Recurrent Mutations of Chromatin-Remodeling Genes and Kinase Receptors in Pheochromocytomas and Paragangliomas Rodrigo A

Published OnlineFirst December 23, 2015; DOI: 10.1158/1078-0432.CCR-15-1841 Biology of Human Tumors Clinical Cancer Research Recurrent Mutations of Chromatin-Remodeling Genes and Kinase Receptors in Pheochromocytomas and Paragangliomas Rodrigo A. Toledo1, Yuejuan Qin1, Zi-Ming Cheng1, Qing Gao1, Shintaro Iwata2, Gustavo M. Silva3, Manju L. Prasad4, I. Tolgay Ocal5, Sarika Rao6, Neil Aronin6, Marta Barontini7, Jan Bruder1, Robert L. Reddick8, Yidong Chen9, Ricardo C.T. Aguiar1,10, and Patricia L.M. Dahia1,11 Abstract Purpose: Pheochromocytomas and paragangliomas (PPGL) G34W mutation of the histone 3.3 gene, H3F3A. Furthermore, are genetically heterogeneous tumors of neural crest origin, but mutations in kinase genes were detected in samples from 15 the molecular basis of most PPGLs is unknown. patients (37%). Among those, a novel germline kinase domain Experimental Design: We performed exome or transcriptome mutation of MERTK detected in a patient with PPGL and medullary sequencing of 43 samples from 41 patients. A validation set of 136 thyroid carcinoma was found to activate signaling downstream of PPGLs was used for amplicon-specific resequencing. In addition, a this receptor. Recurrent germline and somatic mutations were also subset of these tumors was subjected to microarray-based tran- detected in MET, including a familial case and sporadic PPGLs. scription, protein expression, and histone methylation analysis by Importantly, in each of these three genes, mutations were also Western blotting or immunohistochemistry. In vitro analysis of detected in the validation group. In addition, a somatic oncogenic mutants was performed in cell lines. hotspot FGFR1 mutation was found in a sporadic tumor. Results: We detected mutations in chromatin-remodeling Conclusions: This study implicates chromatin-remodeling and genes, including histone-methyltransferases, histone-demethy- kinase variants as frequent genetic events in PPGLs, many of lases, and histones in 11 samples from 8 patients (20%). In which have no other known germline driver mutation. MERTK, particular, we characterized a new cancer syndrome involving MET, and H3F3A emerge as novel PPGL susceptibility genes. Clin PPGLs and giant cell tumors of bone (GCT) caused by a postzygotic Cancer Res; 22(9); 2301–10. Ó2015 AACR. Introduction sympathetic lineage cells of the adrenal medulla and paraganglia, respectively. More than 40% of these tumors are caused by a Pheochromocytomas and paragangliomas (PPGL) are catechol- dominant driver mutation in one of various susceptibility genes amine-secreting tumors of neural crest origin that arise from the involving a broad range of pathways (1). Remarkably, in more than one-third of the patients, the mutation is found in the germline and 1Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas. 2Division of Orthopedic Surgery, is transmitted in an autosomal dominant manner (1, 2). Chiba Cancer Center,Chiba, Japan. 3Department of Biology,Center for To explore new genetic events underlying familial and sporadic Genomics and Systems Biology, New York University, New York, New cases, we sequenced the exomes or transcriptomes of 43 PPGL York. 4Department of Pathology, Yale University, New Haven, Con- necticut. 5Department of Laboratory Medicine and Pathology, Mayo samples from 41 individuals (Supplementary Table S1 and Fig. 1). Clinic Arizona, Scottsdale, Arizona. 6Department of Medicine, Univer- Sequences were generated from 26 tumors, 13 germline samples, sity of Massachusetts Medical School, Worcester, Massachusetts. and 4 tumor–germline pairs. Three separate tumors were from the 7Center for Endocrinological Investigations (CEDIE), Buenos Aires, Argentina. 8Department of Pathology, University of Texas Health same patient. Germline samples were selected from individuals at Science Center at San Antonio, San Antonio, Texas. 9Department of high risk for hereditary PPGL, that is, family history of PPGLs, Biostatistics, University of Texas Health Science Center at San Antonio, disease onset before 30 years of age, and/or multiple tumors San Antonio,Texas. 10South Texas Veterans Health Care System, Audie Murphy VA Hospital, San Antonio, Texas. 