Germline SDHB Research Articles

Insights into the genetic landscape of pheochromocytomas and paragangliomas in a Brazilian cohort.

Germline and somatic drivers are identified in 30% and 40% of pheochromocytomas and paragangliomas (PPGLs), respectively. In this study, we investigated the genetic landscape of PPGLs in a Brazilian cohort. We studied 182 index patients with PPGLs (116 females and 66 males), comprising 118 pheochromocytoma and 70 paraganglioma cases. Our optimized sequencing strategy included SANGER sequencing, targeted next-generation sequencing panel and whole exome sequencing. Germline and somatic pathogenic or likely pathogenic variants in susceptibility genes were identified in 88 (48.4%) and 18 (10.4%) cases, respectively. SDHB was the most frequently affected gene, identified in 30 patients (16.5%), with a germline SDHB exon 1 deletion present in 46.7% of these cases. The Brazilian cohort exhibited a higher rate of germline diagnoses when compared to the European (31%), American (27%), and Chinese (21%) cohorts (p < 0.001). Five germline variants were identified: 1) Three CHEK2 likely pathogenic or pathogenic variants (c.475T>C/p.Tyr159His; c.362G>A/p.Cys121Tyr; c.319+2T>A); and 2) Two BRCA2 pathogenic variants (c.3680_3681delTG/p.Leu1227fs and c.7806-2A>C). These variants are unreported in the Brazilian genomic variant repository. CHEK2 immunostaining was negative in the three tumors, with one case exhibiting CHEK2 loss of heterozygosity. Moreover, the prevalence of CHEK2 or BRCA2 pathogenic or likely pathogenic variants in our cohort was significantly higher compared with to global population databases (p < 0.0001 and p = 0.0004, respectively). Our cohort of PPGLs demonstrated a high frequency of germline diagnoses. Additionally, our findings suggest CHEK2 and BRCA2 as potential susceptibility genes for PPGLs.

Ependymoma Alongside Hereditary Paraganglioma-Phaeochromocytoma Syndrome Due to a SDHB Mutation: A Case Report

Mutations in the SDHB gene cause a characteristic syndrome that includes paragangliomas (PGL) and phaeochromocytomas (PCC). Herein we present a rare case of an ependymoma in a patient with a germline SDHB mutation. A 41-year-old man with a positive family history of PGL/PCC syndrome was found to have the familial SDHB mutation. Screening imaging for paragangliomas revealed an incidental presumed ependymoma originating from the fourth ventricle. He was followed with serial imaging to assess for progression of the lesion. Due to slow, substantial growth of the tumour, and increasing symptoms which included diplopia, unsteadiness, and wide-based gait, he underwent a resection 5 years after the lesion’s identification. Following resection of the tumour, the pathology confirmed the tumour as a posterior fossa type B (PFB) ependymoma of the fourth ventricle. Unfortunately, on Day 26 post-operatively, the patient had a pulmonary embolism and died. His family consented to an autopsy, which revealed the presence of a clinically-silent pituitary neuroendocrine tumour (PitNET). Though ependymomas are not commonly seen in PGL/PCC syndrome, they can occur. This case represents the first molecularly-characterised ependymal tumour described in this tumour predisposition syndrome. Clinicians ought to be aware of the risk of ependymoma in patients with PGL/PCC syndrome and consider this tumour when conducting their screening and follow-up of asymptomatic SDHx mutation carriers.

