Pheochromocytomas And Paragangliomas Research Articles

How to Explore an Endocrine Cause of Hypertension.

Hypertension (HTN) is the most frequent modifiable risk factor in the world, affecting almost 30 to 40% of the adult population in the world. Among hypertensive patients, 10 to 15% have so-called “secondary” HTN, which means HTN due to an identified cause. The most frequent secondary causes of HTN are renal arteries abnormalities (renovascular HTN), kidney disease, and endocrine HTN, which are primarily due to adrenal causes. Knowing how to detect and explore endocrine causes of hypertension is particularly interesting because some causes have a cure or a specific treatment available. Moreover, the delayed diagnosis of secondary HTN is a major cause of uncontrolled blood pressure. Therefore, screening and exploration of patients at risk for secondary HTN should be a serious concern for every physician seeing patients with HTN. Regarding endocrine causes of HTN, the most frequent is primary aldosteronism (PA), which also is the most frequent cause of secondary HTN and could represent 10% of all HTN patients. Cushing syndrome and pheochromocytoma and paraganglioma (PPGL) are rarer (less than 0.5% of patients). In this review, among endocrine causes of HTN, we will mainly discuss explorations for PA and PPGL.

Universal Germline Panel Testing for Individuals With Pheochromocytoma and Paraganglioma Produces High Diagnostic Yield.

BackgroundPractice guidelines to identify individuals with hereditary pheochromocytomas and paragangliomas (PPGLs) advocate for sequential gene testing strategy guided by specific clinical features and predate the routine use of multigene panel testing (MGPT).ObjectiveTo describe results of MGPT for hereditary PPGL in a clinically and ancestrally diverse cohort.SettingCommercial laboratory based in the United States.MethodsClinical data and test results were retrospectively reviewed in 1727 individuals who had targeted MGPT from August 2013 through December 2019 because of a suspicion of hereditary PPGL.ResultsOverall, 27.5% of individuals had a pathogenic or likely pathogenic variant (PV), 9.0% had a variant of uncertain significance, and 63.1% had a negative result. Most PVs were identified in SDHB (40.4%), followed by SDHD (21.1%), SDHA (10.1%), VHL (7.8%), SDHC (6.7%), RET (3.7%), and MAX (3.6%). PVs in FH, MEN1, NF1, SDHAF2, and TMEM127 collectively accounted for 6.5% of PVs. Clinical predictors of a PV included extra-adrenal location, early age of onset, multiple tumors, and positive family history of PPGL. Individuals with extra-adrenal PGL and a positive family history were the most likely to have a PV (85.9%). Restricting genetic testing to SDHB/C/D misses one-third (32.8%) of individuals with PVs.ConclusionOur data demonstrate a high diagnostic yield in individuals with and without established risk factors, a low inconclusive result rate, and a substantial contribution to diagnostic yield from rare genes. These findings support universal testing of all individuals with PPGL and the use of concurrent MGPT as the ideal platform.

Genetic Alterations in Mitochondrial DNA Are Complementary to Nuclear DNA Mutations in Pheochromocytomas

