A multigenomic liquid biopsy biomarker for neuroendocrine tumor disease outperforms CgA and has surgical and clinical utility.

BACKGROUND: Biomarkers are key tools in cancer management. In neuroendocrine tumors (NETs), Chromogranin A (CgA) was considered acceptable as a biomarker. We compared the clinical efficacy of a multigenomic blood biomarker (NETest) to CgA over a 5-year period. PATIENTS AND METHODS: An observational, prospective, cross-sectional, multicenter, multinational, comparative cohort assessment. Cohort 1: NETest evaluation in NETs (n = 1684) and cancers, benign diseases, controls (n = 731). Cohort 2: (n = 1270): matched analysis of NETest/CgA in a sub-cohort of NETs (n = 922) versus other diseases and controls (n = 348). Disease status was assessed by response evaluation criteria in solid tumors (RECIST). NETest measurement: qPCR [upper limit of normal (ULN: 20)], CgA (EuroDiagnostica, ULN: 108 ng/ml). STATISTICS: Mann-Whitney U-test, AUROC, chi-square and McNemar' test. RESULTS: Cohort 1: NETest diagnostic accuracy was 91% (P < 0.0001) and identified pheochromocytomas (98%), small intestine (94%), pancreas (91%), lung (88%), gastric (80%) and appendix (79%). NETest reflected grading: G1: 40 ± 1, G2 (50 ± 1) and G3 (52 ± 1). Locoregional disease levels were lower (38 ± 1) than metastatic (52 ± 1, P < 0.0001). NETest accurately stratified RECIST-assessed disease extent: no disease (21 ± 1), stable (43 ± 2), progressive (62 ± 2) (P < 0.0001). NETest concordance with imaging (CT/MRI/68Ga-SSA-PET) 91%. Presurgery, all NETs (n = 153) were positive (100%). After palliative R1/R2 surgery (n = 51) all (100%) remained elevated. After curative R0-surgery (n = 102), NETest levels were normal in 81 (70%) with no recurrence at 2 years. In the 31 (30%) with elevated levels, 25 (81%) recurred within 2 years. Cohort #2: NETest diagnostic accuracy was 87% and CgA 54% (P < 0.0001). NETest was more accurate than CgA for grading (chi-square = 7.7, OR = 18.5) and metastatic identification (chi-square = 180, OR = 8.4). NETest identified progressive disease (95%) versus CgA (57%, P < 0.0001). Imaging concordance for NETest was 91% versus CgA (46%) (P < 0.0001). Recurrence prediction after surgery was NETest-positive in >94% versus CgA 11%. CONCLUSION: NETest accurately diagnoses NETs and is an effective surrogate marker for imaging, grade, metastases and disease status compared to CgA. A multigenomic liquid biopsy is an accurate biomarker of NET disease.

A systematic review on the genetic analysis of paragangliomas: primarily focused on head and neck paragangliomas.

Head and neck paragangliomas Paragangliomas and pheochromocytomas are rare, mostly benign neuroendocrine tumors, which are embryologically derived from neural crest cells of the autonomic nervous system. Paragangliomas are essentially the extra-adrenal counterparts of pheochromocytomas. As such this family of tumors can be subdivided into head and neck paragangliomas, pheochromocytomas and thoracic and abdominal extra-adrenal paragangliomas. Ten out of fifteen genes that contribute to the development of paragangliomas are more susceptible to the development of head and neck paragangliomas when mutated. Gene expression profiling revealed that pheochromocytomas and paragangliomas can be classified into two main clusters (C1 and C2) based on transcriptomes. These groups were defined according to their mutational status and as such strongly associated with specific tumorigenic pathways. The influence of the main genetic drivers on the somatic molecular phenotype was shown by DNA methylation and miRNA profiling. Certain subunits of succinate dehydrogenase (SDHx), von Hippel-Lindau (VHL) and transmembrane protein 127 (TMEM127) still have the highest impact on development of head and neck paragangliomas. The link between RAS proteins and the formation of pheochromocytoma and paragangliomas is clear due to the effect of receptor tyrosine-protein kinase (RET) and neurofibromatosis type 1 (NF1) in RAS signaling and recent discovery of the role of HRAS. The functions of MYC-associated factor X (MAX) and prolyl hydroxylase 2 (PHD2) mutations in the contribution to the pathogenesis of paragangliomas still remain unclear. Ongoing studies give us insight into the incidence of germline and somatic mutations, thus offering guidelines to early detection. Furthermore, these also show the risk of mistakenly assuming sporadic cases in the absence of definitive family history in head and neck paragangliomas.