11Cancer Therapy and (Supplementary Table S1). Seven patients (17%) had a history of Research Center (CTRC), University of Texas Health Science Center metastatic PPGLs. Here we report novel variants in genes coding at San Antonio, San Antonio, Texas. for tyrosine kinases and chromatin-remodeling proteins in these Note: Supplementary data for this article are available at Clinical Cancer samples. Research Online (http://clincancerres.aacrjournals.org/). Current address for R.A. Toledo: Spanish National Cancer Research Centre Materials and Methods (CNIO), Madrid, Spain. Patient samples Corresponding Author: Patricia L.M. Dahia, Department of Medicine, University One-hundred seventy-seven PPGLs, obtained after written of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MC 7880, informed consent or in an anonymized fashion after deidentifica- San Antonio, TX 78229. Phone: 210-567-4866; Fax: 210-567-1956; E-mail: tion, were collected through a tumor repository approved by the [email protected] University of Texas Health Science Center at San Antonio doi: 10.1158/1078-0432.CCR-15-1841 (UTHSCSA; San Antonio, TX) Institutional Review Board. A Ó2015 American Association for Cancer Research. summary of the main clinical features of this cohort is shown in www.aacrjournals.org 2301 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst December 23, 2015; DOI: 10.1158/1078-0432.CCR-15-1841 Toledo et al. and three unrelated PPGLs, and four sporadic GCTs were used to Translational Relevance quantify variant allele representation. A similar approach was Pheochromocytomas and paragangliomas are genetically used to detect the MET c.2416G>A; p.V806M variant in samples heterogeneous neuroendocrine tumors caused by inherited from three individuals with familial pheochromocytoma without mutations in 40% of the cases. Using exome or transcriptome a known driver mutation, including germline DNA from two sequencing, we identified novel recurrent germline, mosaic, or affected siblings and FFPE from a nonpheochromocytoma tissue somatic mutations in genes encoding chromatin regulators, section from their affected father, along with unrelated samples. including histone and histone modifiers, as well as kinase Primers spanning the two variants contained adaptors to allow for receptors, among which were MERTK, MET, and FGFR1. Some detection of the variant allele at ultra-deep sequencing using of these mutations, in histone 3.3, MERTK, and MET, were Illumina TruSeq Custom Amplicon with slight modification, as associated with co-occurring tumors or familial disease, sug- detailed in Supplementary Methods. The sequenced traces were gesting that they belong to previously unappreciated suscep- annotated using VarScan2 (7) and the specific variants of interest tibility syndromes. These new variants increase the proportion were detected at an average read depth of 67700x (Æ47969SD). of pheochromocytomas and paragangliomas with a known Samples with variant read counts below the threshold of quality genetic basis and broaden the spectrum of genes targeted in detection (below 1%) were considered negative. these tumors. Furthermore, our findings provide new markers for genetic risk assessment. Future studies should define the Sanger sequencing utility of these data in the development of targeted therapeutic Exons spanning the MERTK kinase domain, MET semaphorin, strategies for malignant or inoperable paragangliomas. transcription factor immunoglobin (TIG), juxtamembrane and kinase domains, H3F3A, and H3F3B exon 2 were sequenced in the relevant samples from the exome/transcriptome cohort and in the validation cohort of 136 PPGLs by Sanger sequencing (primers and PCR conditions available upon request). Sequencing was Supplementary Table S1. Forty-three samples from 41 individuals processed at Beckman Genomics and analyzed with Mutation n ¼ were used for whole exome ( 40) or transcriptome sequencing Surveyor (Softgenetics), as previously reported (4). The Mutation n ¼ ( 3). A separate cohort of 136 pheochromocytoma or para- Quantifier tool of Mutation Surveyor was used to measure fre- fi ganglioma tumor only samples was used for veri cation of some quency of the H3F3A c.103 G>T, p.G34W mutation, expressed as of the detected mutations (Supplementary Table S2). Clinical percentage of the mutant allele in the tumor samples, and calcu- features of one patient with associated PPGL and GCT has been lated as described in Supplementary Methods. reported elsewhere (3). Additional details of the samples used in the study are available in Supplementary Data. Differential gene expression analysis of PPGLs Gene expression data generated using the Affymetrix U1332.0 Whole exome and transcriptome sequencing and variant platform were previously reported (GEO accessions GSE2841 and detection GSE19987; ref. 8). Normalized data from the three tumors with Whole-exome sequencing was performed in 40 and RNA H3F3A G34W mutation and from 36 tumors without this muta- sequencing was performed in 3 PPGL samples (Supplementary tion were used for gene set enrichment analysis (GSEA), as Table S1). Of the 43, 26 were tumors, 13 were germline samples detailed in Supplementary Methods. DAVID Bioinformatics – from blood, and four were matched

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