Simple SummaryMitochondrial DNA (mtDNA) alterations have been reported to play important roles in cancer development and metastasis. However, there is scarce information about pheochromocytomas and paragangliomas (PCCs/PGLs) formation. To determine the potential roles of mtDNA alterations in PCCs/PGLs, we analyzed a panel of 26 nuclear susceptibility genes and the entire mtDNA sequence of 77 human tumors, using NGS. We also performed an analysis of copy-number alterations, large mtDNA deletion, and gene/protein expression. Our results revealed that 53.2% of the tumors harbor a mutation in the susceptibility genes and 16.9% harbor complementary mitochondrial mutations. Large deletions and depletion of mtDNA were found in 26% and 87% of tumors, respectively, accompanied by a reduced expression of the mitochondrial biogenesis markers (PCG1α, NRF1, and TFAM). Furthermore, P62 and LC3a gene expression suggested increased mitophagy, which is linked to mitochondrial dysfunction. These finding suggest a complementarity and a potential contributing role in PCCs/PGLs tumorigenesis.Background: Somatic mutations, copy-number variations, and genome instability of mitochondrial DNA (mtDNA) have been reported in different types of cancers and are suggested to play important roles in cancer development and metastasis. However, there is scarce information about pheochromocytomas and paragangliomas (PCCs/PGLs) formation. Material: To determine the potential roles of mtDNA alterations in sporadic PCCs/PGLs, we analyzed a panel of 26 nuclear susceptibility genes and the entire mtDNA sequence of seventy-seven human tumors, using next-generation sequencing, and compared the results with normal adrenal medulla tissues. We also performed an analysis of copy-number alterations, large mtDNA deletion, and gene and protein expression. Results: Our results revealed that 53.2% of the tumors harbor a mutation in at least one of the targeted susceptibility genes, and 16.9% harbor complementary mitochondrial mutations. More than 50% of the mitochondrial mutations were novel and predicted pathogenic, affecting mitochondrial oxidative phosphorylation. Large deletions were found in 26% of tumors, and depletion of mtDNA occurred in more than 87% of PCCs/PGLs. The reduction of the mitochondrial number was accompanied by a reduced expression of the regulators that promote mitochondrial biogenesis (PCG1α, NRF1, and TFAM). Further, P62 and LC3a gene expression suggested increased mitophagy, which is linked to mitochondrial dysfunction. Conclusion: The pathogenic role of these finding remains to be shown, but we suggest a complementarity and a potential contributing role in PCCs/PGLs tumorigenesis.

Loss of SDHB Induces a Metabolic Switch in the hPheo1 Cell Line toward Enhanced OXPHOS.

Background: Enzymes of tricarboxylic acid (TCA) have recently been recognized as tumor suppressors. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) cause pheochromocytomas and paragangliomas (PCCs/PGLs) and predispose patients to malignant disease with poor prognosis. Methods: Using the human pheochromocytoma cell line (hPheo1), we knocked down SDHB gene expression using CRISPR-cas9 technology. Results: Microarray gene expression analysis showed that >500 differentially expressed gene targets, about 54%, were upregulated in response to SDHB knock down. Notably, genes involved in glycolysis, hypoxia, cell proliferation, and cell differentiation were up regulated, whereas genes involved in oxidative phosphorylation (OXPHOS) were downregulated. In vitro studies show that hPheo1 proliferation is not affected negatively and the cells that survive by shifting their metabolism to the use of glutamine as an alternative energy source and promote OXPHOS activity. Knock down of SDHB expression results in a significant increase in GLUD1 expression in hPheo1 cells cultured as monolayer or as 3D culture. Analysis of TCGA data confirms the enhancement of GLUD1 in SDHB mutated/low expressed PCCs/PGLs. Conclusions: Our data suggest that the downregulation of SDHB in PCCs/PGLs results in increased GLUD1 expression and may represent a potential biomarker and therapeutic target in SDHB mutated tumors and SDHB loss of activity-dependent diseases.

Pre-Operative Selective vs Non-Selective α-Blockade in Pheochromocytoma-Paraganglioma Patients Undergoing Surgery: A Meta-Analysis.

The main objective of this systematic review and meta-analysis was to review, assess and report on the studies that have evaluated selective alpha blockade (SAB) vs. non-selective alpha blockade (NSAB) therapy in patients undergoing surgery for pheochromocytomas and paragangliomas (PPGL). We performed a systematic search of electronic databases. A meta-analysis was conducted to examine the effectiveness of the two blockades. RevMan 5.3 was used for the meta-analysis. Of the eight articles that met the inclusion criteria, there was only one randomized control trial. Meta-analysis showed that there was no significant difference between the groups SAB and NSAB with regard to intra-operative systolic blood pressure (SBP) >160 mm Hg (relative risk (RR) 0.95 [95% CI 0.57, 1.56] P = 0·83) and intra-operative vasopressor requirement (RR 1.10 [95% CI 0.96, 1.26] P = 0·16). Meta-analysis revealed that there was a significant difference between the groups (SAB vs NSAB) with respect to post-operative vasopressor requirement (RR 1.66 [95% CI 1.0, 2.74] P = 0·05). There was no significant difference between the groups with respect to post-operative complications (RR 0.84 [95% CI 0.58, 1.22] P = 0·36). In conclusion, as patients blocked selectively may have a higher incidence of vasodilator requirement intra-operatively, NSAB offers some haemodynamic advantage over SAB. However, NSAB’s real clinical benefit cannot be ascertained with the current studies as this difference did not result in any significant advantage over SAB with regard to morbidity or mortality.

