Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma.

The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy.

Genetic bases of pheochromocytoma and paraganglioma.

The genetics of pheochromocytoma and paraganglioma (PPGL) has become increasingly complex over the last two decades. The list of genes involved in the development of these tumors has grown steadily, and there are currently more than 20 driver genes implicated in either the hereditary or the sporadic nature of the disease. Although genetic diagnosis is achieved in about 75-80% of patients, genetic etiology remains unexplained in a significant percentage of cases. Patients lacking a genetic diagnosis include not only those with apparently sporadic PPGL but also patients with a family history of the disease or with multiple tumors, that meet the criteria to be considered as candidates for carrying germline mutations in yet undiscovered genes. Mutations in known PPGL genes deregulate three main signaling pathways (hypoxia, kinase signaling, and Wnt-signaling pathways), which could be the starting point for the development of personalized treatment for PPGL patients. Furthermore, the integration of results from several genomic high-throughput platforms enables the discovery of regulatory mechanisms that cannot be identified by analyzing each piece of information separately. These strategies are powerful tools for elucidating optimal therapeutic options based on molecular biomarkers in PPGL and represent an important step toward the achievement of precision medicine for patients with metastatic PPGL.

A Large Case-Control Study Performed in Spanish Population Suggests That RECQL5 Is the Only RECQ Helicase Involved in Breast Cancer Susceptibility.

Around 50% of the familial breast cancer (BC) cases are estimated to be caused by germline variants in known low-, moderate-, and high-risk susceptibility genes, while the other half is of unknown genetic origin. In the present study, we wanted to evaluate the role of the RECQ helicases, some of which have been studied in the past as candidates, with unclear results about their role in the disease. Using next-generation sequencing (NGS) technology, we analyzed the whole coding sequence of BLM, RECQL1, RECQL4, RECQL5, and WRN in almost 2000 index cases from BC Spanish families that had previously tested negative for the known BC susceptibility genes (BRCAX) and compared the results with the controls extracted from gnomAD. Our results suggest that BLM, RECQL1, RECQL4, and WRN do not play a major role in BC susceptibility. However, in the combined analysis, joining the present results with those previously reported in a series of 1334 BC Spanish patients and controls, we found a statistically significant association between Loss of Function (LoF) variants in RECQL5 and BC risk, with an OR of 2.56 (p = 0.009; 95% CI, 1.18-4.98). Our findings support our previous work and places the RECQL5 gene as a new moderate-risk BC gene.

Comprehensive molecular analysis of immortalization hallmarks in thyroid cancer reveals new prognostic markers.

BACKGROUND: Comprehensive molecular studies on tumours are needed to delineate immortalization process steps and identify sensitive prognostic biomarkers in thyroid cancer. METHODS AND RESULTS: In this study, we extensively characterize telomere-related alterations in a series of 106 thyroid tumours with heterogeneous clinical outcomes. Using a custom-designed RNA-seq panel, we identified five telomerase holoenzyme-complex genes upregulated in clinically aggressive tumours compared to tumours from long-term disease-free patients, being TERT and TERC denoted as independent prognostic markers by multivariate regression model analysis. Characterization of alterations related to TERT re-expression revealed that promoter mutations, methylation and/or copy gains exclusively co-occurred in clinically aggressive tumours. Quantitative-FISH (fluorescence in situ hybridization) analysis of telomere lengths showed a significant shortening in these carcinomas, which matched with a high proliferative rate measured by Ki-67 immunohistochemistry. RNA-seq data analysis indicated that short-telomere tumours exhibit an increased transcriptional activity in the 5-Mb-subtelomeric regions, site of several telomerase-complex genes. Gene upregulation enrichment was significant for specific chromosome-ends such as the 5p, where TERT is located. Co-FISH analysis of 5p-end and TERT loci showed a more relaxed chromatin configuration in short telomere-length tumours compared to normal telomere-length tumours. CONCLUSIONS: Overall, our findings support that telomere shortening leads to a 5p subtelomeric region reorganization, facilitating the transcription and accumulation of alterations at TERT-locus.

