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1 Title Page

2 Telomerase reverse transcriptase promoter mutations in tumors originating

3 from the adrenal gland and extra-adrenal paraganglia

4 5 Thomas G. Papathomas 1, Lindsey Oudijk 1, Ellen C. Zwarthoff 1, Edward Post 1,

6 Floor A. Duijkers 2, Max M. van Noesel 2, Leo J. Hofland 3, Patrick J. Pollard 4,

7 Eamonn R. Maher 5, David F. Restuccia 1, Richard A. Feelders 3, Gaston J.H. Franssen 6, Henri J. Timmers 7, Stefan Sleijfer 8, Wouter W. de Herder 3, Ronald R. de

8

Krijger 1,9, Winand N.M. Dinjens 1, Esther Korpershoek 1

9 10 11 12 The Netherlands

1 Department of Pathology, Josephine Nefkens Institute, Erasmus MC, Rotterdam,

2 Department of Pediatric Oncology-Hematology, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands

13 14 15 3 Sector of Endocrinology, Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands 16 17 4 Cancer Biology and Metabolism Group, Edinburgh Cancer Research UK Centre, 18 Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh 19 EH4 2XR, UK 20 5 Department of Medical Genetics, University of Cambridge, Cambridge, UK 21 Department of Surgery, Erasmus MC, Rotterdam, The Netherlands 22 7 Department of Medicine, Division of Endocrinology, Radboud University Nijmegen

23 Medical Center, Nijmegen, The Netherlands

8 Department of Medical Oncology, Erasmus MC, Rotterdam, The Netherlands 24 25 Department of Pathology, Reinier de Graaf Hospital, Delft, The Netherlands

26

27 Corresponding author:

28 Esther Korpershoek, PhD

Erasmus MC-University Medical Center Rotterdam

29 30 Department of Pathology, Room Ae304

31 Dr Molewaterplein 50

32 3015GE, Rotterdam, The Netherlands

33 Email: e.korpershoek.1@erasmusmc.nl

34 Tel: +3110-7038289

Fax: +3110-7044762

35 36 37

Short title: TERT promoter mutations in ACCs and extra-adrenal PGLs

38 Keywords: TERT promoter mutations; telomerase reverse transcriptase; adrenocortical carcinomas; paragangliomas; neuroblastomas; SDH deficiency

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48 49 50

Abstract

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53 Hotspot mutations in the promoter of the telomerase reverse transcriptase (TERT) gene have been recently reported in human cancers and proposed as a novel mechanism of telomerase activation. To explore TERT promoter mutations in tumors originating from the adrenal gland and extra-adrenal paraganglia, a set of 253 tumors [38 adrenocortical carcinomas (ACCs), 127 pheochromocytomas (PCCs), 18 extra- adrenal paragangliomas (ea PGLs), 37 head and neck paragangliomas (HN PGLs) and 33 peripheral neuroblastic tumors] was selected along with 16 human neuroblastoma (NBL) and 2 ACC cell lines, to assess TERT promoter mutations by Sanger sequencing. All mutations detected were confirmed by a SNAPshot assay. Additionally, 36 gastrointestinal stromal tumors (GISTs) were added to explore an association between TERT promoter mutations and SDH-deficiency. TERT promoter mutations were found in 7 out of 289 tumors and in 3 out of 18 human cell lines; four C228T mutations in 38 ACCs (10.5%), two C228T mutations in 18 ea PGLs (11.1%), one C250T mutation in 36 GISTs (2.8%) and three C228T mutations in 16 human NBL cell lines (18.75%). No mutation was detected in PCCs, HN PGLs, neuroblastic tumors as well as in ACC cell lines. TERT promoter mutations preferentially occurred in SDH-deficient setting (P=0.01) being present in 3/47 (6.4%) SDH-deficient tumors

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69 70 versus 0/171 (0%) of SDH-intact tumors. We conclude that TERT promoter mutations 71 occur in ACCs and ea PGLs. Additionally, preliminary evidence indicates a potential

72 association with the acquisition of TERT promoter mutations in SDH-deficient tumors.

