Development Of Pheochromocytoma Research Articles

Genotype-specific development of MEN 2 constituent components in 683 RET carriers.

The age-specific development of the 3 constituent components of multiple endocrine neoplasia type 2 (MEN 2) is incompletely characterized for many of the >30 causative rearranged during transfection (RET) mutations, which this genetic association study aimed to specify. Included in the study were 683 carriers of heterogeneous RET germline mutations: 53 carriers with 1 highest-risk mutation (codon 918); 240 carriers with 8 different high-risk mutations (codon 634); 176 carriers with 16 different intermediate-risk mutations (codon 609, 611, 618, 620 or 630); and 214 carriers with 6 different low-risk mutations (codon 768, 790, 804 or 891). There was a strong genotype-specific development of MEN 2 constituent components, with distinct age gradients from C cell disease to node-negative MTC, from node-negative to node-positive MTC, from node-positive MTC to pheochromocytoma, and from pheochromocytoma to primary hyperparathyroidism. Primary hyperparathyroidism was not observed among the 53 MEN 2B patients who carried highest-risk mutations (age range 0.5-50 yrs.), of whom no more than 12 (23%) and 3 (6%) carriers were older than age 30 yrs. and 35 yrs., respectively. The age-specific development of MTC differed significantly between the 4 RET risk categories, whereas the age-specific development of pheochromocytoma differed significantly only between the 2 strongest RET risk categories. No significant differences were noted in the development of primary hyperparathyroidism. These findings delineate age-specific disease manifestation corridors for the 3 constituent components of MEN 2 by RET genotype. These corridors are useful for initial risk assessment and organ-specific surveillance of newly identified RET carriers going forward.

SAT266 Transcriptional Changes Associated With Loss Of Function Of Fumarate Hydratase In A Cellular Model Of Pheochromocytoma

Abstract Disclosure: C.L. Hall: None. J.E. Read: None. G. Salsbury: None. E. Lim: None. S. Akker: None. P. Chapple: None. Mutations in fumarate hydratase (FH) predispose to development of pheochromocytomas and paragangliomas (PPGLs), as well as leiomyomas and renal cell carcinoma. Loss of FH function leads to accumulation of fumarate which acts as an oncometabolite to drive tumorigenesis, although the molecular mechanisms leading to the development of these rare tumours are not fully understood. We have used CRISPR/Cas9 genome editing to generate a series of FH knockouts in the PC-12 cell line, derived from MAX-mutated pheochromocytoma in rat. Our knockout cell lines were used as a model to investigate the molecular consequences of loss of FH function. Comparison of the transcriptome of FH knockout cells with isogenic controls identified 1016 differentially expressed genes (adjusted p<0.05 Log2fold±1). This list of differentially expressed genes was then analysed to identify cellular pathways that were dysregulated in the absence of FH. Canonical pathways that were significantly altered, based on transcript expression relative to controls, included axonal signalling pathway (p=7.29x10-11), wound healing signalling pathway (p=1.74x10-07), pulmonary fibrosis idiopathic signalling pathway (p=4.10x10-07) and IL-15 production (p=1.03x10-05). Our results suggest cell adhesion and immune regulation pathways may be relevant to the development of PPGLs with germline mutations in FH, but will require validation in the context of human tissue. We have also performed metabolic flux analysis to identify differences in metabolite profile associated with loss of FH. Interestingly, changes in gene expression, revealed by our transcriptomic analysis, may explain some alterations in cellular metabolism of FH knockout cells. Presentation: Saturday, June 17, 2023

Comprehensive in silico and functional studies for classification of EPAS1/HIF2A genetic variants identified in patients with erythrocytosis.

Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2α-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

A clinical case of composite pheochromocytoma in a child and a literature review

Composite pheochromocytoma (CPh) is a rare malignant neoplasm that occurs in different age ranges. The main treatment for pheochromocytoma is a complete surgical removal of the tumor, while in the case of neuroblastoma, risk-adapted therapy is used, depending on the risk group, including chemotherapy, autologous bone marrow transplantation, radiation therapy, immunotherapy and 131 I-metaiodbenzylguanidine therapy. Due to the rarity of CPh, there are no standard approaches to systemic therapy. The tactics and extent of treatment are determined according to the predominance of the tumor malignant component. This article presents a rare case of the development of composite pheochromocytoma with poorly differentiated neuroblastoma of the left adrenal gland in a 4-year-old child. The patient’s parents gave consent to the use of their child’s data, including photographs, for research purposes and in publications.