Advances in adrenal tumors 2018.

This review aims to provide clinicians and researchers with a condensed update on the most important studies in the field during 2017. We present the academic output measured by active clinical trials and peer-reviewed published manuscripts. The most important and contributory manuscripts were summarized for each diagnostic entity, with a particular focus on manuscripts that describe translational research that have the potential to improve clinical care. Finally, we highlight the importance of collaborations in adrenal tumor research, which allowed for these recent advances and provide structures for future success in this scientific field.

Absence of BRAF mutation in pheochromocytoma and paraganglioma.

Pheochromocytomas and Paragangliomas (PHEO/PARA) are rare endocrine tumors originating from the adrenal medulla. More than 20 genes are involved in the tumorigenesis of these tumors, but a substantial part of the causative genetic events remains unexplained. A recent study has reported the presence of the activating BRAF V600E mutation in PCC, suggesting a role for BRAF activation in tumor development. Other studies have not find this mutation. This study investigates the occurrence of the BRAF V600E mutation in these tumors.A cohort of 64 PHEO/PARA were screened for the BRAF V600E mutation using direct Sanger sequencing and QRT-PCR.All cases investigated displayed wild-type without V600E BRAF mutationTaken together with all previously screened tumors up to date, only 1 V600E BRAF mutation has been found among 427 PCCs. These findings imply that the V600E BRAF mutation is a rare event in PHEO/PARA.

Stress, catecholaminergic system and cancer.

Stress as a modern civilization factor significantly affects our lives. While acute stress might have a positive effect on the organism, chronic stress is usually detrimental and might lead to serious health complications. It is known that stress induced by the physical environment (temperature-induced cold stress) can significantly impair the efficacy of cytotoxic chemotherapies and the anti-tumor immune response. On the other hand, epidemiological evidence has shown that patients taking drugs known as ß-adrenergic antagonists ("ß-blockers"), which are commonly prescribed to treat arrhythmia, hypertension, and anxiety, have significantly lower rates of several cancers. In this review, we summarize the current knowledge about catecholamines as important stress hormones in tumorigenesis and discuss the use of ß-blockers as the potential therapeutic agents.

Attention Deficit Hyperactivity Disorder in Pediatric Patients with Pheochromocytoma and Paraganglioma.

The aim of the study is to evaluate if there is an association between attention deficit hyperactivity disorder (ADHD) and the diagnosis of pheochromocytoma/paraganglioma (PHEO/PGL) in pediatric patients. A case series study of 43 patients under the age of 18 with PHEO/PGL tumors who were evaluated at the National Institute of Health between January 2006 and May 2014 is reported. Prior diagnosis of ADHD and treatment course with stimulant medications was recorded. Patient symptoms, catecholamine and metanephrine levels, tumor characteristics, and genetic analyses for syndromes associated with PHEO/PGL were evaluated. A chi-squared test was used to assess the prevalence of ADHD in the PHEO/PGL patients compared to the general population. Nine out of 43 (21%) of patients diagnosed with PHEO/PGL had been diagnosed with ADHD prior to tumor identification. Four of the 9 patients had been treated with amphetamine, dextroamphetamine, and/or methylphenidate, potentially exacerbating an adrenergic crisis. In addition, 4 patients exhibited hypertension at the initial diagnosis of their PHEO/PGL. Three patients had resolution of their ADHD symptoms after successful surgical removal of PHEO/PGL. Our study found a prevalence of ADHD in 21% of our PHEO/PGL patients, significantly higher than 7.2% seen in the general pediatric population. Symptoms of anxiety and difficulty in concentration in these patients may have been related to their underlying PHEO/PGL and were not recognized as part of the constellation of symptoms in a child with PHEO/PGL. In pediatric patients with hypertension and ADHD symptomatology, an evaluation to rule out PHEO/PGL is warranted prior to treatment with stimulant medications.

