mutations would be negative immunohistochemically for SDHB as well. Four GISTs from patients with Carney-Stratakis syndrome and six from patients with Carney triad were investigated by SDHB immunohistochemistry. Five GISTs with KIT or PDGFRA gene mutations were used as controls. In addition, SDHB immunohistochemistry was performed on 42 apparently sporadic GISTs. In cases in which the SDHB immunohistochemistry was negative, mutational analysis of SDHB, SDHC, and SDHD was performed. All GISTs from patients with Carney-Stratakis syndrome and Carney triad were negative for SDHB immunohistochemically. In one patient with Carney-Stratakis syndrome, a germline SDHB mutation was found (p.Ser92Thr). The five GISTs with a KIT or PDGFRA gene mutation were all immunohistochemically positive for SDHB. Of the 42 sporadic tumors, one GIST was SDHB-negative. Mutational analysis of this tumor did not reveal an SDHx mutation. All SDHB-negative GISTs were located in the stomach, had an epithelioid morphology, and had no KIT or PDGFRA mutations. We show that Carney-Stratakis syndrome-and Carney-triad-associated GISTs are negative by immunohistochemistry for SDHB in contrast to KIT-or PDGFRA-mutated GISTs and a majority of sporadic GISTs. We suggest that GISTs of epithelioid cell morphology are tested for SDHB immunohistochemically. In case of negative SDHB staining in GISTs, Carney-Stratakis syndrome or Carney triad should be considered and appropriate clinical surveillance should be instituted. Modern Pathology (2011) 24, 147-151; doi:10.1038/modpathol.2010.185; published online 1 October 2010

Prediction of malignant behaviour of pheochromocytomas/sympathetic paragangliomas (PCCs/PGLs) is very difficult if not impossible on a histopathological basis. In a familial setting, it is well known that succinate dehydrogenase subunit B (SDHB)-associated PCC/PGL very often metastasise. Recently, absence of SDHB expression as measured through immunohistochemistry was shown to be an excellent indicator of the presence of an SDH germline mutation in PCC/PGL. SDHB loss is believed to lead to tumour formation by activation of hypoxia signals. To clarify the potential use of SDHB immunohistochemistry as a marker of malignancy in PCC/PGL and its association with classic hypoxia signalling we examined SDHB, hypoxia inducible factor-1α (Hif-1α) and its targets CA-9 and GLUT-1 expression on protein level using immunohistochemistry on a tissue micro array on a series of familial and sporadic tumours of 115 patients. Survival data was available for 66 patients. SDHB protein expression was lost in the tumour tissue of 12 of 99 patients. Of those 12 patients, 5 had an SDHB germline mutation, in 4 patients no germline mutation was detected and mutational status remained unknown in parts in 3 patients. Loss of SDHB expression was not associated with increased classic hypoxia signalling as detected by Hif-1α, CA-9 or GLUT-1 staining. Loss of SDHB expression was associated with an adverse outcome. The lack of correlation of SDHB loss with classic hypoxia signals argues against the current hypoxia hypothesis in malignant PCC/PGL. We suggest SDHB protein loss as a marker of adverse outcome both in sporadic and in familial PCC/PGL.

Pheochromocytomas (PCCs) and extra-adrenal sympathetic paragangliomas (sPGLs) are catecholamine-producing tumors occurring in the context of hereditary tumor syndromes, with known germline mutations, and as sporadic tumors. The pathogenesis of sporadic PCC and sPGL is poorly understood, and little is known about intra-tumoral heterogeneity with respect to molecular aberrations. Since knowledge on intra-tumoral heterogeneity is important for understanding the pathogenesis of these tumors, we investigated 12 benign and 8 malignant PCCs and sPGLs for loss of heterozygosity (LOH) on DNA extracted from different regions of each tumor and from metastases. LOH markers were selected on chromosomal regions frequently deleted in PCC, including 1p, 3q, 3p, and 11p. Benign tumors were found to have less intra-tumoral heterogeneity (overall 8%) than malignant tumors (overall 23%), with the highest frequencies for chromosome 1p36 in the benign tumors (17%) and 1p13 and 3q24 in malignant tumors (both 38%). In addition, differences in LOH patterns were detected between paired primary malignant tumors, and their metastases and different LOH patterns were observed in bilateral PCC of a multiple endocrine neoplasia type 2 patient. We demonstrate that malignant PCC and sPGL have more intra-tumoral molecular heterogeneity than benign tumors, which suggests that benign and malignant PCC and sPGL have a different pathogenesis. Endocrine-Related Cancer (2010) 17 653-662