Beneficios del seguimiento telemático en la pérdida de peso de pacientes con sobrepeso y obesidad en tiempos de confinamiento / Benefits of telematic monitoring for weight loss in overweight and obese patients in times of confinement

Introducción: la pandemia originada en 2019 por el SARS-CoV-2 supuso un cambio en los hábitos de alimentación y ejercicio físico por causa de las medidas de confinamiento domiciliario. El seguimiento de pacientes en tratamiento de pérdida de peso mediante una consulta telemática podría ser una herramienta útil para prevenir el fracaso terapéutico.Objetivo:describir la evolución de los parámetros antropométricos de pacientes en seguimiento para pérdida de peso mediante una consulta telemática.Métodos:estudio prospectivo en 2 tiempos (antes y después del confinamiento) de una intervención telemática sobre pacientes adultos en seguimiento habitual por sobrepeso y obesidad. Se analizaron las variables demográficas y los parámetros de composición corporal mediante bioimpendancia. Además se analizaron las diferencias en cuanto a presencia de tratamiento farmacológico adyuvante del tipo de los análogos de la hormona GLP1 (liraglutida o semaglutida). Las variables se estudiaron mediante la prueba de Wilcoxon, la U de Mann-Whitney y la correlación de Spearman. Se consideró la significación si p ≤ 0,05.Resultados:se incluyeron 97 pacientes. Antes del confinamiento, el 42,3 % presentaban sobrepeso (IMC < 30 kg/m2), el 36,1 % tenían obesidad de grado I (IMC = 30-34,9 kg/m2), el 16,4 % la tenían de grado II (IMC = 35-39,9 kg/m2) y el 5,2 % tenían un IMC > 40 kg/m2. El 30,9 % presentaban prediabetes y el 9,3 % tenían diabetes de tipo 2. Entre ambas visitas presenciales, el 81,4 % de los pacientes perdieron un 4,2 ± 3,4 % del peso, con una disminución media significativa de la masa grasa de 3,16 ± 4,4 kg. El grupo en tratamiento farmacológico con análogos de la hormona GLP-1 presentó una pérdida de masa grasa media significativamente superior sin pérdida de masa muscular esquelética significativa. (AU)

Introduction: the pandemic originated by SARS-Cov-2 in 2019 led to eating habits and physical exercise changes due to home confinement measures. The follow-up of patients in treatment for weight loss through telematic consultation could be a useful tool to prevent treatment failure.Objective:to describe the evolution of anthropometric parameters of patients under follow-up for weight loss through telematic consultation.Methods:a two-stage prospective study (before and after confinement) with a telematic intervention in adult patients under regular follow-up for overweight and obesity. Demographic variables and body composition parameters were analyzed by bioimpendance. In addition, the differences in the presence of drug treatment with GLP-1 hormone (liraglutide or semaglutide) adjuvants were also analyzed. The variables were studied using Wilcoxon's test, Mann-Whitney U-test, and Spearman's correlation. Significance was considered for p ≤ 0.05.Results:a total of 97 patients were included, before confinement 42.3 % were overweight (BMI < 30 kg/m2), 36.1 % were obese grade I (BMI = 30-34.9 kg/m2), 16.4 % were obese grade II (BMI = 35-39.9 kg/m2), and 5.2 % had BMI > 40 kg/m2. In all, 30.9 % had prediabetes and 9.3 % had type-2 diabetes. Between both consultations, 81.4 % of patients lost 4.2 ± 3.4 % of their weight, with a significant mean decrease in fat mass of 3.16 ± 4.4 kg. The group on pharmacological treatment with GLP-1 hormone analogs presented a significantly higher average fat loss without significant loss of skeletal muscle mass. Conclusions: telematic monitoring seems to be a useful tool to prevent weight gain in patients with restricted mobility. A telematic intervention that contains dietary advice and exercise, as a reinforcement to hypocaloric diet, helps to achieve weight loss with a predominant fat component. (AU)

[Benefits of telematic monitoring for weight loss in overweight and obese patients in times of confinement]. / Beneficios del seguimiento telemático en la pérdida de peso de pacientes con sobrepeso y obesidad en tiempos de confinamiento.