12488 Exome Sequencing Unravels New Susceptibility Genes For Pheochromocytomas And Paragangliomas

Abstract Disclosure: G.F. Fagundes: None. F.F. Castro: None. L.S. Santana: None. A.F. Afonso: None. A.W. Maciel: None. F.L. Ledesma: None. C.A. Pereira: None. I.C. Soares: None. D.M. Lourenço Junior: None. M.A. Pereira: None. V. Srougi: None. F.Y. Tanno: None. J.L. Chambo: None. M.C. Fragoso: None. A.O. Hoff: None. B.B. Mendonca: None. A. Latronico: None. M.Q. Almeida: None. Background: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors with a high association with hereditary disease, affecting 40% of the cases. PPGL susceptibility genes are categorized into three clusters based on their pathophysiological and metabolomic mechanisms. Somatic oncogenic variants are identified in 30% of the tumors. Most of the germline and somatic drivers are mutually exclusive in PPGLs. Thus, 30% of the PPGLs remain without known genetic causes. Aim: To investigate new susceptibility genes for PPGLs using whole exome sequencing. Methods: We included 145 index patients with PPGLs (93 female and 52 male) with 91 pheochromocytomas (PHEO) and 60 paragangliomas, of which 43 (29.65%) were metastatic. A target next-generation sequencing panel for susceptibility genes was initially performed. Whole exome sequencing was performed in 56 patients with PPGLs (38 were paired with tumor DNA). Results:SDHB was the most frequently affected gene in 29 of 145 patients (20%). Germline SDHB exon 1 deletion was identified in 14 patients (48.27%). Germline variants were identified as follow: RET 22 (15.17%), VHL 13 (8.96%), SDHA 6 (4.14%), SDHD 5 (3.45%), NF1 5 (3.45%), FH 2 (1.38%), MAX 2 (1.38%), DLST 1 (0.69%), TMEM127 1 (0.69%), SDHC 1 (0.69%) and H3F3A 1 (0.69%) case. Somatic variants were identified in 21 tumors: NF1 6 (4.14%), HRAS 5 (3.45%), VHL 2 (1.38%), FGFR1 2 (1.38%), and RET 1 (0.69%). Six variants (five germline and one somatic) were identified in 3 new candidate genes: 1) Three very rare germline CHEK2 likely pathogenic or pathogenic variants (c.475T&amp;gt;C/ p.Tyr159His; c.362G&amp;gt;A/ p.Cys121Tyr; c.319+2T&amp;gt;A) in one metastatic PHEO and two PGLs (one metastatic). The metastatic PGL did not harbor any somatic oncogenic variant, while exome sequencing of a metastatic lesion from the PHEO had a somatic likely oncogenic HRAS variant; 2) Two very rare germline BRCA2 pathogenic variants (c.3680_3681delTG/ p.Leu1227fs; c.7806-2A&amp;gt;C) in a 33-year-old man with non-metastatic PHEO and in a 25-year-old man with metastatic PGL. All germline variants have not been reported in the Brazilian genomic variant repository. Exome sequencing did not reveal any somatic oncogenic driver in both tumors; 3) The somatic GNAS likely oncogenic variant c.601C&amp;gt;T/ p.Arg201Cys in a pheochromocytoma. The prevalence of CHEK2 and BRCA2 pathogenic or likely pathogenic variants in our cohort were significantly higher compared with those in the gnomAD population database (p &amp;lt; 0.0001 and p = 0.0004, respectively). Co-occurrence of germline and somatic drivers were found in 3 cases: FH and FGFR1, DLST and NF1, and CHEK2 and HRAS. In summary, germline and somatic diagnosis were reached in 63.45% and 15.18% of the PPGLs in our cohort, respectively. Conclusion: Our findings support CHEK2, BRCA2 and GNAS as novel susceptibility genes for PPGLs. Support: Sao Paulo Research Foundation (FAPESP) grant 2019/15873-6. Presentation: 6/2/2024

Germline DNA methylation analysis reveals distinct alterations in a large cohort of patients with germline SDHB pathogenic variant

Germline DNA methylation analysis reveals distinct alterations in a large cohort of patients with germline SDHB pathogenic variant

GIANT PROLACTINOMA IN A PATIENT WITH GERMLINE SDHB MUTATION.

GIANT PROLACTINOMA IN A PATIENT WITH GERMLINE SDHB MUTATION.

Development and internal validation of a novel predictive model for SDHB mutations in pheochromocytomas and retroperitoneal paragangliomas.