Phaeochromocytomas (PCCs) are neuro-endocrine tumours of the adrenal medulla that are usually benign, but approximately 10% of patients develop metastases. Malignant PCCs can only be diagnosed with certainty if metastases are present. Here we describe adrenal tumours generated in a Pten conditional knock-out (KO) mouse model. We characterized the molecular alterations in these tumours and compared them with human PCC. Thirty-two of 41 (78%) male Psa-Cre;Pten-loxP/loxP mice presented adrenal tumours that were shown to be PCC by histology and by immunohistochemical staining for enzymes in the catecholamine biosynthetic pathway. In 6 of 17 investigated mice, histological and immunohistochemical evidence was obtained for the presence of PCC lung metastases. Array comparative genomic hybridization (CGH) analysis of the primary tumours showed loss of chromosomes 6 and 19, which are syntenic to human 3p and 11q. Another frequent alteration found was gain of chromosome 15, which is syntenic to human chromosome 5. The molecular aberrations in the mouse model corresponded to the alterations found in a subtype of human PCC, suggesting that the PCC of the Pten KO mice might be representative of human PCC. The mouse model should allow further studies into the pathogenesis of human malignant PCCs and into therapeutic strategies for these tumours. Copyright (C) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic changes in the tumorigenesis of sporadic pheochromocytomas are poorly understood, and there are no good markers to discriminate benign from malignant pheochromocytomas. p53 is a tumor suppressor gene and aberrations in this gene are frequently found in many tumor types. The role of p53 in pheochromocytoma tumorigenesis is unclear, with some studies suggesting that p53 mutations can be used to discriminate benign from malignant pheochromocytomas while other studies do not find such an association. Because most of these investigations were hampered by small series of tumors and the use of varying methods, we have performed a comprehensive analysis of p53 aberrations in a large series of pheochromocytomas. Comparative genomic hybridization analysis of 31 benign and 20 malignant tumors showed loss of the p53 locus at chromosome 17p13.1 in 23/51 (45%) cases, and most of these results were confirmed by fluorescence in situ hybridization. Forty-three tumors, including the malignant tumors and the tumors with loss of the p53 locus, were analyzed for p53 mutations in exons 5-8, but none were found. Furthermore, p53 immunohistochemistry on 35 cases revealed strong nuclear p53 expression in only two pheochromocytoma metastases, all other tumors being negative. We conclude that, although there is frequent loss of the p53 locus on 17p, the p53 gene does not appear to play a major role in pheochromocytoma tumorigenesis.

Thyroid cancer is comparatively rare. Thyroid nodules, on the other hand, are frequently diagnosed as a result of increasing use of diagnostic imaging. Cytological investigation of small nodules that have been found by chance often reveals micropapillary carcinoma that is probably not clinically relevant. The new guideline 'Thyroid cancer' advises that cytological investigation of these non-palpable, incidentally discovered thyroid nodules should only be performed on indication. The standard treatment for patients with papillary or follicular thyroid cancer consists of thyroidectomy followed by, if indicated, lymph-node dissection, ablation therapy with radioactive iodine and TSH-suppression. The extent of this treatment is determined on the basis of known prognostic factors and the results of initial treatment. Targeted systemic therapy is available for patients with metastatic progressive disease. There is more focus on the effects of short- and long-term treatment, in order to optimise quality of life.

Contains fulltext : 50547.pdf (Publisher’s version ) (Closed access) Pheochromocytomas (PCC) are relatively rare neuroendocrine tumors, mainly of the adrenal medulla. They arise sporadically or occur secondary to inherited cancer syndromes, such as multiple endocrine neoplasia type II (MEN2), von Hippel-Lindau disease (VHL), or neurofibromatosis type I (NF1). Loss of 1p is the most frequently encountered genetic alteration, especially in MEN2-related and sporadic PCC. Previous studies have revealed three regions of common somatic loss on chromosome arm 1p, using chromosome-based comparative genomic hybridization (CGH) and LOH analysis. To investigate these chromosomal aberrations with a higher resolution and sensitivity, we performed microarray-based CGH with 13 sporadic and 11 syndrome-related (10 MEN2A-related and 1 NF1-related) tumors. The array consisted of 642 overlapping bacterial artificial chromosome (BAC) clones mapped to 1p11.2-p36.33. Chromosomal deletions on 1p were detected in 18 of 24 cases (75%). Among 9 tumors with partial 1p loss, the deleted region was restricted to 1cen-1p32.3 in six cases (25%), indicating a region of genetic instability. The consensus regions of deletion in this study involved 1cen-1p21.1, 1p21.3-1p31.3, and 1p34.3-1p36.33. In conclusion, these data strongly suggest that chromosome arm 1p is the site for multiple tumor suppressor genes, although the potential candidate genes CDKN2C and PTPRF/LAR are not included in these regions.

Pheochromocytomas (PCC) and paragangliomas (PGL) are tumours occurring in the adrenal medulla and in extra-adrenal paraganglia, respectively. They have long been associated with familial occurrence and several syndromes had been described in which PCC formed an important component, including multiple endocrine neoplasia type 2, von Hippel-Lindau disease and neurofibromatosis type 1. Since the beginning of this millennium, both by the elucidation of specific genes, such as the various succinate dehydrogenase genes, as well as by generic molecular biology approaches, such as The Cancer Genome Atlas (TCGA) initiative, it was shown that the frequency of germline mutations in candidate genes for PCC and PGL has increased to 35–40%. In addition, somatic mutations have been shown to be much more frequent than previously thought, such that now 60–65% of these tumours harbour either a germline or a somatic mutation. This review gives an overview of the various syndromes and the genes involved, concluding with recommendations for genetic testing in the current era of genome wide analysis.