Precision medicine in pheochromocytoma and paraganglioma: current and future concepts.

Pheochromocytoma and paraganglioma (PPGL) are rare diseases but are also amongst the most characterized tumour types. Hence, patients with PPGL have greatly benefited from precision medicine for more than two decades. According to current molecular biology and genetics-based taxonomy, PPGL can be divided into three different clusters characterized by: Krebs cycle reprogramming with oncometabolite accumulation or depletion (group 1a); activation of the (pseudo)hypoxia signalling pathway with increased tumour cell proliferation, invasiveness and migration (group 1b); and aberrant kinase signalling causing a pro-mitogenic and anti-apoptotic state (group 2). Categorization into these clusters is highly dependent on mutation subtypes. At least 12 different syndromes with distinct genetic causes, phenotypes and outcomes have been described. Genetic screening tests have a documented benefit, as different PPGL syndromes require specific approaches for optimal diagnosis and localization of various syndrome-related tumours. Genotype-tailored treatment options, follow-up and preventive care are being investigated. Future new developments in precision medicine for PPGL will mainly focus on further identification of driver mechanisms behind both disease initiation and malignant progression. Identification of novel druggable targets and prospective validation of treatment options are eagerly awaited. To achieve these goals, we predict that collaborative large-scale studies will be needed: Pheochromocytoma may provide an example for developing precision medicine in orphan diseases that could ultimately aid in similar efforts for other rare conditions.

SDH Subunit Mutation Status in Saliva: Genetic Testing in Patients with Pheochromocytoma.

Germline mutations occur in up to 30-40% of pheochromocytoma/paraganglioma, with mutations in the succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD) being the most common. Blood samples are favored for obtaining high quality DNA, however, leukocytes can also be obtained by collecting saliva. The aim of this study was to determine whether SDHB and SDHD gene mutations in patients with pheochromocytoma/paraganglioma could be determined using a salivary sample. Paired blood and salivary samples were collected from 30 patients: 9 SDHB mutation positive, 13 with a SDHD mutation, and 8 without any SDHx mutations. The Oragene DISCOVER kit was used to collect and extract DNA from saliva. Blood DNA was extracted from EDTA blood samples. The DNA purification and concentration were measured by spectrophotometry. The 8 exons of SDHB and the 4 exons of SDHD were amplified and sequenced by PCR-based bidirectional Sanger sequencing. Total DNA yields from blood DNA were similar to those obtained from saliva DNA [mean (±SD) saliva vs. blood DNA concentration 514.6 (±580.8) ng/µl vs. 360.9 (±262.7) ng/µl; p=0.2)]. The purity of the saliva DNA samples was lower than that of blood [mean OD260/OD280 ratio 1.78 (±0.13) vs. 1.87 (±0.04); p=0.001, respectively], indicating more protein contamination in the saliva-extracted DNA. This study shows that salivary DNA collected from patients with pheochromocytoma/paraganglioma is a good alternative for extraction of genomic DNA for its high DNA concentration and acceptable purity and can be used as an alternative to blood derived DNA in screening for SDHB and SDHD mutations.

Genotype-specific differences in the tumor metabolite profile of pheochromocytoma and paraganglioma using untargeted and targeted metabolomics.