To develop and internally validate a novel predictive model for SDHB mutations in pheochromocytomas and retroperitoneal paragangliomas (PPGLs). Clinical data of patients with PPGLs who presented to Peking Union Medical College Hospital from 2013 to 2022 and underwent genetic testing were retrospectively collected. Variables were screened by backward stepwise and clinical significance and were used to construct multivariable logistic models in 50 newly generated datasets after the multiple imputation. Bootstrapping was used for internal validation. A corresponding nomogram was generated based on the model. Sensitivity analyses were also performed. A total of 556 patients with PPGLs were included, of which 99 had a germline SDHB mutation. The prediction model revealed that younger age of onset [Odds ratio (OR): 0.93, 95% CI: 0.91-0.95], synchronous metastasis (OR: 6.43, 95% CI: 2.62-15.80), multiple lesion (OR: 0.22, 95% CI: 0.09-0.54), retroperitoneal origin (OR: 5.72, 95% CI: 3.13-10.47), negative 131I-meta-iodobenzylguanidine (MIBG) (OR: 0.34, 95% CI: 0.15-0.73), positive octreotide scintigraphy (OR: 3.24, 95% CI: 1.25-8.43), elevated 24h urinary dopamine (DA) (OR: 1.72, 95% CI: 0.93-3.17), NE secretory type (OR: 2.83, 95% CI: 1.22- 6.59), normal secretory function (OR: 3.04, 95% CI: 1.04-8.85) and larger tumor size (OR: 1.09, 95% CI: 0.99-1.20) were predictors of SDHB mutations in PPGLs, and showed good and stable predictive performance with a mean area under the ROC curve (AUC) of 0.865 and coefficient of variation of 2.2%. This study provided a novel and useful tool for predicting SDHB mutations by integrating easily obtained clinical data. It may help clinicians select suitable genetic testing methods and make appropriate clinical decisions for these high-risk patients.

Management of phaeochromocytoma and paraganglioma in patients with germline SDHB pathogenic variants: an international expert Consensus statement.

Adult and paediatric patients with pathogenic variants in the gene encoding succinate dehydrogenase (SDH) subunit B (SDHB) often have locally aggressive, recurrent or metastatic phaeochromocytomas and paragangliomas (PPGLs). Furthermore, SDHB PPGLs have the highest rates of disease-specific morbidity and mortality compared with other hereditary PPGLs. PPGLs with SDHB pathogenic variants are often less differentiated and do not produce substantial amounts of catecholamines (in some patients, they produce only dopamine) compared with other hereditary subtypes, which enables these tumours to grow subclinically for a long time. In addition, SDHB pathogenic variants support tumour growth through high levels of the oncometabolite succinate and other mechanisms related to cancer initiation and progression. As a result, pseudohypoxia and upregulation of genes related to the hypoxia signalling pathway occur, promoting the growth, migration, invasiveness and metastasis of cancer cells. These factors, along with a high rate of metastasis, support early surgical intervention and total resection of PPGLs, regardless of the tumour size. The treatment of metastases is challenging and relies on either local or systemic therapies, or sometimes both. This Consensus statement should help guide clinicians in the diagnosis and management of patients with SDHB PPGLs.

SDHB immunohistochemistry for prognosis of pheochromocytoma and paraganglioma: A retrospective and prospective analysis.

Pheochromocytomas and paragangliomas (PCC/PGL) are rare neuroendocrine tumors and can secrete catecholamine. Previous studies have found that SDHB immunohistochemistry (IHC) can predict SDHB germline gene mutation, and SDHB mutation is closely associated with tumor progression and metastasis. This study aimed to clarify the potential effect of SDHB IHC as a predictive marker for tumor progression in PCC/PGL patients. We included PCC/PGL patients diagnosed in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from 2002 to 2014 for retrospective analysis and discovered that SDHB (-) staining patients had poorer prognoses. Then we examined SDHB protein expression by IHC on all tumors in the prospective series, which was composed of patients from 2015 to 2020 in our center. In the retrospective series, the median follow-up was 167 months, and during follow-up, 14.4% (38/264) patients developed metastasis or recurrence, and 8.0% (22/274) patients died. Retrospective analysis revealed that 66.7% (6/9) of participants in the SDHB (-) group and 15.7% (40/255) of those in the SDHB (+) group developed progressive tumors (OR: 10.75, 95% CI: 2.72-52.60, P=0.001), and SDHB (-) was independently associated with poor outcomes after adjusting by other clinicopathological parameters (OR: 11.68, 95% CI: 2.58-64.45, P=0.002). SDHB (-) patients had shorter disease-free survival (DFS) and overall survival (OS) (P<0.001) and SDHB (-) was significantly associated with shorter median DFS (HR: 6.89, 95% CI: 2.41-19.70, P<0.001) in multivariate cox proportional hazard analysis. In the prospective series, the median follow-up was 28 months, 4.7% (10/213) patients developed metastasis or recurrence, and 0.5% (1/217) patient died. For the prospective analysis, 18.8% (3/16) of participants in the SDHB (-) group had progressive tumors compared with 3.6% (7/197) in the SDHB (+) group (RR: 5.28, 95% CI: 1.51-18.47, P=0.009), statistical significance remained (RR: 3.35, 95% CI: 1.20-9.38, P=0.021) after adjusting for other clinicopathological factors. Our findings demonstrated patients with SDHB (-) tumors had a higher possibility of poor outcomes, and SDHB IHC can be regarded as an independent biomarker of prognosis in PCC/PGL.