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

GLOBAL ENDOCRINOLOGY: Geographical variation in the profile of RET variants in patients with medullary thyroid cancer: a comprehensive review.

Genetic variability in humans is influenced by many factors, such as natural selection, mutations, genetic drift, and migrations. Molecular epidemiology evaluates the contribution of genetic risk factors in the etiology, diagnosis, and prevention of a particular disease. Few areas of medicine have been so clearly affected by genetic diagnosis and management as multiple neoplasia type 2 (MEN2), in which activating pathogenic variants in the RET gene results in the development of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism in nearly 98, 50, and 25% of gene carriers, respectively. Here, we aimed to collect RET genotyping data worldwide to analyze the distribution and frequency of RET variants from a global perspective. We show that the mutational spectrum of RET is observed worldwide. The codon 634 variants seem to be the most prevalent, but there are differences in the type of amino acid exchanges among countries and in the frequencies of the other RET codon variants. Most interestingly, studies using haplotype analysis or pedigree linkage have demonstrated that some pathogenic RET variants have been transmitted to offspring for centuries, explaining some local prevalence due to a founder effect. Unfortunately, after almost three decades after the causative role of the germline RET variants has been reported in hereditary MTC, comprehensive genotyping data remain limited to a few countries. The heterogeneity of RET variants justifies the need for a global effort to describe epidemiological data of families with MEN2 to further understand the genetic background and environmental circumstances that affect disease presentation.

Abstract 3132: Tumorigenesis of MEN2 pheochromocytoma

Abstract Backgrounds: Pheochromocytomas are catecholamine-secreting neuroendocrine tumors, which occur sporadically or as part of inherited syndromes, such as multiple endocrine neoplasia type2 (MEN2), caused by deleterious mutations within the RET protooncogene. Although the RET mutation is implicated in the development of MEN2-associated pheochromocytoma, there is a long (typically 40 years) latency period before the disease onset, suggesting the need of the acquisition of secondary hits. However, little is known about such secondary alterations involved in the pathogenesis of MEN2. In this study, we compared genetic profiles of MEN2 pheochromocytoma and its precursor lesion, adrenal medullary hyperplasia (AMH). Materials and methods: Samples were collected from surgical specimens in 6 MEN2 pheochromocytoma cases, including a MEN2B case with RET p.M918T and 5 MEN2A cases with RET p.C634F, p.C634F, p.C634Y, p.E768D and p.C620S, and 1 AMH case with MEN2A carrying RET p.C634Y. Spatially separated samples were collected from a tumor slice (“tumor parts”). Multiple microscale sampling was also performed from apparently normal adrenal medulla (“non-tumor parts”). Somatic mutations and copy number alterations (CNAs) were then evaluated by whole exome sequencing. Result: Regarding “tumor parts”, a total of 20 samples from 6 MEN2 pheochromocytomas was analyzed. The number of somatic mutations per sample was ranging from 2 to 10. No somatic mutations commonly implicated in sporadic pheochromocytomas were detected. In a case with MEN2B, 1p, 3q and 21q losses were shared across all 3 samples, while 11p and 11q losses and 5p, 5q, 12p, 12q and 15q gains were private, suggesting that 1p, 3q and 21q losses occurred early in the tumorigenesis. In 5 MEN2A cases, 1p (80%), 3q (100%), 21q (60%) and 22q (60%) losses were frequently shared across all samples in each case. As for “non-tumor parts”, a total of 12 samples from 2 MEN2 pheochromocytoma cases and 1 AMH case were analyzed. The number of somatic mutations per sample ranged from 0 to 15. In 2 MEN2 pheochromocytoma cases, no somatic mutations were shared across the samples or no CNAs were detected, while 12 mutations were shared in two samples derived from an AMH case. Interestingly, these samples harbored 17p loss alone, while 1p, 3q, 21q or 22q losses were not found. Conclusion and perspective: MEN2 pheochromocytoma and AMH had different genetic characteristics, where 1p, 3q, 21q and 22q losses could have a role in early tumorigenesis. Further studies involving an increasing number of patients with MEN2 pheochromocytoma and AMH are warranted. Citation Format: Tatsuki Ogasawara, Yoichi Fujii, Nobuyuki Kakiuchi, Yusuke Shiozawa, Hiromichi Suzuki, Ryuichi Sakamoto, Yusaku Yoshida, Yuichi Shiraishi, Satoru Miyano, Seishi Ogawa. Tumorigenesis of MEN2 pheochromocytoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3132.

Seven Novel Genes Related to Cell Proliferation and Migration of VHL-Mutated Pheochromocytoma.

Pheochromocytoma, as a neuroendocrine tumor with the highest genetic correlation in all types of tumors, has attracted extensive attention. Von Hipper Lindau (VHL) has the highest mutation frequency among the genes associated with pheochromocytoma. However, the effect of VHL on the proteome of pheochromocytoma remains to be explored. In this study, the VHL knockdown (VHL-KD) PC12 cell model was established by RNA interference (shRNA). We compared the proteomics of VHL-KD and VHL-WT PC12 cell lines. The results showed that the expression of 434 proteins (VHL shRNA/WT > 1.3) changed significantly in VHL-KD-PC12 cells. Among the 434 kinds of proteins, 83 were involved in cell proliferation, cell cycle and cell migration, and so on. More importantly, among these proteins, we found seven novel key genes, including Connective Tissue Growth Factor (CTGF), Syndecan Binding Protein (SDCBP), Cysteine Rich Protein 61 (CYR61/CCN1), Collagen Type III Alpha 1 Chain (COL3A1), Collagen Type I Alpha 1 Chain (COL1A1), Collagen Type V Alpha 2 Chain (COL5A2), and Serpin Family E Member 1 (SERPINE1), were overexpressed and simultaneously regulated cell proliferation and migration in VHL-KD PC12 cells. Furthermore, the abnormal accumulation of HIF2α caused by VHL-KD significantly increased the expression of these seven genes during hypoxia. Moreover, cell-counting, scratch, and transwell assays demonstrated that VHL-KD could promote cell proliferation and migration, and changed cell morphology. These findings indicated that inhibition of VHL expression could promote the development of pheochromocytoma by activating the expression of cell proliferation and migration associated genes.

Metastatic pheochromocytoma and paraganglioma: signs and symptoms related to catecholamine secretion

BackgroundThe presence or future development of metastatic pheochromocytomas or paragangliomas (mPPGLs) can be difficult to diagnose or predict at initial presentation. Since production of catecholamines from mPPGLs is different from non-metastatic tumors (non-mPPGLs), this study aimed to clarify whether presenting catecholamine-related signs and symptoms (cSS) might also differ.MethodsThe study included 249 patients, 43 with mPPGL and 206 with non-mPPGL. Clinical data at the time of biochemical diagnosis (i.e. at entry into the study) were used to generate a cumulative score of cSS for each patient.ResultsPatients with mPPGL were significantly younger (43.3 ± 14 vs. 48.9 ± 16.1 years) and included a lower proportion of females (39.5% vs. 60.7%) than patients with non-mPPGLs. Frequencies of signs and symptoms did not differ between the two groups. Patients with mPPGLs had lower (P < 0.001) urinary excretion of epinephrine (3.5 (IQR, 1.9—6.5) µg/day) than those with non-mPPGLs (19.1 (IQR, 4.3—70.2) µg/day). There was no difference in urinary excretion of norepinephrine. In patients with mPPGLs a high cSS score was associated with high urinary excretion of norepinephrine and normetanephrine. In contrast, in patients with non-mPPGLs, a high cSS was associated with high urinary excretion of epinephrine and metanephrine.ConclusionAlthough presenting signs and symptoms were associated with production of norepinephrine in patients with mPPGLs and of epinephrine in patients with non-mPPGLs, there were no differences in signs and symptoms between the two groups. Therefore, consideration of signs and symptoms does not appear helpful for distinguishing patients with and without mPPGLs.