Introduction: Introduction: the pandemic originated by SARS-Cov-2 in 2019 led to eating habits and physical exercise changes due to home confinement measures. The follow-up of patients in treatment for weight loss through telematic consultation could be a useful tool to prevent treatment failure. Objective: to describe the evolution of anthropometric parameters of patients under follow-up for weight loss through telematic consultation. Methods: a two-stage prospective study (before and after confinement) with a telematic intervention in adult patients under regular follow-up for overweight and obesity. Demographic variables and body composition parameters were analyzed by bioimpendance. In addition, the differences in the presence of drug treatment with GLP-1 hormone (liraglutide or semaglutide) adjuvants were also analyzed. The variables were studied using Wilcoxon's test, Mann-Whitney U-test, and Spearman's correlation. Significance was considered for p ≤ 0.05. Results: a total of 97 patients were included, before confinement 42.3 % were overweight (BMI < 30 kg/m2), 36.1 % were obese grade I (BMI = 30-34.9 kg/m2), 16.4 % were obese grade II (BMI = 35-39.9 kg/m2), and 5.2 % had BMI > 40 kg/m2. In all, 30.9 % had prediabetes and 9.3 % had type-2 diabetes. Between both consultations, 81.4 % of patients lost 4.2 ± 3.4 % of their weight, with a significant mean decrease in fat mass of 3.16 ± 4.4 kg. The group on pharmacological treatment with GLP-1 hormone analogs presented a significantly higher average fat loss without significant loss of skeletal muscle mass. Conclusions: telematic monitoring seems to be a useful tool to prevent weight gain in patients with restricted mobility. A telematic intervention that contains dietary advice and exercise, as a reinforcement to hypocaloric diet, helps to achieve weight loss with a predominant fat component. The presence of drug treatment with GLP-1 hormone analogues appears to significantly help maintain skeletal muscle mass during weight loss.

Introducción: Introducción: la pandemia originada en 2019 por el SARS-CoV-2 supuso un cambio en los hábitos de alimentación y ejercicio físico por causa de las medidas de confinamiento domiciliario. El seguimiento de pacientes en tratamiento de pérdida de peso mediante una consulta telemática podría ser una herramienta útil para prevenir el fracaso terapéutico. Objetivo: describir la evolución de los parámetros antropométricos de pacientes en seguimiento para pérdida de peso mediante una consulta telemática. Métodos: estudio prospectivo en 2 tiempos (antes y después del confinamiento) de una intervención telemática sobre pacientes adultos en seguimiento habitual por sobrepeso y obesidad. Se analizaron las variables demográficas y los parámetros de composición corporal mediante bioimpendancia. Además se analizaron las diferencias en cuanto a presencia de tratamiento farmacológico adyuvante del tipo de los análogos de la hormona GLP1 (liraglutida o semaglutida). Las variables se estudiaron mediante la prueba de Wilcoxon, la U de Mann-Whitney y la correlación de Spearman. Se consideró la significación si p ≤ 0,05. Resultados: se incluyeron 97 pacientes. Antes del confinamiento, el 42,3 % presentaban sobrepeso (IMC < 30 kg/m2), el 36,1 % tenían obesidad de grado I (IMC = 30-34,9 kg/m2), el 16,4 % la tenían de grado II (IMC = 35-39,9 kg/m2) y el 5,2 % tenían un IMC > 40 kg/m2. El 30,9 % presentaban prediabetes y el 9,3 % tenían diabetes de tipo 2. Entre ambas visitas presenciales, el 81,4 % de los pacientes perdieron un 4,2 ± 3,4 % del peso, con una disminución media significativa de la masa grasa de 3,16 ± 4,4 kg. El grupo en tratamiento farmacológico con análogos de la hormona GLP-1 presentó una pérdida de masa grasa media significativamente superior sin pérdida de masa muscular esquelética significativa. Conclusiones: el seguimiento telemático parece una herramienta útil para prevenir la ganancia de peso en los pacientes con restricción de la movilidad. Una intervención telemática que contenga consejo dietético y ejercicio como refuerzo de la dieta hipocalórica ayuda a conseguir perder peso, predominando el componente graso. La presencia de un tratamiento farmacológico con análogos de la hormona GLP-1 parece ayudar significativamente al mantenimiento de la masa muscular esquelética durante la pérdida de peso.

Alterations in SLC4A2, SLC26A7 and SLC26A9 Drive Acid-Base Imbalance in Gastric Neuroendocrine Tumors and Uncover a Novel Mechanism for a Co-Occurring Polyautoimmune Scenario.