Serum Succinate/Fumarate Ratio in Patients With Paraganglioma/Pheochromocytoma Attending an Endocrine Oncogenetic Unit.

Pheochromocytomas and paragangliomas (PPGLs) with SDHx pathogenic variants (PVs) are characterized by a higher intratissular succinate/fumarate ratio (RS/F) than non-SDHx-mutated ones. Also, an increase in serum succinate levels has been reported in patients with germline SDHB or SDHD PV. To assess whether measurement of serum succinate, fumarate levels, and RS/F might aid identification of an SDHx germline PV/likely pathogenic variant (LPV) in patients with PPGL or in asymptomatic relatives; and to guide identification of a PV/LPV among the variants of unknown significance (VUS) identified in SDHx by next-generation sequencing. This prospective monocentric study included 93 patients attending an endocrine oncogenetic unit for genetic testing. Succinate and fumarate were measured in serum by gas chromatography coupled to mass spectrometry. The RS/F was calculated to assess SDH enzymatic function. Diagnostic performance was assessed by receiver operating characteristic analysis. RS/F had a higher discriminant power than succinate alone to identify an SDHx PV/LPV in patients with PPGL. However, SDHD PVs/LPVs are frequently missed. Only RS/F differed between asymptomatic SDHB/SDHD PV/LPV carriers and SDHB/SDHD-linked patients with PPGL. Finally RS/F could be helpful to easily evaluate the functional impact of VUS in SDHx. Measurement of serum RS/F in patients with PPGL and in asymptomatic relatives is a valuable initial workup tool to detect those carrying a germline PV/LPV in SDHx. Its discriminative power is equal or superior to those of succinate measured alone. SDHD PVs/LPVs are less frequently identified by these biochemical tools. Use of RS/F for SDHx VUS reclassification needs to be evaluated further.

Outcomes of systemic treatment in patients with metastatic pheochromocytoma and paraganglioma: A single center retrospective analysis.