Computational Analysis Of Potential Key Genes Associated With Dopamine Neurotransmission In Pheochromocytoma And Paraganglioma

Pheochromocytoma and Paraganglioma (PCPG) are rare and potentially lethal neuroendocrine tumors. PCPG that predominantly or exclusively produce and secrete DA is rarely seen and it has been known that exclusively dopamine-secreting PCPG is related with advanced malignant features and metastases. Up to the present, little has known about the role of dopamine neurotransmission and dopaminergic system in the initiation and progression of PCPG. The genes with significant expression differences between normal tissue and pheochromocytoma and paraganglioma, survival and correlation analysis, CpG islands prediction, and miRNA-target enrichment analysis were performed by several bioinformatics tools. In the present study, it was determined that COMT gene was significantly less expressed in PCPG than in normal tissue and COMT gene showed a remarkable relationship between differential expression with shorter overall survival among the individuals with PCPG (HR=1, p=0.011). MAOA and COMT gene pair was significantly correlated with PCPG (p=0.012; R=0.19) and hsa-miR-5000-5p regulates the expression of both COMT and MAOA genes (p=0.00215, FDR=0.127). Our findings suppose that COMT may potentially be implicated in tumor suppressive mechanism. The expression values of COMT and MAOA genes, and hsa-miR-5000-5p may have the potential to be used in the genetic evaluation of the pathogenesis and prognosis of PCPG. Further in vitro and in vivo studies are required to clarify the molecular mechanism of dopaminergic system in the pathogenesis and prognosis of PCPG.

Protumor Effects of Histone H3-H4 Chaperone Antisilencing Feature 1B Gene on Lung Adenocarcinoma: In Silico and In Vitro Analyses.

Background ASF1B is a member of the histone H3–H4 chaperone antisilencing feature 1 (ASF1). ASF1B reportedly acts as an oncogene in several cancers including, breast cancer and cervical cancer. To date, the role of ASF1B in lung adenocarcinoma (LUAD) is not elucidated. Methods The TCGA database, containing data for 33 cancer types, was used to explore the dysregulation and prognostic value of the ASF1B gene in pan-cancer data. R software packages and public databases/webservers were applied for bioinformatics and statistical analyses. Using in vitro models, immunoprecipitation and immunofluorescence were utilized to investigate if BCAR1 interacted with ASF1B in LUAD. Further, transfection experiments were performed to validate the expression pattern of ASF1B in LUAD and examine its regulating role in tumor-associated processes including tumor cell proliferation and migration. Results ASF1B was found to be significantly elevated in LUAD and the majority of cancer types, except PCPG (pheochromocytoma and paraganglioma). The overexpression of ASF1B was associated with worse prognostic outcomes in most cancer types including LUAD. ASF1B was associated with lymph node metastasis, and in vitro, it promoted the proliferation and migration of LUAD cells. ASF1B knockdown suppressed LUAD cell proliferation and migration and also diminished the expression of cell cycle, metastasis, and EMT signaling-associated proteins. BCAR1 was found positively correlated and interacting with ASF1B, and BCAR1 overexpression reversed the effects of ASF1B knockdown in LUAD cells. Conclusion These findings indicated that ASF1B plays a significant role in the tumor progression of LUAD and BCAR1 mediates the tumor-promotive effects of ASF1B, acting as an intermediate protein. Therefore, the ASF1B/BCAR1 axis might be regarded as a putative therapeutic target for LUAD.

Investigating the role of somatic sequencing platforms for phaeochromocytoma and paraganglioma in a large UK cohort.

ObjectivesPhaeochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumours with malignant potential and a hereditary basis in almost 40% of patients. Germline genetic testing has transformed the management of PPGL enabling stratification of surveillance approaches, earlier diagnosis and predictive testing of at‐risk family members. Recent studies have identified somatic mutations in a further subset of patients, indicating that molecular drivers at either a germline or tumour level can be identified in up to 80% of PPGL cases. The aim of this study was to investigate the clinical utility of somatic sequencing in a large cohort of patients with PPGL in the United Kingdom.Design and PatientsProspectively collected matched germline and tumour samples (development cohort) and retrospectively collected tumour samples (validation cohort) of patients with PPGL were investigated.MeasurementsClinical characteristics of patients were assessed and tumour and germline DNA was analysed using a next‐generation sequencing strategy. A screen for variants within ‘mutation hotspots’ in 68 human cancer genes was performed.ResultsOf 141 included patients, 45 (32%) had a germline mutation. In 37 (26%) patients one or more driver somatic variants were identified including 26 likely pathogenic or pathogenic variants and 19 variants of uncertain significance. Pathogenic somatic variants, observed in 25 (18%) patients, were most commonly identified in the VHL, NF1, HRAS and RET genes. Pathogenic somatic variants were almost exclusively identified in patients without a germline mutation (all but one), suggesting that somatic sequencing is likely to be most informative for those patients with negative germline genetic test results.ConclusionsSomatic sequencing may further stratify surveillance approaches for patients without a germline genetic driver and may also inform targeted therapeutic strategies for patients with metastatic disease.

An open-label, single-arm, multi-center, phase II clinical trial of single-dose [131I]meta-iodobenzylguanidine therapy for patients with refractory pheochromocytoma and paraganglioma

ObjectiveIn this phase II study, we aimed to investigate the efficacy and safety of single-dose [131I]meta-iodobenzylguanidine (131I-mIBG) therapy in patients with refractory pheochromocytoma and paraganglioma (PPGL).Patients and methodsThis study was designed as an open-label, single-arm, multi-center, phase II clinical trial. The enrolled patients were administered 7.4 GBq of 131I-mIBG. Its efficacy was evaluated 12 and 24 weeks later, and its safety was monitored continuously until the end of the study. We evaluated the biochemical response rate as the primary endpoint using the one-sided exact binomial test based on the null hypothesis (≤ 5%).ResultsSeventeen patients were enrolled in this study, of which 16 were treated. The biochemical response rate (≥ 50% decrease in urinary catecholamines) was 23.5% (90% confidence interval: 8.5–46.1%, p = 0.009). The radiographic response rates, determined with CT/MRI according to the response evaluation criteria in solid tumors (RECIST) version 1.1 and 123I-mIBG scintigraphy were 5.9% (0.3%–25.0%) and 29.4% (12.4%–52.2%), respectively. The most frequent non-hematologic treatment-emergent adverse events (TEAEs) were gastrointestinal symptoms including nausea, appetite loss, and constipation, which were, together, observed in 15 of 16 patients. Hematologic TEAEs up to grade 3 were observed in 14 of 16 patients. No grade 4 or higher TEAEs were observed. All patients had experienced at least one TEAE, but no fatal or irreversible TEAEs were observed.ConclusionA single dose 131I-mIBG therapy was well tolerated by patients with PPGL, and statistically significantly reduced catecholamine levels compared to the threshold response rate, which may lead to an improved prognosis for these patients.

Quantifying evidence toward pathogenicity for rare phenotypes: The case of succinate dehydrogenase genes, SDHB and SDHD

PurposeThe weight of the evidence to attach to observation of a novel rare missense variant in SDHB or SDHD in individuals with the rare neuroendocrine tumors, pheochromocytomas and paragangliomas (PCC/PGL), is uncertain. MethodsWe compared the frequency of SDHB and SDHD very rare missense variants (VRMVs) in 6328 and 5847 cases of PCC/PGL, respectively, with that of population controls to generate a pan-gene VRMV likelihood ratio (LR). Via windowing analysis, we measured regional enrichments of VRMVs to calculate the domain-specific VRMV-LR (DS-VRMV-LR). We also calculated subphenotypic LRs for variant pathogenicity for various clinical, histologic, and molecular features. ResultsWe estimated the pan-gene VRMV-LR to be 76.2 (54.8-105.9) for SDHB and 14.8 (8.7-25.0) for SDHD. Clustering analysis revealed an SDHB enriched region (ɑɑ 177-260, P = .001) for which the DS-VRMV-LR was 127.2 (64.9-249.4) and an SDHD enriched region (ɑɑ 70-114, P = .000003) for which the DS-VRMV-LR was 33.9 (14.8-77.8). Subphenotypic LRs exceeded 6 for invasive disease (SDHB), head-and-neck disease (SDHD), multiple tumors (SDHD), family history of PCC/PGL, loss of SDHB staining on immunohistochemistry, and succinate-to-fumarate ratio >97 (SDHB, SDHD). ConclusionUsing methodology generalizable to other gene-phenotype dyads, the LRs relating to rarity and phenotypic specificity for a single observation in PCC/PGL of a SDHB/SDHD VRMV can afford substantial evidence toward pathogenicity.