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Introduction

Telomerase is a ribonucleoprotein complex consisting of the telomerase reverse transcriptase (TERT) catalytic subunit and the telomerase RNA component (TERC). Telomerase is responsible for the addition of telomeric repeats at the end of linear eukaryotic chromosomes, thereby maintaining telomere length (Mocellin et al. 2013). Telomeres have two major functions in normal cells (Blasco & Hahn 2003, Mocellin et al. 2013). Firstly, they function to protect chromosome ends from being recognized as DNA double-strand breaks by the DNA repair machinery that can result in fusion of chromosome ends and gross chromosomal alterations. Secondly, telomeres prevent 3’DNA shortening during cell division that can trigger cellular senescence.

In cancer cells, which display uncontrolled proliferation, maintenance of telomeres is crucial to prevent senescence induction. As a consequence, tumor cells frequently show activation of mechanisms that protect telomeres and confer cellular immortalisation. In over 90% of cases, tumor cells display constitutive telomerase activation (Blasco & Hahn 2003). While there exists evidence that telomerase activity is regulated at various levels including epigenetic mechanisms (Castelo- Branco et al. 2013, Daniel et al. 2012), post-translational modification (Kang et al. 1999, Li et al. 1998) or nuclear translocation (Liu et al. 2001) of TERT, upregulation of TERT at the transcriptional level, via the inappropriate binding of transcription factors like c-myc to the core promoter region (Daniel et al. 2012, Greenberg et al. 1999, Wu et al. 1999), appears to be the primary mechanism yielding telomerase activation.

Consistent with this, recent studies in melanoma have demonstrated that activation of telomerase via transcriptional TERT upregulation can be caused by mutations in

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101 102 103 the core promoter region of TERT (Chr5) with 1,295,228 C>T and 1,295,250 C>T being the two most frequent mutation hotspots (Horn et al. 2013, Huang et al. 2013). Both mutations result in novel binding motifs for E-twenty-six (ETS) transcription 104 factors. This results in enhanced transcription of TERT, demonstrating a novel 105 mechanism contributing to telomerase activation in human cancer (Horn et al. 2013, 106 Huang et al. 2013). Likewise, other studies revealed TERT promoter mutations at varying site-specific frequencies in conjuctival melanoma, non-melanoma skin cancer, bladder cancer, central nervous system (CNS) tumors, thyroid tumors, soft tissue sarcomas, neuroblastomas, hepatocellular carcinomas, renal cell carcinomas (RCCs), mesotheliomas, oral cavity carcinomas, endometrial and ovarian clear cell carcinomas as well as gastrointestinal tract tumors (Arita et al. 2013, Brennan et al. 2013, Goutagny et al. 2013, Griewank et al. 2013, Hurst et al. 2013, Killela et al. 2014, Killela et al. 2013, Koelsche et al. 2014, Koelsche et al. 2013, Landa et al. 2013, Liu et al. 2013, Liu et al. 2013, Nault et al. 2013, Papadopoulos et al. 2013, Pópulo et al. 2014, Qu et al. 2013, Scott et al. 2013, Tallet et al. 2013, Vinagre et al. 2013, Wang et al. 2014, Wu et al. 2013, Zhao et al. 2013).

The prevalence of TERT promoter mutations in follicular cell-derived thyroid cancer indicated that these mutations may be important in endocrine tumorigenesis (Landa et al. 2013, Liu et al. 2013, Liu et al. 2013, Vinagre et al. 2013). Consistent with this, four independent research groups illustrated that more aggressive thyroid cancer subtypes were enriched for these mutations (Landa et al. 2013, Liu et al. 2013, Liu et al. 2013, Vinagre et al. 2013). With regard to adrenocortical carcinoma (ACC), a frequency of 12% was recently shown in a single cohort (Liu et al. 2014). In contrast, no mutations have been observed in parafollicular cell-originated medullary thyroid carcinoma (Killela et al. 2013, Liu et al. 2013, Liu et al. 2013, Vinagre et al.