2 Background: Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising from chromaffin cells of the adrenal medulla or extra-adrenal paraganglia. Although 15-17% of patients develop metastatic disease, there are very limited systemic treatment options. In this study, we aimed to evaluate the treatment landscape and outcome in PPGL patients retrospectively. Methods: Between December 2004 and December 2021, 33 patients were diagnosed with metastatic PPGLs and received systemic treatment at the Department of Oncology, Asan Medical Center, Seoul, South Korea. Metastatic PPGLs included a locally advanced unresectable disease, initially metastatic disease, and recurrent disease. Systemic treatment involves cytotoxic chemotherapy and targeted agents, and efficacy analysis included the objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). Results: The median age at diagnosis was 49 years (range, 15–74), 17 patients were male (52%), and 22 patients were pheochromocytomas (67%). Disease status was recurrent (73%), initially metastatic (21%), and locally advanced (6%). Of 23 patients who underwent germline tests, 14 (61%) were identified with germline mutations, and the most frequent mutation was SDHB germline mutation (n=8, 57%). Of the 33 patients, 18 received chemotherapy with cyclophosphamide, vincristine, and dacarbazine (CVD). The ORR was 22% (4/18; all PR), and the disease control rate (DCR) was 67% (n=12). The median PFS was 7.9 months [95% confidence interval (CI), 1.6-not available (NA)], and the median OS was 36.2 months (95% CI, 5.7-NA). Six patients were treated with sunitinib, and the ORR was 33% (2/6; all PR), and the DCR was 83% (n=5). The median PFS was 14.6 months (95% CI, 3.6-NA), and the median OS was 14.6 months (95% CI, 3.6-NA). In CVD treatment, patients with SDHB/SDHD mutation (either germline or somatic) were significantly associated with superior OS &amp; PFS compared with the non-SDHB/SDHD mutation group, with a median OS of 94.0 months vs. 5.7 months (P = 0.01) and a median PFS of 20.1 months vs. 1.6 months (P = 0.03), respectively. In the SDHB/SDHD mutation group, the ORR was 50% (3/6, all PR), and the DCR was 100% (n=6), but in a non-SDHB/SDHD mutation group, the ORR was 11% (1/9, PR) and the DCR was 44% (n=4). Conclusions: Systemic treatment with CVD and sunitinib provided meaningful disease control, and outcomes were comparable with previous data. Especially, CVD treatment was effective in patients with SDHB/SDHD mutation, and further validation is needed in future studies.

Evidence for a Founder Effect of SDHB Exon 1 Deletion in Brazilian Patients With Paraganglioma.

Limited information is available concerning the genetic spectrum of pheochromocytoma and paraganglioma (PPGL) patients in South America. Germline SDHB large deletions are very rare worldwide, but most of the individuals harboring the SDHB exon 1 deletion originated from the Iberian Peninsula. Our aim was to investigate the spectrum of SDHB genetic defects in a large cohort of Brazilian patients with PPGLs. Genetic investigation of 155 index PPGL patients was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification, and/or target next-generation sequencing panel. Common ancestrality was investigated by microsatellite genotyping with haplotype reconstruction, and analysis of deletion breakpoint. Among 155 index patients, heterozygous germline SDHB pathogenic or likely pathogenic variants were identified in 22 cases (14.2%). The heterozygous SDHB exon 1 complete deletion was the most frequent genetic defect in SDHB, identified in 8 out of 22 (36%) of patients. Haplotype analysis of 5 SDHB flanking microsatellite markers demonstrated a significant difference in haplotype frequencies in a case-control permutation test (P = 0.03). More precisely, 3 closer/informative microsatellites were shared by 6 out of 8 apparently unrelated cases (75%) (SDHB-GATA29A05-D1S2826-D1S2644 | SDHB-186-130-213), which was observed in only 1 chromosome (1/42) without SDHB exon 1 deletion (X2 = 29.43; P < 0.001). Moreover, all cases with SDHB exon 1 deletion had the same gene breakpoint pattern of a 15 678 bp deletion previously described in the Iberian Peninsula, indicating a common origin. The germline heterozygous SDHB exon 1 deletion was the most frequent genetic defect in the Brazilian PPGL cohort. Our findings demonstrated a founder effect for the SDHB exon 1 deletion in Brazilian patients with paragangliomas.

High rates of the SDHB p.Arg46Gln pathogenic variant predisposes New Zealand Māori to phaeochromocytoma/paraganglioma.

Phaeochromocytomas (PCC) and paragangliomas (PGL; together PPGL) are rare tumours of the adrenal medulla or extra-adrenal paraganglia. They may secrete catecholamines with significant cardiovascular effects. Management of PPGL is predominantly surgical, despite the anaesthetic risks related to potential haemodynamic instability. Meticulous pre-treatment and intra-operative management are required to improve cardiovascular outcomes. There are limited local data regarding the incidence of PPGL and the clinical characteristics of individuals diagnosed with these tumours in New Zealand. We undertook a retrospective study investigating the local practice and patient characteristics with an additional focus on intra-operative haemodynamic stability and post-operative outcomes. Electronic patient records were searched for individuals with a diagnosis of PPGL. Clinical records and electronic databases were interrogated for pre-operative, intra-operative and post-operative data points. Particular attention was paid to rates and types of germline mutations, intra-operative haemodynamic stability and post-operative renal and cardiovascular outcomes. We identified 49 individuals with PPGL, of whom 34 were from the local area. This gave a local incidence of PPGL of around five cases per million people per year. Māori were significantly over-represented in our cohort, with this being in part due to high rates of the SDHB R46Q mutation. Over 95% of our cohort met pre-specified pre-operative blood pressure parameters. Intra-operative monitoring revealed a tendency to hypotension, but this did not translate into adverse post-operative outcomes, which were infrequent. Māori were over-represented due to high rates of germline SDHB R46Q mutations. There were few post-operative adverse outcomes in this contemporary cohort.