Precision Medicine in Phaeochromocytoma and Paraganglioma.

Precision medicine is a term used to describe medical care, which is specifically tailored to an individual patient or disease with the aim of ensuring the best clinical outcome whilst reducing the risk of adverse effects. Phaeochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumours with uncertain malignant potential. Over recent years, the molecular profiling of PPGLs has increased our understanding of the mechanisms that drive tumorigenesis. A high proportion of PPGLs are hereditary, with non-hereditary tumours commonly harbouring somatic mutations in known susceptibility genes. Through detailed interrogation of genotype-phenotype, correlations PPGLs can be classified into three different subgroups or clusters. Thus, PPGLs serve as an ideal paradigm for developing, testing and implementing precision medicine concepts in the clinic. In this review, we provide an overview of PPGLs and highlight how detailed molecular characterisation of these tumours provides current and future opportunities for precision oncology.

A novel liquid biopsy (NETest) identifies paragangliomas and pheochromocytomas with high accuracy.

Pheochromocytomas and paragangliomas (PHEOs/PGLs) represent diagnostically challenging and complex neuroendocrine tumors (NETs). Current biomarker tests for PHEOs/PGLs are technically complex or limited. We assessed the diagnostic utility of a NET-specific 51-marker gene blood assay (NETest) in patients with PHEOs/PGLs (n = 81), including ten pediatric patients, and age-/gender-matched controls (n = 142) using a prospective case:control (1:2) analysis. mRNA was measured (qPCR), and results were scaled from 0 to 100 (upper limit of normal < 20). Receiver operating curve (ROC) and non-parametric (Mann-Whitney) tests were used for analyses (two-tailed). All data are presented as mean ± s.e.m. NETest accuracy for PHEO/PGL diagnosis was 100%. PHEO/PGL scores were 70 ± 3 vs 8.5 ± 1 in controls (P < 0.0001), and ROC analysis was 0.99 ± 0.004 (P < 0.0001). Diagnostic metrics were 94% accurate, 100% sensitive, and 92% specific. Imaging correlation with 68Ga-PET-SSA was 100%. NETest levels in PHEOs (n = 26) were significantly (P < 0.0001) elevated (83 ± 4) vs 66 ± 4 in PGLs (n = 40) and mixed PHEOs/PGLs (n = 5: 37 ± 3). Adrenal-derived tumors (n = 30) exhibited higher scores (76 ± 5) than extra-adrenal-derived tumors (66 ± 4, P < 0.05). Cluster 2 tumors exhibited significantly (P = 0.034) elevated NETest levels (n = 4: 92 ± 2) vs cluster 1 tumors (n = 35: 69 ± 4). Regulatory pathway analysis identified elevated RAS-RAF, metastatic, pluripotential, neural and secretory gene cluster levels (P < 0.05) in PHEOs compared to PGLs. Cluster 2 PPGLs exhibited elevated (P = 0.046) levels of growth factor signaling genes compared to cluster 1. The PHEOs/PGLs in the pediatric cohort (n = 10) were all NETest-positive (81 ± 8) and exhibited a gene expression profile spectrum analogous to adults. Circulating NET transcript analysis identifies PHEOs/PGLs with 100% efficacy and is likely to have clinical utility in the diagnosis and management of PHEO/PGL patients.

Genetic Spectrum in a Canadian Cohort of Apparently Sporadic Pheochromocytomas and Paragangliomas (PPGLs): New Data on Multigene Panel Retesting Over Time

Pheochromocytomas (PHEOs) and paragangliomas (PGLs), collectively known as PPGLs, are tumors with a high heritability. The prevalence of germline mutations in apparently sporadic PPGLs varies depending on study population and is unknown in the Canadian population.

