Whole Exome Sequencing Uncovers Germline Variants of Cancer-Related Genes in Sporadic Pheochromocytoma

Hindawi International Journal of Genomics Volume 2018, Article ID 6582014, 9 pages https://doi.org/10.1155/2018/6582014 Research Article Whole Exome Sequencing Uncovers Germline Variants of Cancer-Related Genes in Sporadic Pheochromocytoma Milena Urbini ,1 Margherita Nannini ,2 Annalisa Astolfi ,1 Valentina Indio,1 Valentina Vicennati,3 Matilde De Luca,1 Giuseppe Tarantino,1 Federica Corso,2 Maristella Saponara,2 Lidia Gatto,2 Donatella Santini,4 Guido Di Dalmazi,3 Uberto Pagotto,3 Renato Pasquali,3 Andrea Pession,2 Guido Biasco,1,2 and Maria A. Pantaleo1,2 “Giorgio Prodi” Cancer Research Center, University of Bologna, Bologna, Italy Department of Specialized, Experimental and Diagnostic Medicine, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 3 Endocrinology Unit, Department of Medical and Surgical Sciences, Center for Applied Biomedical Research, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 4 Pathology Unit, S. Orsola-Malpighi Hospital, University of Bologna, Bologna, Italy 1 2 Correspondence should be addressed to Margherita Nannini; Received 6 February 2018; Revised 8 May 2018; Accepted 29 May 2018; Published 19 August 2018 Academic Editor: Atsushi Kurabayashi Copyright © 2018 Milena Urbini et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Pheochromocytomas (PCCs) show the highest degree of heritability in human neoplasms. However, despite the wide number of alterations until now reported in PCCs, it is likely that other susceptibility genes remain still unknown, especially for those PCCs not clearly syndromic. Methods. Whole exome sequencing of tumor DNA was performed on a set of twelve PCCs clinically defined as sporadic. Results. About 50% of PCCs examined had somatic mutations on the known susceptibility VHL, NF1, and RET genes. In addition to these driver events, mutations on SYNE1, ABCC10, and RAD54B genes were also detected. Moreover, extremely rare germline variants were present in half of the sporadic PCC samples analyzed, in particular variants of MAX and SAMD9L were detected in the germline of cases wild-type for mutations in the known susceptibility genes. Conclusions. Additional somatic passenger mutations can be associated with known susceptibility VHL, NF1, and RET genes in PCCs, and a wide number of germline variants with still unknown clinical significance can be detected in these patients. Therefore, many efforts should be aimed to better define the pathogenetic role of all these germline variants for discovering novel potential therapeutic targets for this disease still orphan of effective treatments. 1. Introduction Pheochromocytomas (PCCs) are rare tumors of the autonomic nervous system that arise from the chromaffin tissue of the adrenal medulla [1]. Most of PCCs are benign; however, approximately 10% of cases are malignant and can develop metastases either at the time of diagnosis or even later after several years, with a highly variable clinical course and a 5-year overall survival rate of 50% [2]. PCCs show the highest degree of heritability in human neoplasms and almost 40% of cases occur within heritable syndromes, including multiple endocrine neoplasia type 2 (MEN2), neurofibromatosis type 1 (NF1), von Hippel Lindau (VHL) disease, and hereditary paraganglioma and familial pheochromocytoma [3]. Germline mutations have been identified in more than 15 well-characterized genes, such as VHL, SDHB, SDHD, NF1, and RET [4]. Despite this well-known inherited basis of PCCs and paragangliomas (PGLs), during the past decades somatic mutations with variable frequency in many genes, including EPAS1 (HIF2α), RET, VHL, RAS, NF1, ATRX, and CSDE1 recurrent somatic copy number alterations and several fusion genes, involving MAML3, BRAF, NGFR, and NF1, have been progressively identified [5–16]. Recently a multiplatform 2 International Journal of Genomics Table 1: Patient characteristics. ID Sex Age Tumor size (cm) HIC characteristics PASS score N47 N49 F M 63 65 5.0 4.0 n.a. 3 N50 M 36 4.0 N51 M 46 1.5 N52 M 47 3.5 N53 F 58 5.4 N54 F 46 6.0 N55 F 30 6.0 N56 M 41 1.7 N57 M 35 8.0 N62 M 59 3.0 N63 M 26 7.0 Ki-67 2.4% Positive staining for synaptophysin. Ki-67 3.2% Positive staining for synaptophysin. S100 protein-positive sustentacular cells. Ki-67 2.2% Positive staining for chromogranin A and synaptophysin. S100 proteinpositive sustentacular cells. Negative staining for c-kit, EGFr, p53. Ki-67 0.5% Positive staining for chromogranin A and synaptophysin. S100 proteinpositive sustentacular cells. Negative staining for CD10. Ki-67 0.1% Ki-67 1.8% Positive staining for chromogranin A and synaptophysin. S100 proteinpositive sustentacular cells. Focal positive staining for CD10. Ki-67 0.4% Positive staining for chromogranin A and synaptophysin. S100 proteinpositive sustentacular cells. Ki-67 0.1% Positive staining for synaptophysin. S100 protein-positive sustentacular cells. Negative staining for calretinin. Ki-67 2.2% Positive staining for chromogranin A and synaptophysin. Few S100 proteinpositive sustentacular cells. Negative staining for calretinin and α-inhibin. Ki-67 5.7% Ki-67 1.4%. Positive staining for synaptophysin. S100 protein-positive sustentacular cells. Negative staining for calretinin, MEL-A, and α-inhibin. Ki-67 4% integrated analysis classified PCCs/PGLs into four clinically relevant molecular subtypes: a kinase signaling subtype, a pseudohypoxia subtype, a Wnt-altered subtype driven by MAML3 and CSDE1, and a cortical admixture subtype [16]. Therefore, given this molecular complexity of PCCs, it is likely that other alterations remain still unknown, especially for those PCCs not clearly syndromic. For this purpose, in the present study, we performed whole exome sequencing on a set of 12 clinically sporadic PCCs, with a family history negative for PCCs/PGLs. 2. Materials and Methods 2.1. Patients and Tumor Samples. Fresh tissue specimens of PCC from 12 patients with a family history negative for PCCs/PGLs were collected during the surgical operation, snap-frozen in liquid nitrogen, and stored at −80°C until analysis. Patient characteristics are listed in Table 1. Whole exome sequencing was performed on biological tumor samples on matched peripheral blood samples obtained from all patients. This study was approved by the local institutional ethical committee of S. Orsola-Malpighi hospital (approval number 95/2013/U/Tess). All patients provided written informed consent. 2.2. Whole Exome Sequencing. DNA was extracted from peripheral blood and fresh frozen tissue with DNA mini kit (Qiagen, Milan, Italy) following manufacturer’s instructions. Whole exome sequencing of tumor DNA was performed on HiScanSQ platform in accordance with Nextera Rapid Exome Enrichment protocol (Illumina, (...truncated)