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2013), while these seem to be extremely rare genetic events in pheochromocytomas (PCCs) and paragangliomas (PGLs) (Liu et al. 2014, Vinagre et al. 2013). In the current study, we examined the presence of these mutations in tumor types originating from the adrenal gland and extra-adrenal paraganglia including ACCs, PCCs, ea- and head and neck- (HN-) PGLs, as well as peripheral neuroblastic tumors. Given that TERT promoter mutations occur preferentially in specific genetic backgrounds in various tumors, any association with SDH-deficient status in PCCs, PGLs, and gastrointestinal stromal tumors (GISTs) was explored.

Subjects and Methods

Tumor tissue samples and cell lines

A total of 253 formalin-fixed and paraffin-embedded (FFPE) tumors were selected, including 38 ACC samples (Erasmus MC, Rotterdam, the Netherlands: 35 primary tumors, 2 recurrrences and 1 metastasis), 127 PCCs/ 18 extra-adrenal PGLs/ 37 HN PGLs (Erasmus MC, Rotterdam, the Netherlands: 167 cases; UMC St Radboud, Nijmegen, the Netherlands: 12 cases; and Birmingham, United Kingdom: 3 cases) and 33 peripheral neuroblastic tumors (Erasmus MC, Rotterdam, the Netherlands: 15 neuroblastomas, 8 ganglioneuroblastomas and 10 ganglioneuromas). Tumors with mutations in the SDH-x genes, such as SDHA, SDHB, SDHC, SDHD and SDHAF2, display loss of immunohistochemical staining for SDHB (Korpershoek et al. 2011, van Nederveen et al. 2009). Given that loss of SDHB expression reflects SDH- deficiency (Barletta & Hornick 2012), we will collectively use the term “SDH- deficient” for tumors displaying SDHB immunonegativity. As SDH-deficiency also defines a subset of GISTs similar to the SDH- related PCC/PGL subgroup, an

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additional series of 36 GISTs was examined to explore the relationship between TERT promoter mutations and SDH-deficiency in a non-endocrine tumor type.

All tumor samples were assessed anonymously according to the Proper Secondary Use of Human Tissue code established by the Dutch Federation of Medical Scientific Societies (http://www.federa.org). The Medical Ethical Committee of the Erasmus MC approved the study. Human neuroblastoma (NBL) cell lines: SJNB-12, SJ10 (SJNB-10), SK-N-BE, KCNR, LAN-1, LAN-5, N206, NGP-C4, NMB, TR-14, SH- EP-2/tet2, SJ1 (SJNB-1), SK-N-SH, SH-SY5Y, GI-ME-N and SK-N-AS as well as human ACC cell lines NCI-H295 [Source: ATCC (CRL-2128); method of authentication: STR profiling; passage number: P7] and SW13 [Source: ATCC (CCL- 105); method of authentication: STR profiling; passage number: P2] were also included in the analysis. The NBL cell lines have been originally obtained from the NCI and are molecularly well-characterized/ established in the field of NBL research (Thiele CJ 1998). These cell lines were grown from the original clones and used after less than 35 passages; all have been checked for molecular characteristics in our departmental research laboratory.

DNA isolation and TERT promoter mutation analysis

DNA isolation from tumors was carried out using standard procedures following manual microdissection of all tumor samples to ensure a greater than 80% neoplastic cell content. Standard polymerase chain reaction (PCR) was performed to amplify a 163 bp fragment of the TERT promoter region, covering all previously described mutations [C228T, CC229TT, CC242TT, and C250T, corresponding to nucleotide positions -124, -125, -138 and -145 from the translational start site (UCSC: chr5 nt 1295104)], using forward primer 5’-GTCCTGCCCCTTCACCTT-3’ and reverse

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primer 5’-CAGCGCTGCCTGAAACTC-3’. Subsequently, PCR products were used as templates for direct sequencing using the BigDye Terminator V3.1 cycle sequencing kit (Applied biosystems, Foster City, California). Products were analyzed on the ABI Prism 3130 Genetic Analyzer (Applied Biosystems).