Despite the established role of SDHB/SDHA immunohistochemistry as a valuable tool to identify patients at risk for familial succinate dehydrogenase-related pheochromocytoma/paraganglioma syndromes, the reproducibility of the assessment methods has not as yet been determined. The aim of this study was to investigate interobserver variability among seven expert endocrine pathologists using a web-based virtual microscopy approach in a large multicenter pheochromocytoma/paraganglioma cohort (n=351): (1) 73 SDH mutated, (2) 105 non-SDH mutated, (3) 128 samples without identified SDH-x mutations, and (4) 45 with incomplete SDH molecular genetic analysis. Substantial agreement among all the reviewers was observed either with a two-tiered classification (SDHB kappa= 0.7338; SDHA kappa = 0.6707) or a three-tiered classification approach (SDHB kappa = 0.6543; SDHA kappa = 0.7516). Consensus was achieved in 315 cases (89.74%) for SDHB immunohistochemistry and in 348 cases (99.15%) for SDHA immunohistochemistry. Among the concordant cases, 62 of 69 (similar to 90%) SDHB-/C-/D-/AF2-mutated cases displayed SDHB immunonegativity and SDHA immunopositivity, 3 of 4 (75%) with SDHA mutations showed loss of SDHA/SDHB protein expression, whereas 98 of 105 (93%) non-SDH-x-mutated counterparts demonstrated retention of SDHA/SDHB protein expression. Two SDHD-mutated extra-adrenal paragangliomas were scored as SDHB immunopositive, whereas 9 of 128 (7%) tumors without identified SDH-x mutations, 6 of 37 (similar to 16%) VHL-mutated, as well as 1 of 21 (similar to 5%) NF1-mutated tumors were evaluated as SDHB immunonegative. Although 14 out of those 16 SDHB-immunonegative cases were nonmetastatic, an overall significant correlation between SDHB immunonegativity and malignancy was observed (P=0.00019). We conclude that SDHB/SDHA immunohistochemistry is a reliable tool to identify patients with SDH-x mutations with an additional value in the assessment of genetic variants of unknown significance. If SDH molecular genetic analysis fails to detect a mutation in SDHB-immunonegative tumor, SDHC promoter methylation and/or VHL/NF1 testing with the use of targeted next-generation sequencing is advisable.

Context: Only a small number of case reports has been published on patients with PTHrP-hypersecreting metastatic gastroenteropancreatic (GEP) neuroendocrine tumors (NETs). Objective: The objective of this study was to evaluate the clinical, biochemical, and radiological features, management, and treatment outcome of patients with PTHrP-hypersecreting GEP-NETs. Design: Retrospective case series. Setting: Tertiary referral hospital. Main Outcome Measures: Clinical, biochemical, and radiological features were measured, as well as response to therapy and survival. Patients: Ten patients with PTHrP-secreting GEP-NETs (nine pancreatic and one unknown primary) with a median age of 50.4 years (range, 38.3-61.1) were studied. Multiple endocrine neoplasia type 1 patients were excluded. Results: The median follow-up was 57.2 months (range, 11.6-204.5 mo). Median overall survival was 86.0 months. In total, 51 different treatment interventions and combinations were applied. In seven of the 10 patients, somatostatin analog (SSA) treatment resulted in a temporary normalization of serum calcium levels with a long-term response observed in two patients (up to 35.2 mo). Peptide receptor radiotherapy (PRRT) with radiolabeled SSAs induced long-term responses ranging from 9.0-49.0 months in four of six patients treated with PRRT. Conclusions: Hypersecretion of PTHrP by metastatic GEP-NETs is very rare and seems to be exclusively associated with metastatic pancreatic NETs. PTHrP production has major clinical impact because poorly controllable hypercalcemia is associated with increased morbidity and mortality. The most successful treatment options for PTHrP-producing GEP-NETs are SSAs and PRRT using radiolabeled SSAs. Isotonic saline and bisphosphonates can be considered as supportive therapies.

To the Editor: As many as 25% of pheochromocytomas — catecholamine-producing tumors located along the sympathetic nervous system, including the adrenals — occur in hereditary tumor syndromes that i...

T-cell receptor (TCR) repertoire diversity, thymic output, clonal size and peripheral T-lymphocyte numbers largely depend on intra-thymic and post-thymic T-lymphocyte proliferation. However, quantitative insight into thymocyte and T-lymphocyte proliferation is still lacking. We developed a new TCRG-based TCR excision circle (TREC) assay, the V gamma-J gamma TREC assay, which we used together with an adjusted delta REC-psi J alpha TREC assay to quantify the proliferative history of human thymocyte and T-lymphocyte subpopulations from children and adults. This revealed that thymocytes undergo similar to 6-8 intra-thymic cell divisions from the double negative (DN) 3 developmental stage onwards, which appeared independent of age. Thus thymocyte proliferation after the DN3 developmental stages is stable and therefore not contributing to the reduced thymic output upon ageing. Cord blood naive T lymphocytes had already undergone similar to 2-3 post-thymic cell divisions, which increased to similar to 6-7 cell divisions in naive T lymphocytes of middle-aged adults, indicating the importance of homeostatic naive T-lymphocyte proliferation from a young age onwards in the maintenance of peripheral T-lymphocyte numbers. In conclusion, our data provide quantitative insight into the proliferative history of thymocyte and T-lymphocyte subpopulations and alterations herein upon ageing. This novel TREC assay approach could prove valuable in immune status monitoring in a variety of conditions. (C) 2012 Elsevier Ltd. All rights reserved.