Mutation of the Cell Cycle Regulator p27kip1 Drives Pseudohypoxic Pheochromocytoma Development.

Simple SummaryPheochromocytomas and paragangliomas (PPGLs) can be subdivided into at least three different subgroups associated with different clinical manifestations and depending on the risk to metastasize. A shortage in human tumor material, the lack of a functional human cell line and very limited animal models were major drawbacks for PPGL research and consequently for the development of patient-tailored targeted therapies. We have previously reported that the MENX rat model develops pheochromocytoma with a full penetrance at the age of 8–10 months, however, it was unclear which human group the rat tumors modeled best. In order to characterize the rat pheochromocytomas, we analyzed gene expression, the catecholamine profile, TCA-cycle metabolism, methylation, angiogenesis, histology and mitochondrial ultrastructure. In all aspects, rat MENX pheochromocytomas resemble the features of the human pseudohypoxia group, the most aggressive one and in need of effective therapeutic approaches.Background: Pseudohypoxic tumors activate pro-oncogenic pathways typically associated with severe hypoxia even when sufficient oxygen is present, leading to highly aggressive tumors. Prime examples are pseudohypoxic pheochromocytomas and paragangliomas (p-PPGLs), neuroendendocrine tumors currently lacking effective therapy. Previous attempts to generate mouse models for p-PPGLs all failed. Here, we describe that the rat MENX line, carrying a Cdkn1b (p27) frameshift-mutation, spontaneously develops pseudohypoxic pheochromocytoma (p-PCC). Methods: We compared rat p-PCCs with their cognate human tumors at different levels: histology, immunohistochemistry, catecholamine profiling, electron microscopy, transcriptome and metabolome. The vessel architecture and angiogenic potential of pheochromocytomas (PCCs) was analyzed by light-sheet fluorescence microscopy ex vivo and multi-spectral optoacoustic tomography (MSOT) in vivo. Results: The analysis of tissues at various stages, from hyperplasia to advanced grades, allowed us to correlate tumor characteristics with progression. Pathological changes affecting the mitochrondrial ultrastructure where present already in hyperplasias. Rat PCCs secreted high levels of norepinephrine and dopamine. Transcriptomic and metabolomic analysis revealed changes in oxidative phosphorylation that aggravated over time, leading to an accumulation of the oncometabolite 2-hydroxyglutarate, and to hypermethylation, evident by the loss of the epigenetic mark 5-hmC. While rat PCC xenografts showed high oxygenation, induced by massive neoangiogenesis, rat primary PCC transcriptomes possessed a pseudohypoxic signature of high Hif2a, Vegfa, and low Pnmt expression, thereby clustering with human p-PPGL. Conclusion: Endogenous rat PCCs recapitulate key phenotypic features of human p-PPGLs. Thus, MENX rats emerge as the best available animal model of these aggressive tumors. Our study provides evidence of a link between cell cycle dysregulation and pseudohypoxia.

Metabolism and secretion mechanism of catecholamine syndrome and related treatment strategies

: Catecholamine syndrome, also known as pheochromocytoma, is a kind of tumor that originate from adrenal or extra-adrenal chromaffin tissue, secreting a large amount of catecholamines. Some typical gene mutations such as VHL, RET and NF1 play roles in the development of pheochromocytoma. Besides, headache, sweating, and palpitations are the usual manifestations of pheochromocytoma. Most clinical symptoms such as hypertension, headache, sweating and palpitation are related to excessive catecholamine secretion. Though preoperative pretreatment with α-blockers was routine before pheochromocytoma surgery, it showed a lot of limitations because the blockage took effect after catecholamine releasing. Thus, blocking catecholamine production before its releasing shows great significance. For this purpose, in this review, we described the synthesis and metabolic process of catecholamines, the ion channels associated with catecholamine secretion and the influencing factors of the section of catecholamine. We then discussed the related treatment strategies which based on the metabolism and secretion of catecholamine to lay a foundation for the clinical medication of pheochromocytoma.

HIF2α supports pro-metastatic behavior in pheochromocytomas/paragangliomas.