Autoimmune polyendocrine syndrome (APS) is assumed to involve an immune system malfunction and entails several autoimmune diseases co-occurring in different tissues of the same patient; however, they are orphans of its accurate diagnosis, as its genetic basis and pathogenic mechanism are not understood. Our previous studies uncovered alterations in the ATPase H+/K+ Transporting Subunit Alpha (ATP4A) proton pump that triggered an internal cell acid-base imbalance, offering an autoimmune scenario for atrophic gastritis and gastric neuroendocrine tumors with secondary autoimmune pathologies. Here, we propose the genetic exploration of APS involving gastric disease to understand the underlying pathogenic mechanism of the polyautoimmune scenario. The whole exome sequencing (WES) study of five autoimmune thyrogastric families uncovered different pathogenic variants in SLC4A2, SLC26A7 and SLC26A9, which cotransport together with ATP4A. Exploratory in vitro studies suggested that the uncovered genes were involved in a pathogenic mechanism based on the alteration of the acid-base balance. Thus, we built a custom gene panel with 12 genes based on the suggested mechanism to evaluate a new series of 69 APS patients. In total, 64 filtered putatively damaging variants in the 12 genes of the panel were found in 54.17% of the studied patients and none of the healthy controls. Our studies reveal a constellation of solute carriers that co-express in the tissues affected with different autoimmune diseases, proposing a unique genetic origin for co-occurring pathologies. These results settle a new-fangled genetics-based mechanism for polyautoimmunity that explains not only gastric disease, but also thyrogastric pathology and disease co-occurrence in APS that are different from clinical incidental findings. This opens a new window leading to the prediction and diagnosis of co-occurring autoimmune diseases and clinical management of patients.

Analysis of Telomere Maintenance Related Genes Reveals NOP10 as a New Metastatic-Risk Marker in Pheochromocytoma/Paraganglioma.

One of the main problems we face with PPGL is the lack of molecular markers capable of predicting the development of metastases in patients. Telomere-related genes, such as TERT and ATRX, have been recently described in PPGL, supporting the association between the activation of immortalization mechanisms and disease progression. However, the contribution of other genes involving telomere preservation machinery has not been previously investigated. In this work, we aimed to analyze the prognostic value of a comprehensive set of genes involved in telomere maintenance. For this study, we collected 165 PPGL samples (97 non-metastatic/63 metastatic), genetically characterized, in which the expression of 29 genes of interest was studied by NGS. Three of the 29 genes studied, TERT, ATRX and NOP10, showed differential expression between metastatic and non-metastatic cases, and alterations in these genes were associated with a shorter time to progression, independent of SDHB-status. We studied telomere length by Q-FISH in patient samples and in an in vitro model. NOP10 overexpressing tumors displayed an intermediate-length telomere phenotype without ALT, and in vitro results suggest that NOP10 has a role in telomerase-dependent telomere maintenance. We also propose the implementation of NOP10 IHC to better stratify PPGL patients.

Guía para el manejo y la prevención de la insuficiencia suprarrenal aguda / SEEN guidelines for the management and prevention of acute adrenal insufficiency

La insuficiencia suprarrenal aguda (ISA) es una urgencia médica potencialmente letal cuya prevención y tratamiento deberían ser conocidos por todos los profesionales médicos. La ISA es una condición infradiagnosticada debido a la inespecificidad de los síntomas de presentación, pero su diagnóstico y tratamiento con glucocorticoides es vital. Puede ser desencadenada por una deficiencia de novo en la síntesis de cortisol o secundaria a la omisión del tratamiento hormonal sustitutivo con corticoides o al ajuste inadecuado de la dosis requerida en situaciones de estrés en el paciente ya diagnosticado. La prevención de la ISA disminuye de forma significativa la mortalidad cardiovascular y por infecciones de los pacientes con insuficiencia renal y mejora su calidad de vida. Por ello, es fundamental la adecuada educación del paciente, sus familiares y del personal sanitario. El Grupo de Trabajo de Patología Suprarrenal del Área de Conocimiento de Neuroendocrinología de la Sociedad Española de Endocrinología y Nutrición (SEEN) ha elaborado, a partir de una propuesta de la Junta Directiva de la SEEN, esta guía para el óptimo manejo de la insuficiencia suprarrenal en fase aguda. Esta guía tiene el objetivo de ser eminentemente práctica y dar recomendaciones orientadas a todos los profesionales sanitarios que pueden estar involucrados en el diagnóstico, tratamiento y la prevención de la ISA. Así mismo, pretende facilitar pautas de actuación para el paciente y sus familiares en su manejo y prevención