The purpose of this study was to give an overview on hereditary syndromes associated with head and neck paragangliomas (HNPGs).Our methods were the review and discussion of the pertinent literature.About one third of all patients with HNPGs are carriers of germline mutations. Hereditary HNPGs have been described in association with mutations of 10 different genes. Mutations of the genes succinate dehydrogenase subunit D (SDHD), succinate dehydrogenase complex assembly factor 2 gene (SDHAF2), succinate dehydrogenase subunit C (SDHC), and succinate dehydrogenase subunit B (SDHB) are the cause of paraganglioma syndromes (PGLs) 1, 2, 3, and 4. Succinate dehydrogenase subunit A (SDHA), von Hippel-Lindau (VHL), and transmembrane protein 127 (TMEM127) gene mutations also harbor the risk for HNPG development. HNPGs in patients with rearranged during transfection (RET), neurofibromatosis type 1 (NF1), and MYC-associated factor X (MAX) gene mutations have been described very infrequently.All patients with HNPGs should be offered a molecular genetic screening. This screening may usually be restricted to mutations of the genes SDHD, SDHB, and SDHC. Certain clinical parameters can help to set up the order in which those genes should be tested.

ContextPrimary pigmented nodular adrenocortical disease (PPNAD) can lead to steroid hormone overproduction. Mutations in the cAMP protein kinase A regulatory subunit type 1A (PRKAR1A) are causative of PPNAD. Steroidogenesis in PPNAD can be modified through a local glucocorticoid feed-forward loop.ObjectiveInvestigation of regulation of steroidogenesis in a case of PPNAD with virilization.Materials and methodsA 33-year-old woman presented with primary infertility due to hyperandrogenism. Elevated levels of testosterone and subclinical ACTH-independent Cushing's syndrome led to the discovery of an adrenal tumor, which was diagnosed as PPNAD.In vivoevaluation of aberrantly expressed hormone receptors showed no steroid response to known stimuli. Genetic analysis revealed a PRKAR1A protein-truncating Q28X mutation. After adrenalectomy, steroid levels normalized. Tumor cells were cultured and steroidogenic responses to ACTH and dexamethasone were measured and compared with those in normal adrenal and adrenocortical carcinoma cells. Expression levels of 17β-hydroxysteroid dehydrogenase (17β-HSD) types 3 and 5 and steroid receptors were quantified in PPNAD, normal adrenal, and adrenal adenoma tissues.ResultsIsolated PPNAD cells, analogous to normal adrenal cells, showed both increased steroidogenic enzyme expression and steroid secretion in response to ACTH. Dexamethasone did not affect steroid production in the investigated types of adrenal cells. 17β-HSD type 5 was expressed at a higher level in the PPNAD-associated adenoma compared with control adrenal tissue.ConclusionPPNAD-associated adenomas can cause virilization and infertility by adrenal androgen overproduction. This may be due to steroidogenic control mechanisms that differ from those described for PPNAD without large adenomas.

Thyroid abnormalities are found in 70% of patients with primary hyperparathyroidism. The simultaneous occurrence of a parathyroid adenoma and non-medullary thyroid carcinoma is rare.A parathyroid adenoma was suspected in a 39-year-old man with hypertension and a previous history of nephrolithiasis on the basis of laboratory results and scintigraphic investigations of the thyroid gland. Histopathological investigation of perioperatively obtained frozen sections of 2 nodules, however, revealed lymph node metastases of a papillary thyroid carcinoma; thyroid carcinoma was confirmed by studying a biopsy of the thyroid gland. Immunohistochemical staining of this biopsy for parathyroid hormone was negative; a parathyroid adenoma could not be confirmed. Total thyroidectomy and exploration of the parathyroid glands followed. Parathyroid hormone and calcium values declined following extirpation of the thyroid gland. Histopathological investigation of the thyroid gland revealed an intrathyroidally situated parathyroid adenoma and a multifocal papillary thyroid carcinoma.The case history emphasises the importance of thorough evaluation of the thyroid gland in patients with primary hyperparathyroidism to reveal or exclude simultaneous presentation of parathyroid adenoma and thyroid carcinoma.

Congenital diaphragmatic hernia (CDH) is associated with lung hypoplasia and pulmonary hypertension and has high morbidity and mortality rates. The cause and pathophysiology of CDH are not fully understood. However, impaired angiogenesis appears to play an important role in the pathophysiology of CDH. Therefore, we examined different components of an important pathway in angiogenesis: hypoxia-inducible factors (HIFs); HIF regulators von Hippel-Lindau (VHL) and prolyl 3-hydroxylase (PHD3); and HIF target genes vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR-2). Quantitative polymerase chain reaction of lung tissue showed a significantly decreased expression of VEGF-A mRNA in the alveolar stage of lung development in CDH patients compared with matched control patients. In the canalicular stage, no differences for VEGF-A were seen between the lungs of CDH patients and those of control patients. Other components of angiogenesis (VHL, HIF-1 alpha, HIF-2 alpha, HIF-3 alpha, VEGFR-2 mRNA, PHD3 protein) that were analyzed showed no differences in expression between CDH and control patients, independent of the developmental stage. A lower expression of VEGF mRNA in CDH patients in the alveolar stage, possibly as a result of downregulation of HIF-2 alpha might indicate a role for these factors in the pathophysiology of CDH.