Mutations that drive the stabilization of hypoxia inducible factor 2α (HIF2α) and downstream pseudohypoxic signaling are known to predispose to the development of pheochromocytomas and paragangliomas (PPGLs). However, any role of HIF2α in predisposition to metastatic disease remains unclear. To assess such a role we combined gene-manipulations in pheochromocytoma cell lines with retrospective analyses of patient data and gene expression profiling in tumor specimens. Among 425 patients with PPGLs identified with mutations in tumor-susceptibility genes, those with tumors due to activation of pseudohypoxic pathways had a higher frequency of metastatic disease than those with tumors due to activation of kinase-signaling pathways, even without inclusion of patients with mutations in SDHB (18.6% vs 4.3% in, P < 0.0001). Three out of nine (33%) patients with gain-of-function mutations in HIF2α had metastatic disease. In cell line studies, elevated expression of HIF2α enhanced cell proliferation and led to increased migration and invasion capacity. Moreover, HIF2α expression in HIF2α-deficient cells resulted in increased cell motility, diffuse cluster formation and emergence of pseudopodia indicating changes in cell adhesion and cytoskeletal remodeling. In a mouse liver metastasis model, Hif2a enhanced the metastatic load. Transcriptomics data revealed alterations in focal adhesion and extracellular matrix-receptor interactions in HIF2α-mutated PPGLs. Our translational findings demonstrate that HIF2α supports pro-metastatic behavior in PPGLs, though other factors remain critical for subsequent transition to metastasis. We identified LAMB1 and COL4A2 as new potential therapeutic targets for HIF2α-driven PPGLs. Identified HIF2α downstream targets might open a new therapeutic window for aggressive HIF2α-expressing tumors.

SAT-222 Aggressive Phenotype Pheochromocytoma Associated with NF-1 and BRCA Mutation

IntroductionPheochromocytomas are catecholamine-secreting tumors of the adrenal medulla that occur sporadically or with familial association. Familial predilection occurs in those with multiple endocrine neoplasia type 2, von Hippel-Lindau disease, and neurofibromatosis Type 1 (NF-1). To date, more than 21 gene mutations have been identified that are involved in the development of pheochromocytomas. However, co-existence of two different mutations such as NF-1 and BRCA gene mutations with pheochromocytoma has not been well described.Clinical CaseA 40 year-old woman with NF-1 and recently diagnosed left breast invasive ductal carcinoma, estrogen positive, progesterone positive, HER-2 negative BRCA2 positive breast cancer and right breast lobular carcinoma in situ underwent staging CT scan. A 2.7 cm left adrenal incidentaloma with heterogeneous texture and 69–100 HU was discovered. Symptom review revealed chronic headaches and increasing frequency of anxiousness and irritability. She had no personal or family history of hypertension. Laboratory analysis showed elevated catecholamine levels included plasma normetanephrine of 2.8 nmol/L (normal <0.9), metanephrine of 0.9 nmol/L (normal <0.5), 24 hour urine metanephrine 498 mcg/24 hr (normal <180) and 24 hour urine normetanephrine of 1,152 mcg/24 hr (normal <451). Findings were consistent with adrenal pheochromocytoma.She was immediately started on phenoxybenzamine and encouraged to increase fluid intake to expand intravascular volume before surgery. She underwent laparoscopic left partial adrenalectomy fourteen days later. Surgical pathology revealed a 3.0 cm pheochromocytoma in a background of nodular hyperplasia of the medulla, described as an aggressive tumor phenotype based on morphological features.ConclusionsIt has been estimated that 1.5–14% of adrenal incidentalomas are pheochromocytomas. While only 3% of NF-1 patients have pheochromocytomas, 20–50% of NF-1 patients with concurrent hypertension have underlying chromaffin tumors. This association is significant when compared to the pheochromocytoma incidence of 0.1% in all hypertensive individuals. Interestingly, germline mutations of BRCA-1 associated protein-1 gene have been reported in patients with paragangliomas, therefore it may be plausible to consider interactions between NF-1 and breast cancer BRCA-2 gene mutations, resulting in an unusual and more aggressive pheochromocytoma phenotype, even when detected at an early stage, as in this case. It is also important to recognize and adequately treat pheochromocytoma prior to having surgery to avoid possible intraoperative hypertensive crisis, a concept that was underscored in this particular case when the patient was relatively asymptomatic and entirely normotensive preoperatively.