CONTEXT AND OBJECTIVE: Pheochromocytomas and paragangliomas (PGLs) are neuroendocrine tumors of sympathetic or parasympathetic paraganglia. Nearly 40% of PGLs are caused by germline mutations. The present study investigated the effect of genetic alterations on metabolic networks in PGLs. DESIGN: Homogenates of 32 sporadic PGLs and 48 PGLs from patients with mutations in SDHB, SDHD, SDHAF-2, VHL, RET, and NF-1 were subjected to proton ((1)H) nuclear magnetic resonance (NMR) spectroscopy at 500 MHz for untargeted and HPLC tandem mass spectrometry for targeted metabolite profiling. RESULTS: (1)H NMR spectroscopy identified 28 metabolites in PGLs of which 12 showed genotype-specific differences. Part of these results published earlier reported low complex II activity (P < .0001) and low ATP/ADP/AMP content (P < .001) in SDH-related PGLs compared with sporadics and PGLs of other genotypes. Extending these results, low levels of N-acetylaspartic acid (NAA; P < .05) in SDH tumors and creatine (P < .05) in VHL tumors were observed compared with sporadics and other genotypes. Positive correlation was observed between NAA and ATP/ADP/AMP content (P < .001) and NAA and complex II activity (P < .0001) of PGLs. Targeted purine analysis in PGLs showed low adenine in cluster 1 compared with cluster 2 tumors (SDH P < .0001; VHL P < .05) whereas lower levels (P < .05) of guanosine and hypoxanthine were observed in RET tumors compared with SDH tumors. Principal component analysis (PCA) of metabolites could distinguish PGLs of different genotypes. CONCLUSIONS: The present study gives a comprehensive picture of alterations in energy metabolism in SDH- and VHL-related PGLs and establishes the interrelationship of energy metabolism and amino acid and purine metabolism in PGLs.

HIF signaling pathway in pheochromocytoma and other neuroendocrine tumors.

Hypoxia-inducible factors (HIFs) are transcription factors controlling energy, iron metabolism, erythropoiesis, and development. Dysregulation of these proteins contributes to tumorigenesis and cancer progression. Recent findings revealed the important role of HIFs in the pathogenesis of neuroendocrine tumors, especially pheochromocytoma (PHEO) and paraganglioma (PGL). PHEOs and PGLs are catecholamine-producing tumors arising from sympathetic- or parasympathetic-derived chromaffin tissue. To date, eighteen PHEO/PGL susceptibility genes have been identified. Based on the main signaling pathways, PHEOs/PGLs have been divided into two clusters, pseudohypoxic cluster 1 and cluster 2, rich in kinase receptor signaling and protein translation pathways. Recent data suggest that both clusters are interconnected via the HIF signaling and its role in tumorigenesis is supported by newly described somatic and germline mutations in HIF2A gene in patients with PHEOs/PGLs associated with polycythemia, and in some of them also with somatostatinoma. Moreover, HIFalpha signaling has also been shown to be upregulated in neuroendocrine tumors other than PHEO/PGL. Some of these tumors are components of hereditary tumor syndromes which can be associated with PHEO/PGL, but also in ileal carcinoids or melanoma. HIF signaling appears to be one of the crucial players in tumorigenesis, which could suggest new therapeutic approaches for treatment of neuroendocrine tumors.

Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline

"OBJECTIVE: The aim was to formulate clinical practice guidelines for pheochromocytoma and paraganglioma (PPGL). PARTICIPANTS: The Task Force included a chair selected by the Endocrine Society Clinical Guidelines Subcommittee (CGS), seven experts in the field, and a methodologist. The authors received no corporate funding or remuneration. EVIDENCE: This evidence-based guideline was developed using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system to describe both the strength of recommendations and the quality of evidence. The Task Force reviewed primary evidence and commissioned two additional systematic reviews. CONSENSUS PROCESS: One group meeting, several conference calls, and e-mail communications enabled consensus. Committees and members of the Endocrine Society, European Society of Endocrinology, and Americal Association for Clinical Chemistry reviewed drafts of the guidelines. CONCLUSIONS: The Task Force recommends that initial biochemical testing for PPGLs should include measurements of plasma free or urinary fractionated metanephrines. Consideration should be given to preanalytical factors leading to false-positive or false-negative results. All positive results require follow-up. Computed tomography is suggested for initial imaging, but magnetic resonance is a better option in patients with metastatic disease or when radiation exposure must be limited. (123)I-metaiodobenzylguanidine scintigraphy is a useful imaging modality for metastatic PPGLs. We recommend consideration of genetic testing in all patients, with testing by accredited laboratories. Patients with paraganglioma should be tested for SDHx mutations, and those with metastatic disease for SDHB mutations. All patients with functional PPGLs should undergo preoperative blockade to prevent perioperative complications. Preparation should include a high-sodium diet and fluid intake to prevent postoperative hypotension. We recommend minimally invasive adrenalectomy for most pheochromocytomas with open resection for most paragangliomas. Partial adrenalectomy is an option for selected patients. Lifelong follow-up is suggested to detect recurrent or metastatic disease. We suggest personalized management with evaluation and treatment by multidisciplinary teams with appropriate expertise to ensure favorable outcomes."