Context: Pheochromocytoma-paraganglioma syndrome is caused by mutations in SDHB, SDHC, and SDHD, encoding subunits of succinate dehydrogenase (SDH), and in SDHAF2, required for flavination of SDHA. A recent report described a patient with an abdominal paraganglioma, immunohistochemically negative for SDHA, and identified a causal germline mutation in SDHA. Objective: In this study, we evaluated the significance of SDHA immunohistochemistry in the identification of new patients with SDHA mutations. Setting: This study was performed in the Erasmus Medical Center in Rotterdam (The Netherlands) and the Université Paris Descartes in Paris (France). Methods: We investigated 316 pheochromocytomas and paragangliomas for SDHA expression. Sequence analysis of SDHA was performed on all tumors that were immunohistochemically negative for SDHA and on a subset of tumors immunohistochemically positive for SDHA. Results: Six tumors were immunohistochemically negative for SDHA. Four tumors from Dutch patients showed a germline c.91C→T SDHA gene mutation (p.Arg31X). Another tumor (from France) carried a germline SDHA missense mutation c.1753C→T (p.Arg585Trp). Loss of the wild-type SDHA allele was confirmed by loss of heterozygosity analysis. Sequence analysis of 35 SDHA immunohistochemically positive tumors did not reveal additional SDHA mutations. Conclusions: Our results demonstrate that SDHA immunohistochemistry on paraffin-embedded tumors can reveal the presence of SDHA germline mutations and allowed the identification of SDHA-related tumors in at least 3% of patients affected by apparently sporadic (para)sympathetic paragangliomas and pheochromocytomas.

Pheochromocytomas (PCC) and paragangliomas (PGL) are tumors arising from neural crest-derived chromaffin cells. PCC are located in the adrenal medulla and produce, with few exceptions, catecholamines. Sympathetic PGL (sPGL, previously also designated extraadrenal PCC) also produce catecholamines but arise in the chromaffin tissue of the sympathetic trunk that is situated along the proximal aorta reaching down to the abdominal aorta and the urinary bladder. In the developing embryo, the organs of Zuckerkandl, which serve as catecholamine-releasing organs during development of the fetal adrenals, can be identified in the sympathetic trunk, which is an important site for sPGL to occur, especially in young patients. Together, PCC and sPGL have an incidence that varies between 2 and 8 per million.

Context: Von Hippel-Lindau (VHL) disease, caused by germline mutations in the VHL gene, is a hereditary tumor syndrome manifested by hemangioblastomas, clear cell renal cell carcinomas, and pheochromocytomas. In addition, a multitude of other rare tumors, including parasympathetic paragangliomas, can occur and even be the sole manifestation of VHL disease. The VHL gene is a bona fide tumor suppressor gene with biallelic inactivation contributing to tumor formation. However, in parasympathetic paragangliomas occurring in VHL disease, biallelic inactivation of the VHL gene has not been demonstrated to date. Design: The head and neck paragangliomas of two VHL patients were analyzed for mutations by direct sequencing of the VHL gene. In addition loss of heterozygosity analysis was performed for three microsatellite loci near the VHL gene. To rule out other underlying genetic causes of the parasympathetic paragangliomas, mutation analysis of the SDHB, SDHC, and SDHD genes was also performed. Results: Apart from germline VHL mutations, no additional mutations were found in the paraganglioma-related tumor suppressor genes SDHB, SDHC, and SDHD. Analysis of paraganglioma tissue revealed loss of the VHL wild-type allele in both tumors, indicating that in these tumors biallelic VHL gene inactivation occurred. Conclusions: These findings indicate that parasympathetic paragangliomas in VHL disease, although rare, are part of the syndrome and related to VHL gene inactivation. Clinicians should be aware of the potential occurrence of parasympathetic paragangliomas in VHL disease. (J Clin Endocrinol Metab 94: 4367-4371, 2009)

Contains fulltext : 80017.pdf (Publisher’s version ) (Closed access) BACKGROUND: Phaeochromocytomas and paragangliomas are neuro-endocrine tumours that occur sporadically and in several hereditary tumour syndromes, including the phaeochromocytoma-paraganglioma syndrome. This syndrome is caused by germline mutations in succinate dehydrogenase B (SDHB), C (SDHC), or D (SDHD) genes. Clinically, the phaeochromocytoma-paraganglioma syndrome is often unrecognised, although 10-30% of apparently sporadic phaeochromocytomas and paragangliomas harbour germline SDH-gene mutations. Despite these figures, the screening of phaeochromocytomas and paragangliomas for mutations in the SDH genes to detect phaeochromocytoma-paraganglioma syndrome is rarely done because of time and financial constraints. We investigated whether SDHB immunohistochemistry could effectively discriminate between SDH-related and non-SDH-related phaeochromocytomas and paragangliomas in large retrospective and prospective tumour series. METHODS: Immunohistochemistry for SDHB was done on 220 tumours. Two retrospective series of 175 phaeochromocytomas and paragangliomas with known germline mutation status for phaeochromocytoma-susceptibility or paraganglioma-susceptibility genes were investigated. Additionally, a prospective series of 45 phaeochromocytomas and paragangliomas was investigated for SDHB immunostaining followed by SDHB, SDHC, and SDHD mutation testing. FINDINGS: SDHB protein expression was absent in all 102 phaeochromocytomas and paragangliomas with an SDHB, SDHC, or SDHD mutation, but was present in all 65 paraganglionic tumours related to multiple endocrine neoplasia type 2, von Hippel-Lindau disease, and neurofibromatosis type 1. 47 (89%) of the 53 phaeochromocytomas and paragangliomas with no syndromic germline mutation showed SDHB expression. The sensitivity and specificity of the SDHB immunohistochemistry to detect the presence of an SDH mutation in the prospective series were 100% (95% CI 87-100) and 84% (60-97), respectively. INTERPRETATION: Phaeochromocytoma-paraganglioma syndrome can be diagnosed reliably by an immunohistochemical procedure. SDHB, SDHC, and SDHD germline mutation testing is indicated only in patients with SDHB-negative tumours. SDHB immunohistochemistry on phaeochromocytomas and paragangliomas could improve the diagnosis of phaeochromocytoma-paraganglioma syndrome. FUNDING: The Netherlands Organisation for Scientific Research, Dutch Cancer Society, Vanderes Foundation, Association pour la Recherche contre le Cancer, Institut National de la Sante et de la Recherche Medicale, and a PHRC grant COMETE 3 for the COMETE network.