Acute adrenal insufficiency (AAI) is a potentially fatal medical emergency whose prevention and treatment should be known by all medical professionals. AAI is an underdiagnosed condition because of its non-specific symptoms, but its diagnosis and early treatment with glucocorticoids is vital. It may be triggered by a de novo deficiency in cortisol synthesis or occur secondarily to omission of hormone replacement therapy (corticosteroids) or inadequate adjustment of the dose required in stress situations in patients previously diagnosed with adrenal insufficiency. AAI prevention significantly decreases death from cardiovascular diseases and infections in patients with adrenal insufficiency, and also improves their quality of life. Adequate education of patients, relatives, and all healthcare professionals is therefore essential. Therefore, the Adrenal Disorders Group of the Neuroendocrinology Area of the Spanish Society of Endocrinology and Nutrition (SEEN) has prepared, at the proposal of the SEEN's board, a guideline for optimal management of acute adrenal insufficiency. The guideline is intended to provide practical recommendations for all healthcare professionals who may be involved in the diagnosis, treatment, and prevention of AAI. It is also intended to provide patients and their families with action guidelines for AAI management and prevention

SEEN guidelines for the management and prevention of acute adrenal insufficiency. / Guía para el manejo y la prevención de la insuficiencia suprarrenal aguda.

Acute adrenal insufficiency (AAI) is a potentially fatal medical emergency whose prevention and treatment should be known by all medical professionals. AAI is an underdiagnosed condition because of its non-specific symptoms, but its diagnosis and early treatment with glucocorticoids is vital. It may be triggered by a de novo deficiency in cortisol synthesis or occur secondarily to omission of hormone replacement therapy (corticosteroids) or inadequate adjustment of the dose required in stress situations in patients previously diagnosed with adrenal insufficiency. AAI prevention significantly decreases death from cardiovascular diseases and infections in patients with adrenal insufficiency, and also improves their quality of life. Adequate education of patients, relatives, and all healthcare professionals is therefore essential. Therefore, the Adrenal Disorders Group of the Neuroendocrinology Area of the Spanish Society of Endocrinology and Nutrition (SEEN) has prepared, at the proposal of the SEEN's board, a guideline for optimal management of acute adrenal insufficiency. The guideline is intended to provide practical recommendations for all healthcare professionals who may be involved in the diagnosis, treatment, and prevention of AAI. It is also intended to provide patients and their families with action guidelines for AAI management and prevention.

Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/mTOR axis in metastatic pheochromocytoma/paraganglioma.

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.

Recurrent Germline DLST Mutations in Individuals with Multiple Pheochromocytomas and Paragangliomas.

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.

Prognosis of Malignant Pheochromocytoma and Paraganglioma (MAPP-Prono Study): A European Network for the Study of Adrenal Tumors Retrospective Study.

BACKGROUND: Malignant pheochromocytoma and paraganglioma (MPP) are characterized by prognostic heterogeneity. Our objective was to look for prognostic parameters of overall survival (OS) in MPP patients. PATIENTS AND METHODS: Retrospective multicenter study of MPP characterized by a neck-thoraco-abdomino-pelvic CT or MRI at the time of malignancy diagnosis in European centers between 1998 and 2010. RESULTS: One hundred sixty-nine patients from 18 European centers were included. Main characteristics of patients with MPP were: primary pheochromocytoma in 53% of patients; tumor- or hormone-related symptoms in 57% or 58% of cases; positive plasma or urine hormones in 81% of patients; identification of a mutation in SDHB in 42% of cases. Metastatic sites included bone (64%), lymph node (40%), lung (29%), and liver (26%); mean time between initial and malignancy diagnosis was 43 months (range, 0 to 614). Median follow-up was 68 months and median survival 6.7 years. Using univariate analysis, better survival was associated with head and neck paraganglioma, age <40 years, metanephrines less than fivefold the upper limits of the normal range, and low proliferative index. In multivariate analysis, hypersecretion [hazard ratio 3.02 (1.65 to 5.55); P = 0.0004] was identified as an independent significant prognostic factor of worst OS. CONCLUSIONS: Our results do not confirm SDHB mutations as a major prognostic parameter in MPP and suggest additional key molecular events involved in MPP tumor progression. Aside from SDHB mutation, the biology of aggressive MPP remains to be understood.

Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients.