Locally invasive recurrence or metastasis of pheochromocytoma into the liver?—clinicopathological challenges

Pheochromocytomas (PCC) are rare and functional neuroendocrine tumors developing from adrenal chromaffin cells. Predicting malignant behavior especially in the absence of metastasis can be quite challenging even in the era of improved understanding of the molecular mechanisms involved in PCCs. Currently, two histopathological grading systems Pheochromocytoma of the Adrenal Gland Scaled Score (PASS) and Grading of Adrenal Pheochromocytoma and Paraganglioma (GAPP) score are used in clinical practice, but these are subject to significant interobserver variability. Some of the most useful clinical factors associated with malignancy are large size ([4–5 cm), and genetic features such as presence of SDHB germline mutations. Local invasion is uncommon in PCC and metastasis seen in 10 to 17% but higher in germline mutations and when this occurs management can be challenging. Here, we report on a case with challenges faced by the pathologist and clinicians alike in diagnosis and management of PCC recurrence.

Extensive patient-to-patient single nucleus transcriptome heterogeneity in pheochromocytomas and paragangliomas.

Pheochromocytoma, neuroendocrine tumor, single cell RNA-sequencing, transcriptome, heterogeneity, SDHB, RET, paraganglinoma; Pheochromocytomas (PC) and paragangliomas (PG) are rare neuroendocrine tumors with varied genetic makeup and are associated with high cardiovascular morbidity and a variable risk of malignancy. The source of the transcriptional heterogeneity of the disease and the underlying biological processes that determine the outcome of PCPG remain largely unclear. We focused on PCPG tumors with germline SDHB and RET mutations, which represent distinct prognostic groups with worse or better prognoses, respectively. We applied single-nuclei RNA sequencing (snRNA-seq) to tissue samples from 11 patients and found high patient-to-patient transcriptome heterogeneity in neuroendocrine tumor cells. The tumor microenvironment also showed heterogeneous profiles, mainly contributed by macrophages of the immune cell clusters and Schwann cells of the stroma. By performing non-negative matrix factorization, we identified common transcriptional programs active in RET and SDHB, as well as distinct modules, including neuronal development, hormone synthesis and secretion, and DNA replication. Similarities between the transcriptomes of the tumor cells and those of the chromaffin- and precursor cell types suggests different developmental stages at which PC and PG tumors appear to be arrested.

Papillary Thyroid Cancer and a TERT Promotor Mutation-positive Paraganglioma in a Patient With a Germline SDHB Mutation.

PurposeAbout 40% of paragangliomas (PGL) are due to germline mutations in one of several susceptibility genes. These genes rarely predispose to other non-PGL tumors. Here, we describe and functionally characterize a germline SDHB mutation in a patient who developed a BRAFV600E mutation-positive papillary thyroid cancer (PTC) and a TERT promotor mutation-positive PGL.Experimental designA 28-year-old asymptomatic man was discovered incidentally to have a large left-sided mid-abdominal PGL and PTC. He underwent resection of the PGL and total thyroidectomy and neck dissection followed by I-131 adjuvant therapy for PTC. The histopathology revealed a high-grade PGL and a tall cell-variant PTC with lymph node metastases (T1b N1b M0). He soon developed PGL spinal metastases that have been rapidly progressing and is currently being treated with Lu177-dotatate therapy. Family screening revealed a positive SDHB mutation in the mother, a son, and a brother.ResultsIn addition to the heterozygous SDHB germline mutation (c.688C>T, p.Arg230Cys), molecular analysis revealed a somatic TERT promotor mutation (C228T) in PGL (negative in PTC) and a somatic BRAFV600E mutation in PTC (negative in PGL). Functional studies showed a higher proliferation rate in the mutant compared with the wild-type SDHB.ConclusionGermline SDHB mutations rarely occur in patients with PTC and may contribute to its aggressiveness. Somatic TERT promotor mutations rarely occur in PGL and contribute to its aggressiveness and metastatic potential.