Morphologic determination of the malignant potential of adrenal pheochromocytoma is a challenging problem in surgical pathology. A multiparameter Pheochromocytoma of the Adrenal Gland Scaled Score (PASS) wits recently developed based on a comprehensive study of a single institutional cohort of 100 cases. Assignment of a PASS was proposed to be useful for identifying pheochromocytomas with potential to metasta-size, which defines malignancy according to the current World Health Organization terminology. A PASS is derived by evaluating multiple morphologic parameters to obtain a scaled score based on the summed weighted importance of each. Despite the proposal of this system several years ago, few studies have since examined its robustness and, in particular, the potential for observer variation inherent in the interpretation and assessment of these morphologic criteria. We further examined the utility of PASS by reviewing an independent single institutional cohort of adrenal pheochromocytomas as evaluated by 5 multi-institutional pathologists with at least 10 years experience in endocrine pathology. We found significant interobserver and intraobserver variation in assignment of PASS with variable interpretation of the underlying components. We consequently suggest that PASS requires further refinement and validation. We cannot currently recommend its use for clinical prognostication.

Contains fulltext : 80209.pdf (Publisher’s version ) (Open Access) Pheochromocytomas (PCC) are catecholamine-producing tumors arising from the adrenal medulla that occur either sporadically or in the context of hereditary cancer syndromes, such as multiple endocrine neoplasia type 2 (MEN2), von Hippel-Lindau disease (VHL), neurofibromatosis type 1, and the PCC-paraganglioma syndrome. Conventional comparative genomic hybridization studies have shown loss of 1p and 3q in the majority of sporadic and MEN2-related PCC, and 3p and 11p loss in VHL-related PCC. The development of a submegabase tiling resolution array enabled us to perform a genome-wide high-resolution analysis of 36 sporadic benign PCC. The results show that there are two distinct patterns of abnormalities in these sporadic PCC, one consisting of loss of 1p with or without concomitant 3q loss in 20/36 cases (56%), the other characterized by loss of 3p with or without concomitant 11p loss in 11/36 (31%). In addition, we found loss of chromosome 22q at high frequency (35%), as well as the novel finding of high frequency chromosome 21q loss (21%). We conclude that there appear to be two subgroups of benign sporadic PCC, one of which has a pattern of chromosomal abnormalities that is comparable with PCC from patients with MEN2 and the other that is comparable with the PCC that arise in patients with VHL disease. In addition, genes on 21q and 22q might play a more important role in PCC pathogenesis than had been assumed thus far.

About 30% of phaeochromocytomas (PCCs), sympathetic paragangliomas (sPGLs) and parasympathetic paragangliomas (pPGLs) are due to a familial syndrome. In half of these patients the presentation is syndromic or accompanied by a positive family history. However, over 10% of patients with clinically sporadic disease are still affected by an inheritable disease. Patients with multiple and/or bilateral tumours or disease onset at a young age are at an increased likelihood of such a syndrome and require genetic counselling and DNA testing. A genotype–phenotype correlation is emerging: multiple endocrine neoplasia type 2 (MEN-2) and von Hippel – Lindau disease-associated adrenal PCCs are often bilateral. Extra-adrenal (malignant) sPGLs are more typical in SDHB families. Multiple pPGLs (sometimes together with PCCs) occur in the setting of SDHD mutations, and SDHC families suffer from familial singular pPGLs. Some familial tumours also show typical histological features which should be looked for and reported by the pathologist. We review endocrine tumour syndromes with PCCs and/or PGLs, and summarize their genetic background and clinical and morphological features, and give recommendations for genetic testing.

Background: Head and neck paragangliomas (HNPs) occur as sporadic or familial entities, the latter mostly in association with germline mutations of the SDHB, SDHC, or SDHD(SDHx) genes. Heritable non-SDHx HNP might occur in von Hippel-Lindau disease (VHL, VHL gene), multiple endocrine neoplasia type 2 (MEN2, RET gene), and neurofibromatosis type 1 (NF1, NF1 gene). Reports of non-SDHx HNP presentations are scarce and guidance for genetic testing nonexistent. Patients and Methods: An international consortium registered patients with HNPs and performed mutation analyses of the SDHx, VHL, and RET genes. Those with SDHx germline mutations were excluded for purposes of this study. Personal and family histories were evaluated for paraganglial tumors, for the major tumor manifestations, and for family history of VHL, MEN2, or NF1. Results: Twelve patients were found to have hereditary non-SDHx HNPs of a total of 809 HNP and 2084 VHL registrants, 11 in the setting of germline VHL mutations and one of a RET mutation. The prevalence of hereditary HNP is five in 1000 VHL patients and nine in 1000 non-SDHx HNP patients. Comprehensive literature review revealed previous reports of HNP sin five VHL, two MEN2, and one NF1 patient. Overall, 11 here presented HNP cases, and four previously reported VHL-HNPs had lesions characteristic for VHL and/or a positive family history for VHL. Conclusions: Our observations provide evidence that molecular genetic testing for VHL or RET germline mutations in patients with HNP should be done only if personal and/or family history shows evidence for one of these syndromes. (J Clin Endocrinol Metab 94: 1938-1944, 2009)