Deep Membrane Proteome Profiling Reveals Overexpression of Prostate-Specific Membrane Antigen (PSMA) in High-Risk Human Paraganglioma and Pheochromocytoma, Suggesting New Theranostic Opportunity.

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors arising from chromaffin cells of adrenal medulla or sympathetic or parasympathetic paraganglia, respectively. To identify new therapeutic targets, we performed a detailed membrane-focused proteomic analysis of five human paraganglioma (PGL) samples. Using the Pitchfork strategy, which combines specific enrichments of glycopeptides, hydrophobic transmembrane segments, and non-glycosylated extra-membrane peptides, we identified over 1800 integral membrane proteins (IMPs). We found 45 “tumor enriched” proteins, i.e., proteins identified in all five PGLs but not found in control chromaffin tissue. Among them, 18 IMPs were predicted to be localized on the cell surface, a preferred drug targeting site, including prostate-specific membrane antigen (PSMA), a well-established target for nuclear imaging and therapy of advanced prostate cancer. Using specific antibodies, we verified PSMA expression in 22 well-characterized human PPGL samples. Compared to control chromaffin tissue, PSMA was markedly overexpressed in high-risk PPGLs belonging to the established Cluster 1, which is characterized by worse clinical outcomes, pseudohypoxia, multiplicity, recurrence, and metastasis, specifically including SDHB, VHL, and EPAS1 mutations. Using immunohistochemistry, we localized PSMA expression to tumor vasculature. Our study provides the first direct evidence of PSMA overexpression in PPGLs which could translate to therapeutic and diagnostic applications of anti-PSMA radio-conjugates in high-risk PPGLs.

Local-Regional Recurrence of Pheochromocytoma/Paraganglioma: Characteristics, Risk Factors and Outcomes.

ObjectiveTo study the characteristics, risk factors, and outcomes of local-regional recurrence of pheochromocytoma and paraganglioma (PPGL).MethodsClinical data of 96 PPGL patients with local-regional recurrence and 112 patients without recurrence were retrospectively analyzed.ResultsRecurrent patients exhibited a median recurrence time of 6.0 (4.0, 9.0) years after resection of the primary tumor. SDHB mutation [HR 4.1 (1.7, 9.5), p=0.001), primary tumor size ≥5cm [HR 2.3 (1.1, 4.7), p=0.028], and average Ki-67 count ≥3% in the primary tumor [HR 2.6 (1.4, 4.9), p=0.003] were independent predictors for recurrence of PPGL. Primary tumor sizes ≥5cm [HR 5.1 (1.7, 15.3), p=0.003] and average Ki-67 counts ≥3% of the primary tumor [HR 2.4 (1.1, 5.2), p=0.035] were independent predictors for recurrence of pheochromocytoma, while SDHB mutation [HR 4.6 (1.5, 13.9), p=0.007] was a predictor for paraganglioma recurrence. Among recurrent patients, 47% (45/96) had multiple nodules in recurrent sites, and 58% (56/96) had metastases, with 20% (19/96) being implantation metastases. The risk of metastases (42% vs. 25%, p=0.030) and death (15% vs. 8%, p=0.003) was significantly increased in untreated patients after recurrence compared with treated patients.ConclusionLong-term follow-up is necessary for all PPGL patients. Risk factors for recurrence of pheochromocytoma and paraganglioma differ, with primary tumor size and average Ki-67 count representing independent predictors for pheochromocytoma patients and SDHB mutations predicting paraganglioma recurrence. Although the treatment of recurrence can be difficult, patients should be treated once recurrence is detected as it decreases the risk of metastases and death.

Prognostic factors for refractory pheochromocytoma and paraganglioma after 131I-metaiodobenzylguanidine therapy.

