Oral Presentation 5th International Symposium on Phaeochromocytoma and Paraganglioma 2017

Contribution of targeted next-generation sequencing (NGS) to improve detection of germline and somatic mutations in pheochromocytomas and paragangliomas (#20)

Nelly BURNICHON 1 2 , Laurene BEN AIM 2 , Mathilde PADILLA-GIROLA 2 , Caroline TRAVERS 2 , Nelly LE POTTIER 2 , Mathieu MADELAINE 2 , Alexandre BUFFET 1 , Judith FAVIER 1 , Anne-Paule GIMENEZ-ROQUEPLO 1 2
  1. Team 13, UMR970, Paris-Cardiovascular Research Center, INSERM, Paris, France
  2. Georges Pompidou european Hospital, PARIS, 75, France

Context

Since the first description of germline mutations in patients with paraganglioma or pheochromocytoma (PPGL), over 15 susceptibility genes have been identified. It is currently accepted that up to 60% of PPGL are associated with inherited or somatic mutation in these driver genes. Due to the high heritability, genetic testing has been recommended in all patients with PPGLs.

In this context, the use of Next-Generation Sequencing (NGS) represents a particularly relevant technique allowing a simultaneous screening of all PPGL genes of interest.

 

Material

A retrospective cohort including 202 tumour DNAs was analysed in order to validate the NGS protocol.

Since transfer to clinical practice, 461 leukocyte DNAs, 51 frozen-tumour DNAs and 10 FFPE-DNAs were sequenced.

 Methods

We developed and optimized a custom amplicon-based NGS panel (SDH MASTR Plus v2 kit, Multiplicom) using a MiSeq (Illumina) instrument. The targeted panel included 17 genes (SDHA, SDHAF2, SDHB, SDHC, SDHD, RET, VHL, NF1, TMEM127, EGLN1, EGLN2, FH, MAX, MDH2, EPAS1, ATRX and HRAS). Primer design was compatible for DNA extracted from FFPE tissues. This panel allowed sequencing of 451 amplicons covering 182 exons. Data were analysed using SeqNext (JSI) and PolyDiag software. Presence of variant of interest was confirmed by Sanger sequencing. Pathogenicity of variants was evaluated by immunohistochemistry whenever possible.

 

Results

All mutations previously found using Sanger method1 were confirmed by NGS (n=133). In the prospective study, 121 germline and 73 somatic variants of interest (class 3 to 5) were identified. Mutations were found in all tested genes. Functional studies were helpful for classification of 61 variants.

 

Conclusion

The use of targeted NGS approach improves the detection of mutations responsible for PPGL, especially somatic and mosaic mutations. Notably, the analysis of unselected PPGL tumour samples allows to clarify the implication of the different genes in development of sporadic PPGL.

  1. Castro-Vega*, Letouzé*, Burnichon* et al. Multi-omics analysis defines core genomic alterations in pheochromocytomas and paragangliomas. Nat Commun. 2015.