Pheochromocytomas (PCCs) are rare tumors that arise from chromaffin tissue in the adrenal medulla, but can also occur in the abdomen outside the adrenals and are then called sympathetic paragangliomas (sPGLs). According to the literature, between 15 and 25% of apparently sporadic adrenal PCC and sPGL are caused by germline mutations in RET, von Hippel–Lindau disease (VHL), succinate dehydrogenase subunit B (SDHB), or subunit D SDHD. However, few studies have addressed the mutationfrequency of these candidate genes in selected subgroups of PCC andsPGL, such as bilateral adrenal PCC or extra-adrenal sPGL, and none have looked at somatic mutations by analyzing tumor tissue. Therefore, we have investigated the occurrence of germline and somatic mutations in RET, VHL, SDHB, and SDHD in comparatively large series of bilateral adrenal PCC (n = 33 patients) and sPGL (n = 26 patients), with the aim of determining the mutation frequency of each of these genes and to establish a genetic testing algorithm. Twenty-one RET, two VHL germline, and one SDHD mutations were found in the patients with bilateral adrenal PCC. In sPGL, one novel SDHB germline and one novel SDHB somatic mutation were observed. In addition, two SDHD germline mutations were found. We conclude that germline RET mutations are predominantly found in bilateral PCC, and that somatic and germline SDHB and SDHD mutations usually occur in sPGL, which has practical consequences for genetic testing algorithms. We suggest that sequential mutation analysis should be directed first at RET, followed by VHL and SDHD for patients with bilateral adrenal PCC at diagnosis, and at SDHB and SDHD for patients with sPGL.

Pheochromocytomas (PCCs) are rare tumors arising from neural crest-derived chromaffin cells. The majority of these tumors are located in the adrenals and gives rise to catecholamine overproduction. In at least 10% of the cases the tumors are located outside the adrenal gland, in extra-adrenal sites like the bladder and the organ of Zuckerkandl. Recent investigations have found mutations in succinate dehydrogenase subunit B (SDHB), the gene coding for subunit B of the respiratory chain complex II. Mutations in the SDHB gene, with additional loss of the wild-type allele, result in loss of function of respiratory complex II and appear to correlate with extra-adrenal location of PCCs. Also, a link has been established between malignant behavior and inactivating mutations of SDHB. In this article we review the published SDHB gene mutations, as well as the location and behavior of the resulting PCCs.

Pheochromocytomas (PCCs) are rare catecholamine-producing tumors of the adrenal gland which may also occur elsewhere in the abdomen and are then called paragangliomas. A proportion of PCCs occurs in hereditary cancer syndromes, including multiple endocrine neoplasia Type 2 (MEN2), caused by mutations in the RET proto-oncogene, von Hippel-Lindau (VHL) disease, caused by VHL gene abnormalities, and the pheochromocytoma-paraganglioma (PCC-PGL) syndrome, caused by mutations in SDHB and SDHD. Since a proportion of PCCs occurs in children we hypothesized that germline mutations in RET, VHL, succinate dehydrogenase subunit B (SDHB), and subunit D (SDHD) occur more frequently in the pediatric age range. From our single-institution collection of PCCs, we have selected 10 cases that occurred in individuals up to 18 years of age at diagnosis. In these, we have performed mutation analysis on normal and tumor tissues for exons 10, 11, and 16 of RET and for the entire coding sequence of VHL, SDHB, and SDHD. The 10 patients include 7 boys and 3 girls, with an average age of 15.5 years (range 9-18 years). Two patients had germline RET exon 11 mutations (C634R) and 1 patient had an R64P germline mutation in the VHL gene. In the remaining 7 patients there was one patient from a family fulfilling the clinical criteria for VHL disease. All tumors were benign (average follow-up: 12 years) and were located in the adrenal. From our findings we conclude that (a) a large proportion (40%) of pediatric PCC patients is diagnosed in the context of inherited cancer syndromes, and (b) candidate gene analysis appears to be indicated to detect germline mutations.