Long-term Functional and Oncologic Outcomes of Partial Adrenalectomy for Pheochromocytoma

ObjectiveTo evaluate the recurrence and functional outcomes in a primarily hereditary cohort of patients undergoing partial adrenalectomy for pheochromocytoma. MethodsA retrospective review from a prospectively managed database of patients undergoing partial adrenalectomy from 1995 to 2018 at the National Cancer Institute was performed. Local recurrence was defined as imaging evidence of a recurrent or de novo lesion on the operative side. Steroid dependency was defined as requiring daily steroid replacement at time of last follow-up. ResultsOne hundred and twenty-four partial adrenalectomies, removing 162 tumors, were performed in 107 patients. Most patients had a known hereditary predisposition to develop bilateral, multifocal, and recurrent pheochromocytoma. Median tumor size was 2 cm (interquartile range (IQR) 1.5-2.8). Median follow-up was 60 months (IQR 13-131). Local recurrence occurred in 17 patients (15.8%) and were managed with active surveillance or surgery. A single patient (1/106, 0.9%) developed metastatic spread of pheochromocytoma approximately 14 years after his first of 2 partial adrenalectomies and remains alive under active surveillance. Median time to recurrence was 71 months (IQR 26-127) with 10 patients (9.3%) requiring daily steroid replacement at time of last follow-up. ConclusionPartial adrenalectomy offers excellent oncologic and functional outcomes, sparing most patients from lifelong steroid replacement therapy. Recurrences can be easily managed with repeat surgery or active surveillance via functional work-up and imaging. Partial adrenalectomy remains the recommended surgical management for patients pre-disposed to development of bilateral, multifocal and recurrent pheochromocytoma.

Comparison of Pheochromocytoma-Specific Morbidity and Mortality Among Adults With Bilateral Pheochromocytomas Undergoing Total Adrenalectomy vs Cortical-Sparing Adrenalectomy

Large studies investigating long-term outcomes of patients with bilateral pheochromocytomas treated with either total or cortical-sparing adrenalectomies are needed to inform clinical management. To determine the association of total vs cortical-sparing adrenalectomy with pheochromocytoma-specific mortality, the burden of primary adrenal insufficiency after bilateral adrenalectomy, and the risk of pheochromocytoma recurrence. This cohort study used data from a multicenter consortium-based registry for 625 patients treated for bilateral pheochromocytomas between 1950 and 2018. Data were analyzed from September 1, 2018, to June 1, 2019. Total or cortical-sparing adrenalectomy. Primary adrenal insufficiency, recurrent pheochromocytoma, and mortality. Of 625 patients (300 [48%] female) with a median (interquartile range [IQR]) age of 30 (22-40) years at diagnosis, 401 (64%) were diagnosed with synchronous bilateral pheochromocytomas and 224 (36%) were diagnosed with metachronous pheochromocytomas (median [IQR] interval to second adrenalectomy, 6 [1-13] years). In 505 of 526 tested patients (96%), germline mutations were detected in the genes RET (282 patients [54%]), VHL (184 patients [35%]), and other genes (39 patients [7%]). Of 849 adrenalectomies performed in 625 patients, 324 (52%) were planned as cortical sparing and were successful in 248 of 324 patients (76.5%). Primary adrenal insufficiency occurred in all patients treated with total adrenalectomy but only in 23.5% of patients treated with attempted cortical-sparing adrenalectomy. A third of patients with adrenal insufficiency developed complications, such as adrenal crisis or iatrogenic Cushing syndrome. Of 377 patients who became steroid dependent, 67 (18%) developed at least 1 adrenal crisis and 50 (13%) developed iatrogenic Cushing syndrome during median (IQR) follow-up of 8 (3-25) years. Two patients developed recurrent pheochromocytoma in the adrenal bed despite total adrenalectomy. In contrast, 33 patients (13%) treated with successful cortical-sparing adrenalectomy developed another pheochromocytoma within the remnant adrenal after a median (IQR) of 8 (4-13) years, all of which were successfully treated with another surgery. Cortical-sparing surgery was not associated with survival. Overall survival was associated with comorbidities unrelated to pheochromocytoma: of 63 patients who died, only 3 (5%) died of metastatic pheochromocytoma. Patients undergoing cortical-sparing adrenalectomy did not demonstrate decreased survival, despite development of recurrent pheochromocytoma in 13%. Cortical-sparing adrenalectomy should be considered in all patients with hereditary pheochromocytoma.