Progress and challenges in experimental models for pheochromocytoma and paraganglioma.

Experimental models for pheochromocytoma and paraganglioma are needed for basic pathobiology research and for preclinical testing of drugs to improve treatment of patients with these tumors, especially patients with metastatic disease. The paucity of models reflects the rarity of the tumors, their slow growth, and their genetic complexity. While there are no human cell line or xenograft models that faithfully recapitulate the genotype or phenotype of these tumors, the past decade has shown progress in development and utilization of animal models, including a mouse and a rat model for SDH-deficient pheochromocytoma associated with germline Sdhb mutations. There are also innovative approaches to preclinical testing of potential treatments in primary cultures of human tumors. Challenges with these primary cultures include how to account for heterogeneous cell populations that will vary depending on the initial tumor dissociation and how to distinguish drug effects on neoplastic vs normal cells. The feasible duration for maintaining cultures must also be balanced against time required to reliably assess drug efficacy. Considerations potentially important for all in vitro studies include species differences, phenotype drift, changes that occur in the transition from tissue to cell culture, and the O2 concentration in which cultures are maintained.

Mutation Profile of Aggressive Pheochromocytoma and Paraganglioma with Comparison of TCGA Data.

In pheochromocytoma and paraganglioma (PPGL), germline or somatic mutations in one of the known susceptibility genes are identified in up to 60% patients. However, the peculiar genetic events that drive the aggressive behavior including metastasis in PPGL are poorly understood. We performed targeted next-generation sequencing analysis to characterize the mutation profile in fifteen aggressive PPGL patients and compared accessible data of aggressive PPGLs from The Cancer Genome Atlas (TCGA) with findings of our cohort. A total of 115 germline and 34 somatic variants were identified with a median 0.58 per megabase tumor mutation burden in our cohort. The most frequent mutation was SDHB germline mutation (27%) and the second frequent mutations were somatic mutations for SETD2, NF1, and HRAS (13%, respectively). Patients were subtyped into three categories based on the kind of mutated genes: pseudohypoxia (n = 5), kinase (n = 5), and unknown (n = 5) group. In copy number variation analysis, deletion of chromosome arm 1p harboring SDHB gene was the most frequently observed. In our cohort, SDHB mutation and pseudohypoxia subtype were significantly associated with poor overall survival. In conclusion, subtyping of mutation profile can be helpful in aggressive PPGL patients with heterogeneous prognosis to make relevant follow-up plan and achieve proper treatment.

Long Intergenic Non Coding RNA and MicroRNA Profiles of Pheochromocytoma and Paraganglioma as Prognostic Biomarkers

Abstract microRNAs (miRNAs) and long intergenic noncoding RNAs (lincRNAs) have been reported as important markers for many cancers. In search of new markers for the metastatic or aggressive phenotypes in the neuroendocrine tumor pheochromocytomas and paragangliomas (PCPG), we analyzed the non-coding transcriptome from patient gene expression data in The Cancer Genome Atlas. We used differential expression analysis and an elastic-net machine-learning model to identify miRNA and lincRNA transcriptomic signature specific to PCPG molecular subtypes. Similarly, miRNAs and lincRNAs specific to aggressive PCPGs were identified, and univariate and multivariate analysis were performed for identifying factors associated with metastasis-free survival. Upregulation of 13 lincRNAs and 4 miRNAs was found to be associated with aggressive/metastatic PCPGs. RT-PCR validation in tumor samples from PCPG patients confirmed the overexpression of 4 miRNAs and 4 lincRNAs in metastatic compared to non-metastatic PCPGs. Kaplan-Meier analysis identified 3 miRNAs and 5 lincRNAs as prognostic markers for metastasis-free survival of patients in PCPGs. In a multivariate Cox regression analysis combining these miRNA and lincRNA expression signatures with the previously identified clinically relevant parameters like SDHB germline mutation, ATRX somatic mutation, tumor location and hormone secretion phenotypes, we identified the miRNA miR-182 and lincRNA HIF1A-AS2 as independent predictors of poor metastasis-free survival. We formulated a risk-score model using multivariate analysis of lincRNA and miRNA expression profiles, presence of SDHB and ATRX mutations, tumor location, and hormone secretion phenotypes. Stratification of PCPG patients with this risk-score showed significant differences in metastasis-free survival.