TERT promoter mutations were confirmed by a SNAPshot assay using the ABI Prism SNAPshot Multiplex Kit (Applied Biosystems) as previously described (Allory et al. 2013). In brief, after the multiplex SNAPshot reaction, products were treated with shrimp alkaline phosphatase to remove excess dideoxynucleotide triphosphates, and subsequently were labeled and separated in a 25-minute run on 36-cm-long capillaries in an automatic sequencer (ABI Prism 3130 Genetic Analyzer, Applied Biosystems). GeneScan Analysis Software version 3.7 (Applied Biosystems) was used for data analysis. All experimental conditions are available on request. Probe sequences of the SNAPshot reaction are displayed in Supplementary Table S1.

SDHB/SDHA Immunohistochemistry (IHC), Mutation Screening and Loss of Heterozygosity (LOH) Analysis

SDH (IHC and/or mutation) status was known for 218 PCCs, extra-adrenal PGLs, HN PGLs, and GISTs. To investigate the SDH-status of the ACC samples included in the current study, these were arranged in a tissue microarray (TMA) format along with additional adrenocortical adenomas (ACAs), normal adrenal tissue and control tissue samples (38 ACC samples, 17 ACAs, 5 normal adrenal tissue and 12 control tissue samples) using an automated TMA constructor (ATA-27 Beecher Instruments, Sun Prairie, WI, USA) available at the Department of Pathology, Erasmus MC. For each tumoral case, representative areas were selected and marked on a haematoxylin and eosin (H&E)-stained slide. Accordingly, two tissue cores with a diameter of 1 mm

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were extracted from ‘donor’ block and brought into the ‘recipient’ paraffin block at predefined coordinates. SDHA and SDHB immunostainings were performed on 4-5 um TMA sections with mouse monoclonal Ab14715 antibody (Mitosciences, Abcam; 1:500 dilution) against SDHA and rabbit polyclonal HPA002868 antibody (Sigma- Aldrich Corp .; 1:400 dilution) against SDHB on an automatic Ventana Benchmark Ultra System (Ventana Medical Systems Inc. Tuscon, AZ, USA). If the internal control (granular staining in endothelial cells) was positive, slides were considered informative. From SDHB immunonegative/SDHA immunopositive ACCs, (i) the entire SDHA, SDHB, SDHC, SDHD, and SDHAF2 coding sequences were assessed at the germline and somatic level for mutations using an Ion AmpliSeq Custom Panel that was sequenced on the Ion Torrent Personal Genome Machine (PGM; Life Technologies) on 10 nanograms of FFPE tumor DNA according to the manufacturer’s protocols. In short, libraries were made using the Ion AmpliSeq 2.0 Library Kit. Template was prepared using the Ion OneTouch Template Kit and sequencing was performed with the Ion PGM Sequencing 200 Kit v2.0 on an Ion 316v2 chip. Data were analyzed with Torrent Suite Software, version 3.6.2 (Life Technologies). Annotation of variant calls was performed with Annovar (http://www.openbioinformatics.org/annovar/) (Wang et al. 2010) and facilitated using an in-house galaxy platform/server on which Annovar wrapper was installed (Blankenberg et al. 2010, Giardine et al. 2005, Goecks et al. 2010, Hiltemann et al. 2014). The variants with a read frequency higher than 10%, not known as common polymorphisms according to 1000G2012 April and ESP6500, nonsynonymous with a minimum of 5 forward/reverse variant read and 100 total depth read were retained as interesting ones (mutations) (sequences of all primers and probes are available upon request); and large intragenic deletions using multiplex ligation-dependent probe

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amplification (MLPA) assay with a commercially available kit (SALSA MLPA P226- B2; MRC Holland, Amsterdam, The Netherlands) and (ii) LOH analysis was performed for polymorphic microsatellite markers flanking the SDHB, SDHC, SDHD, and SDHAF2 genes as previously described (Papathomas et al. 2013).