Given the rarity of refractory pheochromocytoma and paraganglioma (PPGL), outcomes and prognostic factors after 131I-metaiodobenzylguanidine (131I-mIBG) treatment still remain unclear. Therefore, this study evaluated whether baseline characteristics at initial 131I-mIBG therapy and imaging response to repeated 131I-mIBG therapy could be prognostic factors for refractory PPGL. All patients [n = 59 (male/female = 35/24), median age; 49.3years] with refractory PPGL who received 131I-mIBG therapy at our institution between September 2009 and September 2019 were retrospectively reviewed for the effects of the following factors on overall survival: age, sex, hypertension, diabetes mellitus, palpitations, constipation, cancer pain, catecholamines values, past history of therapy (external beam radiation for bone metastasis, operation, and chemotherapy), metastasis sites, and response to 131I-mIBG treatments. Throughout the follow-up period, 18 patients died from disease exacerbation. The estimated 5- and 10-year survival rates were 79.4% and 67.2% from the initial diagnoses of refractory PPGL and 68.5% and 49.9% from the first 131I-mIBG therapy, respectively. The multivariate Cox proportional hazards model showed that progressive disease (PD) [hazard ratio (HR) 96.3, P = 0.011] and constipation (HR 8.2, P = 0.024) were adverse prognostic factors for overall survival after initial 131I-mIBG therapy. The log-rank test demonstrated that PD in response to 131I-mIBG therapies (P < 0.0001) and constipation (P < 0.01) were correlated with poor survival rates. Response to repeated 131I-mIBG treatment can be a strong predictor of prognosis after initial 131I-mIBG therapy for refractory PPGL. Repeated 131I-mIBG therapy may be a good option for controlling refractory PPGL.

Targeting the Redox Balance Pathway Using Ascorbic Acid in sdhb Zebrafish Mutant Larvae

Simple SummaryThus far, no curative therapies are available for malignant SDHB-associated phaeochromocytomas and paragangliomas (PPGLs). Therapy development is severely hampered by the limited availability of suitable animal models. In this study, we investigated the potential of the sdhbrmc200 zebrafish model to study SDHB-associated PPGLs using a drug screening approach. One of the key features of cancer initiation and progression is redox imbalance. First, we identified increased reactive oxygen species levels in homozygous sdhbrmc200 larvae at baseline. Next, we tested the effect of anti- and pro-oxidant ascorbic acid (Vitamin C) on these larvae. We validated the sdhbrmc200 zebrafish model as a powerful drug screening tool to provide valuable insights into pathomechanisms, which may lead to novel therapeutic targets and therapy development in the future.Patients with mutations in the β-subunit of the succinate dehydrogenase (SDHB) have the highest risk to develop incurable malignant phaeochromocytomas and paragangliomas (PPGLs). Therapy development is hindered by limited possibilities to test new therapeutic strategies in vivo. One possible molecular mechanism of SDHB-associated tumorigenesis originates in an overproduction of reactive oxygen species (ROS) due to mitochondrial dysfunction. Ascorbic acid (Vitamin C) has already been shown to act as anti-cancer agent in several clinical trials for various types of cancer. In this study, the potential of the sdhbrmc200 zebrafish model to study SDHB-associated PPGLs using a drug screening approach was investigated. First, we identified increased basal ROS levels in homozygous sdhb larvae compared to heterozygous and wild-type siblings. Using a semi high-throughput drug screening, the effectiveness of different dosages of anti- and pro-oxidant Vitamin C were assessed to evaluate differences in survival, ROS levels, and locomotor activity. Low-dosage levels of Vitamin C induced a decrease of ROS levels but no significant effects on lifespan. In contrast, high-dosage levels of Vitamin C shortened the lifespan of the homozygous sdhbrmc200 larvae while not affecting the lifespan of heterozygous and wild-type siblings. These results validated the sdhbrmc200 zebrafish model as a powerful drug screening tool that may be used to identify novel therapeutic targets for SDHB-associated PPGLs.

Quantitation of plasma metanephrines using isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS): a candidate reference measurement procedure and its application to evaluating routine ID-LC/MS/MS methods.

