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Context: SDHB mutations are found in an increasing number of neoplasms, most notably in paragangliomas and pheochromocytomas (PPGLs). SDHB-PPGLs are slow-growing tumors, but ∼50% of them may develop metastasis. The molecular basis of metastasis in these tumors is a long-standing and unresolved problem. Thus, a better understanding of the biology of metastasis is needed., Objective: This study aimed to identify gene methylation changes relevant for metastatic SDHB-PPGLs., Design: We performed genome-wide profiling of DNA methylation in diverse clinical and genetic PPGL subtypes, and validated protocadherin γ-C3 (PCDHGC3) gene promoter methylation in metastatic SDHB-PPGLs., Results: We define an epigenetic landscape specific for metastatic SDHB-PPGLs. DNA methylation levels were found significantly higher in metastatic SDHB-PPGLs than in SDHB-PPGLs without metastases. One such change included long-range de novo methylation of the PCDHA, PCDHB, and PCDHG gene clusters. High levels of PCDHGC3 promoter methylation were validated in primary metastatic SDHB-PPGLs, it was found amplified in the corresponding metastases, and it was significantly correlated with PCDHGC3 reduced expression. Interestingly, this epigenetic alteration could be detected in primary tumors that developed metastasis several years later. We also show that PCDHGC3 down regulation engages metastasis-initiating capabilities by promoting cell proliferation, migration, and invasion., Conclusions: Our data provide a map of the DNA methylome episignature specific to an SDHB-mutated cancer and establish PCDHGC3 as a putative suppressor gene and a potential biomarker to identify patients with SDHB-mutated cancer at high risk of metastasis who might benefit from future targeted therapies., (Copyright © 2019 Endocrine Society.)

Background: Succinate dehydrogenase subunit B (SDHB) immunohistochemistry was considered a valuable tool to identify patients with inherited paraganglioma/pheochromocytoma (PGL/PCC). However, previous studies jointly analyzed 2 related but clinically distinct entities, parasympathetic head and neck paragangliomas (HNPGLs) and sympathetic PCCs/PGLs. Additionally, a role for hypoxia inducible factor-1α (HIF-1α) as a biomarker for succinate dehydrogenase (SDHx)-mutated tumors has not been studied. Here, we evaluated the utility of SDHB/HIF-1α proteins in HNPGLs and PCCs/PGLs as clinically useful biomarkers., Methods: The SDHB/succinate dehydrogenase subunit A (SDHA)/HIF-1α immunohistochemistry analysis was performed in 158 genetically defined patients., Results: Similarly to PCCs/PGLs, SDHB immune-negativity correlated with SDHx-mutations in HNPGLs (P < .0001). The HIF-1α stabilization was associated with SDHx-mutations in HNPGLs (P = .020), not in PCCs/PGLs (P = .319). However, 25% of SDHx-HNPGLs lacked HIF-1α positive cells., Conclusion: As in PCCs/PGLs, SDHB immunohistochemistry in HNPGLs is a valuable method for identification of candidates for SDHx-genetic testing. On the contrary, although SDHx mutations may favor HIF-1α stabilization in HNPGLs, this is not a clinically useful biomarker., (© 2018 Wiley Periodicals, Inc.)

The hypoxia-inducible factor 1α (HIF-1α) and its microRNA target, miR-210, are candidate tumor-drivers of metabolic reprogramming in cancer. Neuroendocrine neoplasms such as paragangliomas (PGLs) are particularly appealing for understanding the cancer metabolic adjustments because of their associations with deregulations of metabolic enzymes, such as succinate dehydrogenase (SDH), and the von Hippel Lindau (VHL) gene involved in HIF-1α stabilization. However, the role of miR-210 in the pathogenesis of SDH-related tumors remains an unmet challenge. Herein is described an in vivo genetic analysis of the role of VHL, HIF1A and SDH on miR-210 by using knockout murine models, siRNA gene silencing, and analyses of human tumors. HIF-1α knockout abolished hypoxia-induced miR-210 expression in vivo but did not alter its constitutive expression in paraganglia. Normoxic miR-210 levels substantially increased by complete, but not partial, VHL silencing in paraganglia of knockout VHL-mice and by over-expression of p76del-mutated pVHL. Similarly, VHL-mutated PGLs, not those with decreased VHL-gene/mRNA dosage, over-expressed miR-210 and accumulate HIF-1α in most tumor cells. Ablation of SDH activity in SDHD-null cell lines or reduction of the SDHD or SDHB protein levels elicited by siRNA-induced gene silencing did not induce miR-210 whereas the presence of SDH mutations in PGLs and tumor-derived cell lines was associated with mild increase of miR-210 and the presence of a heterogeneous, HIF-1α-positive and HIF-1α-negative, tumor cell population. Thus, activation of HIF-1α is likely an early event in VHL-defective PGLs directly linked to VHL mutations, but it is a late event favored but not directly triggered by SDHx mutations. This combined analysis provides insights into the mechanisms of HIF-1α/miR-210 regulation in normal and tumor tissues potentially useful for understanding the pathogenesis of cancer and other diseases sharing similar underpinnings.