Carbonic anhydrase 9 immunohistochemistry as a tool to predict or validate germline and somatic VHL mutations in pheochromocytoma and paraganglioma-a retrospective and prospective study.

The development of pheochromocytomas and paragangliomas is strongly linked to the presence of germline mutations in more than 15 predisposing genes. Among them, germline and somatic VHL mutations account for ~10% of all cases. In contrast with SDHA and SDHB immunohistochemistries that are routinely used to validate SDHx gene mutations, there is no such tool available for VHL mutations. The aim of this study was to evaluate whether CA9 immunostaining could be used as a tool to predict the presence or validate the pathogenicity of VHL gene mutations in paraganglioma. Immunohistochemistry for CA9 was performed on 207 tumors. A retrospective series of 100 paragangliomas with known mutation status for paraganglioma susceptibility genes was first investigated. Then, a prospective series of 107 paragangliomas was investigated for CA9 immunostaining followed by germline and/or somatic genetic testing of all paraganglioma susceptibility genes by next-generation sequencing. Cytosolic CA9 protein expression was heterogeneous in the different samples. However, we observed that a membranous CA9 staining was almost exclusively observed in VHL-related cases. Forty two of 48 (88%) VHL-mutated samples showed a CA9 membranous immunostaining. Positive cells were either isolated, varying from 1 or 2 cells (5% of cases) to 10-20 cells per tumor block (35% of cases), grouped in areas of focal positivity representing between 1 and 20% of the tissue section (35% of cases), or widely distributed on 80-100% of the tumor sections (25% of samples). In contrast, 142/159 (91%) of non-VHL-mutated tumors presented no membrane CA9 localization. Our results demonstrate that VHL gene mutations can be predicted or validated reliably by an easy-to-perform and low-cost immunohistochemical procedure. CA9 immunohistochemistry on paragangliomas will improve the diagnosis of VHL-related disease, which is important for the surveillance and therapeutic management of paraganglioma patients, and in case of germline mutation, their family members.

RESULTS OF GENETIC TESTING IN PATIENTS WITH PHEOCHROMOCYTOMA/PARAGANGLIOMA

Objective:More than 20 genes associated with the development of pheochromocytoma or paraganglioma (PPGL) have been described so far. In previous studies, mutation rate of involved genes reached up to 30–40% depending of studied populations. Aim of the study was to search for mutations of PPGL-relate

Fibroblast deficiency of insulin-like growth factor 1 receptor type 1 (IGF1R) impairs initial steps of murine pheochromocytoma development

Insulin-like growth factor 1 (IGF1) has a critical role in maintaining tumor phenotype and survival of already transformed murine pheochromocytoma (pheo) cells (MPC4/30) and it is required for the initial establishment of these tumors. However, the role of local IGF1/IGF1R system in tumor microenvironment has not been fully understood. In vivo, by subcutaneous injection of pheo cells in heterozygous IGF1R knockout mice (L/n), we found that the time of noticeable tumor appearance was delayed, and incidence was decreased in L/n group compared to control (L/L) mice. Once established, tumor proliferation, vascularization or growth rate did not differ between groups. In vitro, fibroblast from L/n and L/L mice were cultured to generate conditioned media (CM) and differential matrixes on which pheo cells were seeded. Proliferation rate was higher when pheo cells were cultured with CM, or in differential matrix generated by L/L murine fibroblasts. A diminished fibronectin (FN) expression and secretion from L/n fibroblast was associated with decreased expression of integrin subunits in tumor cells. Also, soluble factors as IGF1 and insulin-like growth factor binding protein 2 (IGFBP2) were reduced. Our data suggest that IGF1 signaling through IGF1R may contribute to tumor cells anchorage and survival by interaction with both matrix and soluble factors produced by tumor microenvironment fibroblasts.