Should every patient diagnosed with a phaeochromocytoma have a ¹²³ I-MIBG scintigraphy?

Localization of phaeochromocytomas and paragangliomas (PPGLs) should involve functional imaging as anatomical imaging modalities can either fail to locate the tumour or can be suboptimal due to an anatomical abnormality or previous surgery. Functional imaging is particularly useful to fully delineate the extent of disease using the whole-body scan and the evaluation of multifocality, metastatic or recurrent disease. An increasing number of radiolabeled tracers have become available for tumour visualization during the past decade. (123) I-meta-iodobenzylguanidine scintigraphy is the most widely used functional imaging modality, and its sensitivity to identify chromaffin cell tumours varies from 85 to 88% for phaeochromocytomas and 56-76% for paragangliomas, while specificity ranges between 70 and 100% and 84-100%, respectively.

Current and future therapeutic approaches for metastatic pheochromocytoma and paraganglioma: focus on SDHB tumors.

As a result of intense genetic studies of families with specific mutations, the road to better therapeutic intervention for pheochromocytoma (PHEOs) and parangangliomas (PGLs) has more recently become populated with several promising molecular targets. Consequently a change in paradigm from a previous view on nonspecific therapy has shifted towards more selective molecular targeted therapies. In particular, malignant PHEOs/PGLs, more specifically the tumors that result from mutations in succinate dehydrogenase subunit B (SDHB), are a clear concern, and novel therapies should be developed to address this problem. Here we summarize current and future therapeutic approaches.

Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.

An update on the genetics of pheochromocytoma.

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are rare neuroendocrine tumors. About 30% or more of them are thought to be of inherited origin due to germ-line mutations in at least 10 well-characterized genes. There are data linking specific genotypes of these tumors to specific locations, typical biochemical phenotypes or future clinical behaviors. Conversely, clinical features, catecholamine production and immunohistochemistry evaluation can help with the proper order of genetic testing for PHEO and PGL. The identification of a germ-line mutation can lead to an early diagnosis, appropriate treatment, regular surveillance and better prognosis not only for the patient but also for their family members. Moreover, the latest discoveries in molecular pathogenesis of these tumors will provide an important basis for future personalized therapy.

[Hereditary pheochromocytoma and paraganglioma]. / Hereditární feochromocytom a paragangliom.

Pheochromocytomas and paragangliomas are tumors arising from chromaffin cells. These tumors produce catecholamines and are typically found with symptoms and signs that may include hypertension (persistent or episodic), palpitations, headache and sweating. So far, 10 different genes have been associated with both tumors and other genes are expected to be detected. Pheochromocytoma and paraganglioma can occur as a part of genetic syndromes - familial paragangliomas (SDH genes, SDHAF2 gene), von Hippel-Lindau syndrome (VHL gene), multiple endocrine neoplasia type 2 (RET gene), and neurofibromatosis type 1 (NF1 gene). These tumors may be the first and only manifestation of these genetic syndromes. Patients with SDHB mutations are at high risk to develop malignant disease and unfortunately current therapeutic options for malignant form of disease are poor. Genetic testing plays a key role in the management of these tumors and therefore not only index patients with pheochromocytoma but also relatives should be tested. Management of this disease requires multidisciplinary cooperation and should be performed in the specialized medical centres.