PURPOSE: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype. METHODS: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach. RESULTS: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL. CONCLUSION: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.

Gain-of-function mutations in DNMT3A in patients with paraganglioma.

PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.

Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer-Predisposing Mutations in Pheochromocytomas and Paragangliomas.

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes.Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases.Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1 A variant in GOT2, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio.Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315-24. ©2017 AACR.

PheoSeq: A Targeted Next-Generation Sequencing Assay for Pheochromocytoma and Paraganglioma Diagnostics.

Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.

The mTORC1 Complex Is Significantly Overactivated in SDHX-Mutated Paragangliomas.

AIM: We aimed at exploring the activation pattern of the mTOR pathway in sporadic and hereditary pheochromocytomas (PCCs) and paragangliomas (PGLs). METHODS: A total of 178 PCCs and 44 PGLs, already characterized for the presence of germline mutations in VHL, RET, NF1, MAX, SDHA, SDHB, SDHC, and SDHD as well as somatic mutations in VHL, RET, H-RAS, and MAX, were included in 5 tissue microarrays and tested using immunohistochemistry for mTOR and Rictor as well as the phosphorylated forms of mTOR, p70S6K, AMPK, AKT, 4EBP1, S6, and Raptor. RESULTS: The positive correlation among most of the molecules investigated proved the functional activation of the mTOR pathway in PCCs/PGLs. Total mTOR, p-S6K and p-S6, and mTORC1-associated molecules p-Raptor and p-AMPK were all significantly overexpressed in PGLs rather than in PCCs, and in the head and neck rather than in abdominal locations. None of the markers, except for the low expression of p-mTOR, was associated with malignancy. Cluster 1 PCCs/PGLs had higher total mTOR, p-Raptor, and p-S6 expression than cluster 2 PCCs/PGLs. In contrast, p-mTOR and mTORC2-associated molecule Rictor were significantly overexpressed in cluster 2 tumors. Within cluster 1, molecules active in the mTORC1 complex were significantly overexpressed in SDHX- as compared to VHL-mutated tumors. CONCLUSION: In summary, the mTOR pathway is activated in a high proportion of PCCs/PGLs, with a preferential overactivation of the mTORC1 complex in PGLs of the head and neck and/or harboring SDHX mutations.

Targeted Sequencing Reveals Low-Frequency Variants in EPHA Genes as Markers of Paclitaxel-Induced Peripheral Neuropathy.

Purpose: Neuropathy is the dose-limiting toxicity of paclitaxel and a major cause for decreased quality of life. Genetic factors have been shown to contribute to paclitaxel neuropathy susceptibility; however, the major causes for interindividual differences remain unexplained. In this study, we identified genetic markers associated with paclitaxel-induced neuropathy through massive sequencing of candidate genes.Experimental Design: We sequenced the coding region of 4 EPHA genes, 5 genes involved in paclitaxel pharmacokinetics, and 30 Charcot-Marie-Tooth genes, in 228 cancer patients with no/low neuropathy or high-grade neuropathy during paclitaxel treatment. An independent validation series included 202 paclitaxel-treated patients. Variation-/gene-based analyses were used to compare variant frequencies among neuropathy groups, and Cox regression models were used to analyze neuropathy along treatment.Results: Gene-based analysis identified EPHA6 as the gene most significantly associated with paclitaxel-induced neuropathy. Low-frequency nonsynonymous variants in EPHA6 were present exclusively in patients with high neuropathy, and all affected the ligand-binding domain of the protein. Accumulated dose analysis in the discovery series showed a significantly higher neuropathy risk for EPHA5/6/8 low-frequency nonsynonymous variant carriers [HR, 14.60; 95% confidence interval (CI), 2.33-91.62; P = 0.0042], and an independent cohort confirmed an increased neuropathy risk (HR, 2.07; 95% CI, 1.14-3.77; P = 0.017). Combining the series gave an estimated 2.5-fold higher risk of neuropathy (95% CI, 1.46-4.31; P = 9.1 × 10-4).Conclusions: This first study sequencing EPHA genes revealed that low-frequency variants in EPHA6, EPHA5, and EPHA8 contribute to the susceptibility to paclitaxel-induced neuropathy. Furthermore, EPHA's neuronal injury repair function suggests that these genes might constitute important neuropathy markers for many neurotoxic drugs. Clin Cancer Res; 23(5); 1227-35. ©2016 AACR.