Background: Head and neck paragangliomas (HNPGLs) are rare tumors associated with the parasympathetic nervous system. Most are sporadic, but about one third result from germline mutations in succinate dehydrogenase (SDH) genes (SDHB, SDHC, SDHD, SDHA, or SDHAF2). Although a molecular connection between SDH dysfunction and tumor development is still unclear, the most accepted hypothesis proposes a central role of the pseudohypoxic pathway. SDH dysfunction induces abnormal stabilization of the hypoxia-inducible factors (HIFs) that regulate target genes involved in proliferation, apoptosis, angiogenesis, and metabolism. The involvement of these pathways in the development of sporadic HNPGLs is presently unknown., Objective: To get some insights into the hypoxic/pseudohypoxic molecular basis of HNPGLs, we attempted to define the gene, microRNA (miRNA), and HIF-1α expression patterns that distinguish tumors from normal paraganglia tissue., Design: Genome microarray and TaqMan low-density arrays were used to analyze gene and miRNA expression, respectively, in 17 HNPGL tumor tissues and three normal human carotid bodies. Twelve HNPGLs were used for validation of data. HIF-1α, SDHB, and iron-sulfur cluster scaffold protein (ISCU) protein expression was analyzed by immunohistochemistry., Results: We found activation of a canonical HIF-1α-related gene expression signaling only in a subset of HNPGLs from patients that did not harbor germline or somatic SDH mutations. The pseudohypoxic signature consisted in the overexpression of both HIF-1α-target genes and the HIF-1α-inducible miRNA, miR-210, and down-regulation of the miR-210 target gene, ISCU1/2. A decreased level of the iron-sulfur-containing protein SDHB was found by immunohistochemical analysis performed in two of these tumors., Conclusions: Collectively, this study unveiled a putative signaling axis of HIF-1α/miRNA-210/ISCU in a subset of HNPGLs that could have an impact on SDHB protein stability by a mechanism independent of SDH mutations, thus providing a foundation to better understand the functional interplay between HIF, miR-210, and mitochondria and its relevance in the pathogenesis of HNPGLs.

Neuroendocrine neoplasms (NENs) encompass tumors arising from neuroendocrine cells in various organs, including the gastrointestinal tract, pancreas, adrenal gland, and paraganglia. Despite advancements, accurately predicting the aggressiveness of gastroenteropancreatic (GEP) NENs based solely on pathological data remains challenging, thereby limiting optimal clinical management. Our previous research unveiled a crucial link between hypermethylation of the protocadherin PCDHGC3 gene and neuroendocrine tumors originating from the paraganglia and adrenal medulla. This epigenetic alteration was associated with increased metastatic potential and succinate dehydrogenase complex (SDH) dysfunction. Expanding upon this discovery, the current study explored PCDHGC3 gene methylation within the context of GEP‐NENs in a cohort comprising 34 cases. We uncovered promoter hypermethylation of PCDHGC3 in 29% of GEP‐NENs, with a significantly higher prevalence in gastrointestinal (GI) neuroendocrine carcinomas (NECs) compared with both pancreatic (Pan) NECs and neuroendocrine tumors (NETs) of GI and Pan origin. Importantly, these findings were validated in one of the largest multi‐center GEP‐NEN cohorts. Mechanistic analysis revealed that PCDHGC3 hypermethylation was not associated with SDH mutations or protein loss, indicating an SDH‐independent epigenetic mechanism. Clinically, PCDHGC3 hypermethylation emerged as a significant prognostic factor, correlating with reduced overall survival rates in both patient cohorts. Significantly, whereas PCDHGC3 hypermethylation exhibited a strong correlation with TP53 somatic mutations, a hallmark of NEC, its predictive value surpassed that of TP53 mutations, with an area under the curve (AUC) of 0.95 (95% CI 0.83–1.0) for discriminating GI‐NECs from GI‐NETs, highlighting its superior predictive performance. In conclusion, our findings position PCDHGC3 methylation status as a promising molecular biomarker for effectively stratifying patients with GI‐NENs. This discovery has the potential to advance patient care by enabling more precise risk assessments and tailored treatment strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Metastatic pheochromocytoma and paraganglioma (PPGL) have poor prognosis and limited therapeutic options. The recent advent