Mutational Profile of Metastatic Pheochromocytoma and Paraganglioma

Background: In pheochromocytoma and paraganglioma (PPGL), germline and somatic mutations in one of the known susceptibility genes are present in about 60% of tumors. However, the genetic events that drive the malignant progression of the disease are yet poorly understood. We aimed to evaluate the mutational profiles of metastatic PPGLs by targeted next-generation sequencing (NGS) to characterize the genetic events in metastatic PPGLs. Methods: Among the previously reported metastatic PPGL series from Asan Medical Center (AMC), Seoul, Korea, fifteen patients with available formalin-fixed, paraffin embedded (FFPE) archival samples for targeted exome sequencing were enrolled in this study. We also analyzed accessible data of aggressive PPGLs from The Cancer Genome Atlas (TCGA) and compared with findings of AMC samples. Results: A total of 115 germline and 34 somatic variants were identified in AMC cohort. Tumors of AMC cohort had median 0.58 per megabase tumor mutation burden. Most frequently mutated mutations were SDHB germline mutation (27%), and SETD2, NF1, HRAS somatic mutations (13%). Genes are subtyped into pseudohypoxia group (n=5), kinase group (n=5) and unknown (n=5) group. In unknown subgroup, two samples showed ATRX mutations and one accompanied SETD2 mutation. In copy number variation analysis, the most frequently observed pattern was deletion of 1p arm where SDHB is present. In survival analysis, SDHB mutation and pseudohypoxia subtype was significantly associated with poor prognosis. Conclusion: The analysis of NGS from patients with metastatic PPGLs demonstrated rare genetic events as well as well-known mutations. The pseudohypoxia subtype presented poor prognosis than kinase or unknown subtypes. Subjects who had no deletion in 1p arm showed favorable treatment response. Further studies to discover driver events and markers of metastasis are warranted.

Genetic and clinical aspects of paediatric pheochromocytomas and paragangliomas.

Few and conflicting reports have characterized the genetics of paediatric pheochromocytomas and paragangliomas (PPGLs). This study aimed to investigate the clinical and genetic features of Brazilian children with PPGL. This study included 25 children (52% girls) with PPGL. The median age at diagnosis was 15years (4-19). The median time of follow-up was 145months. The genetic investigation was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification and/or target next-generation sequencing panel. Of the 25 children with PPGL, 11 (44%), 4 (16%), 2 (8%), 1 (4%) and 7 (28%) had germline VHL pathogenic variants, SDHB, SDHD, RET and negative genetic investigation, respectively. Children with germline VHL missense pathogenic variants were younger than those with SDHB or SDHD genetic defects [median (range), 12 (4-16) vs. 15.5 (14-19)years; P=.027]. Moreover, 10 of 11 cases with VHL pathogenic variants had bilateral pheochromocytoma (six asynchronous and four synchronous). All children with germline SDHB pathogenic variants presented with abdominal paraganglioma (one of them malignant). The two cases with SDHD pathogenic variants presented with head and neck paraganglioma. Among the cases without a genetic diagnosis, 6 and 2 had pheochromocytoma and paraganglioma, respectively. Furthermore, metastatic PPGL was diagnosed in four (16%) of 25 PPGL. Most of the paediatric PPGL were hereditary and multifocal. The majority of the affected genes belong to pseudohypoxic cluster 1, with VHL being the most frequently mutated. Therefore, our findings impact surgical management and surveillance of children with PPGL.