SUN-032 Exome Sequencing Reveals that Pathogenic RET Variants Occur at Higher Prevalence Than Previously Recognized: Data from a US Health System Biobank

Background: Multiple Endocrine Neoplasia Syndrome Type 2 (MEN2) is an inherited endocrine disorder characterized by the development of pheochromocytoma, medullary thyroid carcinoma (MTC) and parathyroid neoplasia. MEN2 is autosomal dominant and occurs from activating missense variants in the RET proto-oncogene. The estimated prevalence of MEN2 is thought to be 1 per 30,000 in the general population, and although rare, the identification of MEN2 can aid with early screening and prevention for patients and their families. Aim: The aim of our study was to investigate the prevalence and clinical manifestations of pathogenic RET variants in the multi-ethnic BioMe Biobank. Methods: We used exome sequencing and electronic health record (EHR) data from BioMe Biobank participants, who are recruited from primary care and specialty clinics across a single U.S. Health System. We evaluated exome sequence data from 30,000 BioMe participants for RET variants designated as pathogenic for MEN2 according to the clinical genetics database ClinVar. We reviewed EHR data for carriers of pathogenic RET variants. Results: In the BioMe Biobank, 5 ClinVar pathogenic variants were identified in the RET gene. Fifteen participants were heterozygous carriers of these variants; 5 were of self-reported African ancestry, 5 of Hispanic/Latino ancestry, 2 of European ancestry, and 3 of other or unknown ancestry. The age range of carriers was 23-78 years, and 60% were female. The estimated prevalence of pathogenic RET variant carriers in BioMe was ~1 in 2000. Based on EHRs, only 3 carriers (20%) were identified from usual clinical care as having the diagnosis of MEN2A. All 3 carriers with diagnosed MEN2A had the 10:43114500:T:C variant (rs75076352) and had a history of MTC and pheochromocytoma. One of the 3 carriers had a history of elevated parathyroid hormone levels and renal calculi without hypercalcemia. Two of the 3 carriers had a documented family history of MEN2A. Of the 12 pathogenic RET variant carriers without known MEN2, 9 (75%) were diagnosed with hypertension. 7 (58%) carried the 10:43119548:G:A variant (rs79658334). Of these 7 carriers, one had a thyroid nodule, three had autoimmune thyroid disease, one had an adrenal cortical adenoma with primary hyperaldosteronism and hypokalemic periodic paralysis, and two had no known endocrine conditions. Conclusions: These results from the BioMe Biobank reveal a higher prevalence of pathogenic RET variants than previously recognized. Carriers of pathogenic RET variants demonstrate highly variable expressivity for traits for which earlier diagnoses might improve outcomes; these findings have implications for cascade testing of family members. Genomic screening for pathogenic RET variants will likely improve our understanding of MEN2 on a more molecular basis.

Retrospective evaluation of toceranib phosphate (Palladia\xae) use in the treatment of inoperable, metastatic, or recurrent canine pheochromocytomas: 5 dogs (2014\u20132017)

BackgroundEffective treatment options for inoperable, metastatic, or recurrent canine pheochromocytomas are lacking. In humans, specific germline mutations exist that drive the development of pheochromocytomas. Pharmaceutical blockade of these abnormalities with small molecule inhibitors are an effective treatment strategy. Similar mutations may exist in the dog, and thus, treatment with similar small molecule inhibitors may provide a survival advantage. The purpose of this study was to assess the role of toceranib phosphate in the treatment of inoperable, metastatic, or recurrent canine pheochromocytomas.ResultsRetrospectively, medical records of dogs that had a diagnosis or suspect diagnosis of a pheochromocytoma were reviewed for information regarding response to toceranib phosphate and overall outcome. Five dogs were identified that fit the inclusion criteria. All five experienced clinical benefit (1 partial response, 4 stable disease). Progression-free interval (PFI) for the dog with the partial response was 61 weeks. PFI for the two dogs with stable measurable disease were 36 weeks and 28 weeks. PFI in the two dogs with stable metastatic disease were at least 11 weeks and 18 weeks.ConclusionsBased on this limited series of dogs, the results suggest that toceranib may have biological activity in dogs with primary and metastatic pheochromocytomas. Larger studies are needed to define the use and response to toceranib in dogs with gross, microscopic, and metastatic pheochromocytoma.