Purpose We examined the value of SDHD mutation screening in patients presenting with apparently sporadic and familial pheochromocytoma for the identification of SDHD-related pheochromocytomas. Patients and Methods This retrospective study involved 126 patients with adrenal or extra-adrenal pheochromocytomas, including 24 patients with a family history of multiple endocrine neoplasia 2, von Hippel-Lindau disease, neurofibromatosis type 1, or paraganglioma (PGL). Conformation-dependent gel electrophoresis and sequence determination analysis of germline and tumor DNA were used to identify SDHD alterations. The clinical and molecular characteristics of sporadic and hereditary tumors were compared. We reviewed the literature and compared our results with those from previously published studies. Results Pathogenic germline SDHD mutations were identified in three patients: two (2.0%) of the 102 apparently sporadic pheochromocytoma patients and one patient with a family history of PGL. These patients presented with multifocal disease (two of three multifocal patients) or with a single adrenal tumor (one of 82 patients). In the literature, mutations are mostly found in patients ≤ 35 years of age or presenting with multifocal or extra-adrenal disease. All patients with an SDHD mutation developed extra-adrenal tumors (pheochromocytomas or PGLs) at presentation or during follow-up. Conclusion SDHD gene mutations in patients presenting with apparently sporadic adrenal pheochromocytoma are rare. We recommend SDHD mutation screening for patients presenting with a family history of pheochromocytoma or PGL, multiple tumors, isolated adrenal or extra-adrenal pheochromocytomas, and age ≤ 35 years. Analysis of SDHD can also help to distinguish synchronous primary tumors from abdominal metastases.

Experimental and observational evidence suggests that chronic hypoxic stimulation can induce parasympathetic paraganglioma. This is emphasized by the identification of germline mutations in genes of the mitochondrial succinate dehydrogenase enzyme complex II in hereditary paraganglioma. Because of inactivating mutations in the succinate dehydrogenase subunit B (SDHB), C (SDHC), or D (SDHD) gene, the paraganglia undergo a chronic hypoxic stimulus leading to proliferation of the paraganglionic cells. Hypoxia is a known inducer of p53 up-regulation, which triggers cell cycle arrest and apoptosis. Inactivation of the p53 pathway, by gene mutation or by MDM2 overexpression, would enable cells to escape from cell cycle arrest and apoptosis and could contribute to tumorigenesis. To determine whether p53 inactivation plays a role in paraganglioma tumorigenesis, we investigated a series of 43 paragangliomas from 41 patients (of whom 24 patients harbored a germline SDHD mutation) for mutations in p53 exons 5-8 by PCR-SSCP. In addition, these tumors were investigated for p53 and MDM2 protein expression by immunohistochemistry, and the results were compared with clinical data and the presence of SDHD mutations. No aberrations in p53 exons 5-8 were found. The immunohistochemical experiments showed nuclear p53 expression in 15 tumors. Three tumors were positive for MDM2 that were also positive for p53. There was no correlation between p53 and MDM2 expression and clinical data or SDHD status. Given the fact that hypoxia induces p53 expression and regarding the absence of p53 mutations, these results suggest that p53 inactivation does not play a major role in the tumorigenesis of hereditary and sporadic paragangliomas.

Pheochromocytomas (PCC) are catecholamine-producing tumors that are, by definition, located in the adrenal medulla. Extra-adrenal catecholamine-producing tumors are called paragangliomas (PGL), which should be distinguished from head and neck paragangliomas, which are of parasympathetic origin. As is true for many (neuro)endocrine tumors, but unlike most other epithelial tumors, histopathological analysis does not allow a distinction to be made between PCC and PGL that will follow a benign course and those that have metastasized or will do so, a condition associated with poor prognosis. Therefore, many studies have been undertaken in the past decade, with the aim of providing a marker or a set of markers that allows clinical behavior in PCC and PGL to be predicted. Despite promising results in some areas, such as histopathological scoring systems, the use of the MIB-1 labeling index, and the analysis of telomerase activity, no single test or combination of tests has thus far yielded sufficiently high sensitivity and specificity to result in widespread acceptance in every day clinical practice. The relative rarity of PCC and PGL combined with a frequency of malignancy from as low as 2% up to 25% has hampered the power of past research and can only be overcome by multicenter collaborative efforts. In this article, recent attempts at marker detection, such as those mentioned above, as well as emerging knowledge on the molecular abnormalities in benign and malignant PCC and PGL will be presented.

Background: Adrenal Cushing's syndrome caused by ACTH-independent macronodular adrenocortical hyperplasia (AIMAH) can be accompanied by aberrant responses to hormonal stimuli. We investigated the prevalence of adrenocortical reactions to these stimuli in a large cohort of AIMAH patients, both in vivo and in vitro.Methods: In vivo cortisol responses to hormonal stimuli were studied in 35 patients with ACTH-independent bilateral adrenal enlargement and (sub-) clinical hypercortisolism. In vitro, the effects of these stimuli on cortisol secretion and steroidogenic enzyme mRNA expression were evaluated in cultured AIMAH and other adrenocortical cells. Arginine-vasopressin (AVP) receptor mRNA levels were determined in the adrenal tissues.Results: Positive serum cortisol responses to stimuli were detected in 27/35 AIMAH patients tested, with multiple responses within individual patients occurring for up to four stimuli. AVP and metoclopramide were the most prevalent hormonal stimuli triggering positive responses in vivo. Catecholamines induced short-term cortisol production more often in AIMAH cultures compared to other adrenal cells. Short-and long-term incubation with AVP increased cortisol secretion in cultures of AIMAH cells. AVP also increased steroidogenic enzyme mRNA expression, among which an aberrant induction of CYP11B1. AVP type 1a receptor was the only AVPR expressed and levels were high in the AIMAH tissues. AVPR1A expression was related to the AVP-induced stimulation of CYP11B1.Conclusions: Multiple hormonal signals can simultaneously induce hypercortisolism in AIMAH. AVP is the most prevalent eutopic signal and expression of its type 1a receptor was aberrantly linked to CYP11B1 expression.