of immunotherapies showing remarkable clinical efficacies against various cancer types offers the possibility of novel opportunities also for metastatic PPGL. Most PPGLs are pathogenically linked to inactivating mutations in genes encoding different succinate dehydrogenase (SDH) subunits. This causes activation of the hypoxia‐inducible factor 2 (HIF2)‐mediated transcriptional program in the absence of decreased intratumoral oxygen levels, a phenomenon known as pseudohypoxia. Genuine hypoxia in a tumor creates an immunosuppressive tumor microenvironment. However, the impact of pseudohypoxia in the immune landscape of tumors remains largely unexplored. In this study, tumoral expression of programmed death‐ligand 1 (PD‐L1) and HIF2α and tumor infiltration of CD8 T lymphocytes (CTLs) were examined in PPGL specimens from 102 patients. We assessed associations between PD‐L1, CTL infiltration, HIF2α expression, and the mutational status of SDH genes. Our results show that high PD‐L1 expression levels in tumor cells and CTL tumor infiltration were more frequent in metastatic than nonmetastatic PPGL. However, this phenotype was negatively associated with SDH mutations and high HIF2α protein expression. These data were validated by analysis of mRNA levels of genes expressing PD‐L1, CD8, and HIF2α in PPGL included in The Cancer Genome Atlas database. Further, PD‐L1 and CD8 expression was lower in norepinephrine than epinephrine‐secreting PPGL. This in silico analysis also revealed the low PD‐L1 or CD8 expression levels in tumors with inactivating mutations in VHL or activating mutations in the HIF2α‐coding gene, EPAS1, which, together with SDH‐mutated tumors, comprise the pseudohypoxic molecular subtype of PPGL. These findings suggest that pseudohypoxic tumor cells induce extrinsic signaling toward the immune cells promoting the development of an immunosuppressive environment. It also provides compelling support to explore the differential response of metastatic PPGL to immune checkpoint inhibitors. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Simple Summary: Thoraco-abdominal paraganglioma and pheochromocytoma (PPGL) are pathogenically linked to mutations in SDH genes. Loss-of-function of SDH has been associated with stabilization of HIF2α, which otherwise would be degraded via oxygen-sensitive mechanisms. SDH dysfunction also predispose to the development of paragangliomas arising at the carotid body or other head and neck paraganglia (HNPGL). Although PPGL and HNPGL share similar features, they have certain clinical and genetic peculiarities. By comparison of HIF2α expression in HNPGL and PPGL, we found that functional HIF2α is overexpressed in 80% of PPGLs, including those with SDH mutations as compared with non-tumor tissue. However, HIF2α is already highly expressed in the carotid body under physiologic conditions, and it is not overexpressed in HNPGL. These data suggest that selective HIF2α inhibitors already in clinical trials may benefit a wide spectrum of PPGL. Hypoxia-inducible factors (HIF) 2α and 1α are the major oxygen-sensing molecules in eukaryotic cells. HIF2α has been pathogenically linked to paraganglioma and pheochromocytoma (PPGL) arising in sympathetic paraganglia or the adrenal medulla (AM), respectively. However, its involvement in the pathogenesis of paraganglioma arising in the carotid body (CB) or other parasympathetic ganglia in the head and neck (HNPGL) remains to be defined. Here, we retrospectively analyzed HIF2α by immunohistochemistry in 62 PPGL/HNPGL and human CB and AM, and comprehensively evaluated the HIF-related transcriptome of 202 published PPGL/HNPGL. We report that HIF2α is barely detected in the AM, but accumulates at high levels in PPGL, mostly (but not exclusively) in those with loss-of-function mutations in VHL and genes encoding components of the succinate dehydrogenase (SDH) complex. This is associated with upregulation of EPAS1 and the HIF2α-regulated genes COX4I2 and ADORA2A. In contrast, HIF2α and HIF2α-regulated genes are highly expressed in CB and HNPGL, irrespective of VHL and SDH dysfunctions. We also found that HIF2α and HIF1α protein expressions are not correlated in PPGL nor HNPGL. In addition, HIF1α-target genes are almost exclusively overexpressed in VHL-mutated HNPGL/PPGL. Collectively, the data suggest that involvement of HIF2α in the physiology and tumor pathology of human paraganglia is organ-of-origin-dependent and HIF1α-independent. [ABSTRACT FROM AUTHOR]