Pheochromocytoma: implications in tumorigenesis and the actual management.

Pheochromocytomas and paragangliomas are rare neuroendocrine catecholamine producing tumors with varied clinical presentations, biochemistries and genetic makeup. These features outline the complexity and the difficulties in studying and understanding the oncogenesis of these tumors. The study of families with genetically inherited mutations in pheochromocytoma susceptibility genes has greatly enhanced our understanding of the pathophysiology and mechanisms of oncogenesis of the disease, and consequently changed our clinical approach. Several molecular pathways and mutations in their important regulatory proteins have been identified. Such mutations are responsible for the dysregulation of metabolic pathways involved in oxygen and nutrient sensing, apoptosis regulation, cell proliferation, migration and invasion. The knowledge derived from the study of these pathways will be fundamental in the future clinical management of these patients. As a rare disease that often masks its clinical presentation, the diagnosis is frequently missed and a high level of suspicion is required. Management of this disease requires a multidisciplinary team approach and will be discussed along with advances in its treatment.

Genetic screening for von Hippel-Lindau gene mutations in non-syndromic pheochromocytoma: low prevalence and false-positives or misdiagnosis indicate a need for caution.

Genetic testing of tumor susceptibility genes is now recommended in most patients with pheochromocytoma or paraganglioma (PPGL), even in the absence of a syndromic presentation. Once a mutation is diagnosed there is rarely follow-up validation to assess the possibility of misdiagnosis. This study prospectively examined the prevalence of von Hippel-Lindau (VHL) gene mutations among 182 patients with non-syndromic PPGLs. Follow-up in positive cases included comparisons of biochemical and tumor gene expression data in 64 established VHL patients, with confirmatory genetic testing in cases with an atypical presentation. VHL mutations were detected by certified laboratory testing in 3 of the 182 patients with non-syndromic PPGLs. Two of the 3 had an unusual presentation of diffuse peritoneal metastases and substantial increases in plasma metanephrine, the metabolite of epinephrine. Tumor gene expression profiles in these 2 patients also differed markedly from those associated with established VHL syndrome. One patient was diagnosed with a partial deletion by Southern blot analysis and the other with a splice site mutation. Quantitative polymerase chain reaction, multiplex ligation-dependent probe amplification, and comparative genomic hybridization failed to confirm the partial deletion indicated by certified laboratory testing. Analysis of tumor DNA in the other patient with a splice site alteration indicated no loss of heterozygosity or second hit point mutation. In conclusion, VHL germline mutations represent a minor cause of non-syndromic PPGLs and misdiagnoses can occur. Caution should therefore be exercised in interpreting positive genetic test results as the cause of disease in patients with non-syndromic PPGLs.

Current and future anatomical and functional imaging approaches to pheochromocytoma and paraganglioma.

After establishing a biochemical diagnosis, pheochromocytomas and extra-adrenal paragangliomas (PPGLs) can be localized using different anatomical and functional imaging modalities. These include computed tomography, magnetic resonance imaging, single-photon emission computed tomography (SPECT) using 123I-metaiodobenzylguanidine or 111In-DTPA-pentetreotide, and positron emission tomography (PET) using 6-[18F]-fluorodopamine (18F-FDA), 6-[18F]-fluoro-l-3,4-dihydroxyphenylalanine (18F-DOPA), and 2-[18F]-fluoro-2-deoxy-d-glucose. We review the currently available data on the performance of anatomical imaging, SPECT, and PET for the detection of (metastatic) PPGL as well as parasympathetic head and neck paragangliomas. We show that there appears to be no 'gold-standard' imaging technique for all patients with (suspected) PPGL. A tailor-made approach is warranted, guided by clinical, biochemical, and genetic characteristics. In the current era of a growing number of PET tracers, PPGL imaging has moved beyond tumor localization towards functional characterization of tumors.