Abstract
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.
Highlights
Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases
This study aimed to perform genetic screening of all PPGL major genes, as well as MDH2 and three minor genes classically involved in PPGL (EGLN1, EGLN2, and multiple endocrine neoplasia type 1 (MEN1)), using two customized targeted gene panels (TGPs) in 491 DNA samples [obtained from blood, saliva, formalin-fixed paraffin-embedded (FFPE) tissue, and frozen tumor] from 453 PPGL index patients recruited in an international effort to validate their use in the clinical setting
FFPE tumor slides were evaluated for succinate dehydrogenase complex iron sulfur subunit B (SDHB)-immunohistochemistry (SDHB-IHC) using anti-SDHB rabbit polyclonal antibody
Summary
Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. 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 directed toward formalinfixed paraffin-embedded tissue. 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. Pheochromocytomas (PCCs) and paragangliomas (PGLs), altogether PPGLs, are rare neuroendocrine tumors arising from adrenal gland (PCCs), sympathetic thoracoabdominal paraganglia, and head and neck parasympathetic paraganglia
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