RESUMO
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.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Anidrase Carbônica IX/análise , Paraganglioma/genética , Feocromocitoma/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Neoplasias das Glândulas Suprarrenais/diagnóstico , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Feocromocitoma/diagnóstico , Estudos Prospectivos , Estudos RetrospectivosRESUMO
Paragangliomas are neuroendocrine tumors frequently associated with mutations in RET, NF1, VHL, and succinate dehydrogenase (SDHx) genes. Methylome analysis of a large paraganglioma cohort identified three stable clusters, associated with distinct clinical features and mutational status. SDHx-related tumors displayed a hypermethylator phenotype, associated with downregulation of key genes involved in neuroendocrine differentiation. Succinate accumulation in SDH-deficient mouse chromaffin cells led to DNA hypermethylation by inhibition of 2-OG-dependent histone and DNA demethylases and established a migratory phenotype reversed by decitabine treatment. Epigenetic silencing was particularly severe in SDHB-mutated tumors, potentially explaining their malignancy. Finally, inactivating FH mutations were identified in the only hypermethylated tumor without SDHx mutations. These findings emphasize the interplay between the Krebs cycle, epigenomic changes, and cancer.