Integrative genetic characterization and phenotype correlations in pheochromocytoma and paraganglioma tumours

Abstract

Background: About 60% of Pheochromocytoma (PCC) and Paraganglioma (PGL) patients have either germline or somatic mutations in one of the 12 proposed disease causing genes; SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, RET, NF1, TMEM127, MAX and H-RAS. Selective screening for germline mutations is routinely performed in clinical management of these diseases. Testing for somatic alterations is not performed on a regular basis because of limitations in interpreting the results.

Aim: The purpose of the study was to investigate genetic events and phenotype correlations in a large cohort of PCC and PGL tumours.

Methods: A total of 101 tumours from 89 patients with PCC and PGL were re-sequenced for a panel of 10 disease causing genes using automated Sanger sequencing. Selected samples were analysed with Multiplex Ligation-dependent Probe Amplification and/or SNParray.

Results: Pathogenic genetic variants were found in tumours from 33 individual patients (37%), 14 (16%) were discovered in constitutional DNA and 16 (18%) were confirmed as somatic. Loss of heterozygosity (LOH) was observed in 1/1 SDHB, 11/11 VHL and 3/3 NF1-associated tumours. In patients with somatic mutations there were no recurrences in contrast to carriers of germline mutations (P = 0.022). SDHx/VHL/EPAS1 associated cases had higher norepinephrine output (P = 0.03) and lower epinephrine output (P<0.001) compared to RET/NF1/H-RAS cases.

Conclusion: Somatic mutations are frequent events in PCC and PGL tumours. Tumour genotype may be further investigated as prognostic factors in these diseases. Growing evidence suggest that analysis of tumour DNA could have an impact on the management of these patients.

Figure 1. Chromatograms exported from CLC Genomics Workbench 5.5 displaying (A) Pathogenic genetic variants available in constitutional DNA, (B) confirmed somatic variants and (C) 30 base pair somatic deletion in VHL.

Figure 2. Detected copy number events from SNP array as displayed by Nexus copy number 7.0.

Results are separated by chromosome and presented for each individual tumour with patient id at the left margin. Presented data constitute cases harbouring pathogenic genetic variants in VHL (n = 11) as well as patients with clinical criteria of NF1 (n = 3). Data is also presented for patient 36 that harboured a VHL mutation of unknown significance. Colour annotation indicates copy number loss (red), copy number gain (blue), loss of heterozygosity (yellow) and allelic imbalance (magenta). Arrows indicates VHL (chromosome 3) and NF1 (chromosome 17) loci. Loss of heterozygosity at VHL locus was detected in 11/11 tumours with pathogenic VHL mutations and in 3/3 tumours from patients with clinical criteria of NF1.

Figure 3. Box plots illustrating preoperative levels of urinary (A) norepinephrine and (B) epinephrine stratified accordingly to genotype clusters.