Accurate measurement of plasma metanephrines (MNs) including metanephrine (MN) and normetanephrine (NMN) is crucial for the screening and diagnosis in pheochromocytomas and paragangliomas (PPGLs). Although the number of laboratories using liquid chromatography tandem mass spectrometry (LC-MS/MS) method to measure MNs has been increasing rapidly, those laboratory-developed assays showed incomparable results. There are no reference measurement procedures (RMPs) or reference materials (RMs) for MNs in Joint Committee for Traceability in Laboratory Medicine (JCTLM), which hindered the standardization of MNs measurement. We established a candidate RMP (cRMP) based on isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for plasma MNs measurement. Plasma samples were spiked with MN-D3 and NMN-D3 as internal standards; protein precipitation and ion-exchange solidphase extraction (SPE) were performed to extract samples, eventually analyzed by LC-MS/MS. The cRMP was applied to evaluate two routine ID-LC/MS/MS methods through split-sample comparisons. Fifty-three individual patient samples were determined by cRMP and two routine ID-LC/MS/MS methods; results were analyzed by ordinary linear regression and Bland-Altman plots. The cRMP exhibited desirable imprecision, with intra-run and total imprecision (coefficient variation, CV) for MN being 0.79-1.36% and 1.53-1.87% and for NMN being 1.10-1.34% and 1.15-1.64%. The analytical recoveries of MN and NMN ranged from 98.3 to 101.7% and from 98.5 to 101.9%, respectively. Significant calibrator biases and sample-specific deviations were observed in method comparison. An accurate, precise, and reliable cRMP for plasma MNs was developed, and RMs with value assigned following the cRMP would help minimize the calibration bias and improve the comparability of different measuring systems.

A UK national audit of the laboratory investigation of phaeochromocytoma and paraganglioma.

Phaeochromocytomas and paragangliomas (PPGL) are catecholamine secreting tumours associated with significant morbidity and mortality. Timely diagnosis and management are essential. A range of laboratory tests can be utilised in the investigation of PPGL. There is scope for significant variation in practice between centres. We aimed to investigate how the laboratory investigation of PPGL is performed in laboratories across the United Kingdom. A questionnaire consisting of 21 questions was circulated to Clinical Biochemistry laboratories in the United Kingdom via the Association for Clinical Biochemistry and Laboratory Medicine office. The survey was designed to allow audit against Endocrine Society Guidelines on the Investigation and Management of PPGL and to obtain information on other important aspects not included in these guidelines. Responses were received from 58 laboratories and the data were compiled. The majority of laboratories use either urine or plasma metanephrines in first-line testing for PPGL, although a number of different combinations of biochemistry tests are utilised in different centres. All laboratories measuring metanephrines or catecholamines in-house use LC or LC-MS/MS methods. There are some marked differences between laboratories in urine metanephrines reference ranges used and sample requirements. There is evidence of good practice in UK laboratories (as assessed against Endocrine Society Guidelines) such as widespread use of urine/plasma metanephrines and appropriate analytical methodologies used. However, there is also evidence of variations in practice in some areas that should be addressed.

Recurrent Germline Mutations of CHEK2 as a New Susceptibility Gene in Patients with Pheochromocytomas and Paragangliomas.

Purpose Recently, pheochromocytomas and paragangliomas (PPGLs) have been strongly suspected as hereditary tumors, as approximately 40% of patients carry germline mutations. In the cancers where defects occur to corrupt DNA repair and facilitate tumorigenesis, a CHEK2 strong association has been observed. Therefore, the purpose of this study was to investigate the effect of CHEK2 mutations for its possible pathogenicity in PPGLs. Methods Four patients with CHEK2 mutations were recruited, as previously detected by the whole exome sequencing. Sanger sequencing was used to verify the germline mutations as well as the loss of heterozygosities (LOHs) in their somatic DNAs. Immunohistochemistry was used to analyze the expression of CHEK2 and its downstream target p53 Ser20 (phosphorylated p53). Results The average age of studied patients was 44.25 ± 11.18 years, at the time diagnosis. One patient had multiple tumors which recurred quickly, while two patients had distant metastasis. None of the patient had any relevant family history. Four germline CHEK2 mutations were identified (c.246_260del; c.715G > A; c.1008+3A > T; and c.1111C > T). All the patients were predicted to have either pathogenic or suspected pathogenic mutations. There was no LOH of CHEK2 gene in somatic DNAs found. Additionally, neither CHEK2 proteins nor its downstream target p53 Ser20 were expressed in the tumor tissues. The inactivation of CHEK2 leads to the decrease in the p53 phosphorylation, which might promote tumorigenesis. Conclusions For the first time, CHEK2 was identified as a susceptibility gene for PPGLs. However, the penetrance of CHEK2 gene with genotype-phenotype correlation needs to be investigated.