RNA extraction and TERT Expression Analysis by Quantitative Real-Time PCR (qRT-PCR)

Total mRNA was extracted from human primary adrenal tissue (one ACC 234 harboring a TERT promoter mutation, two ACCs without TERT promoter mutation, one ACA and two normal adrenocortical tissue samples) or cell pellets (HEK and SW13 cell lines) using Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA) and the RNA containing supernatant purified using RNeasy spin colums (Qiagen Benelux B.V., Venlo, Netherlands). First strand cDNA synthesis was performed on 200ng total RNA using qScript cDNA Supermix (Quanta Biosciences, Gaithersburg, MD), followed by a TERT-specific and hypoxanthine phosphoribosyltransferase 1 (HPRT)-pre-amplification using PerfeCTa PreAmp SuperMix (Quanta Biosciences, Gaithersburg, MD). The PreAmp product was diluted and used to assess human telomerase expression in all samples by qRT-PCR in triplicate using TaqMan (Applied Biosystems, Foster City, CA) gene expression assays. TERT (TERT Hs00972656_m1) was measured relative to HPRT expression. The relative amount of RNA was calculated with the 2-delta delta CT method. Fold changes in gene expression were determined by comparing expression levels of tumor tissue or cell lines with normal adrenocortical tissue. No RNA was available to test the remaining tumors endowed with the C228T and C250T mutations.

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Statistical analysis

Statistical analysis was conducted with SPSS (IBM®SPPS Statistics, version 20) and performed on a series of 218 tumors (PCCs/PGLs/GISTs) of known SDH-status. The Fisher’s exact test was used to determine the relationships between the presence of a TERT promoter mutation and SDH-deficiency. Statistical differences were considered significant if P <0.05.

Results

Prevalence of TERT promoter mutations in various human tumors and cell lines

TERT promoter mutations were found in 7 out of 289 tumors investigated with C228T being the most frequent substitution. There were four C228T mutations in 38 ACCs (10.5%), two C228T mutations in 18 extra-adrenal PGLs (11.1%) and one C250T mutation in 36 GISTs (2.8%). Clinicopathological and genetic data of these patients are detailed in Table 1, while representative somatic TERT promoter mutations (C228T and C250T) detected both by Sanger sequencing and a SNAPshot assay are displayed in Figure 1. Six of 7 TERT promoter-mutated tumors were metastatic (Table 1). Although 3 of 4 mutation-positive ACCs were characterized by highly aggressive biologic behaviour, we could not perform proper survival analysis due to the limited number of these cases. Mutations were not detected in any of 127 PCCs, 37 HN PGLs, and 33 peripheral neuroblastic tumors. The TERT promoter mutation C228T was found in 3 out of 16 (18.8%) human NBL cell lines (SJNB-10, SJNB-12 and SK-N-BE), while no mutations were present in the 2 ACC cell lines (Supplementary Table S2).

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Enrichment of TERT promoter mutations in SDH-deficient tumors

Given that a subset of PCCs, PGLs, and GISTs is associated with germline SDH-x mutations and/or loss of SDHB immunoexpression (collectively known as SDH- deficient tumors) and 3 out of 47 (6.4%) SDH-deficient tumors harbored a TERT promoter mutation, we analyzed the relationship between the SDH-deficient status and the presence of TERT promoter mutations. It was shown that TERT promoter mutations occur preferentially in SDH-deficient tumors (6.4% vs 0%; p = 0.01).

Loss of SDHB expression in TERT promoter-mutated ACC

Of 55 adrenocortical tumor samples, one ACC harbouring a TERT C228T mutation was SDHB immunonegative/ SDHA immunopositive. SDHB/SDHA IHC was re- performed on whole-tissue sections in all four TERT promoter-mutated ACCs and accordingly confirmed the aforementioned finding. Mutational analysis did not reveal any pathogenic germline or somatic SDHB/C/D/AF2 mutations, while large intragenic SDHB, SDHD and SDHAF2 deletions were detected only at the somatic level. Being consistent with the latter, LOH analysis revealed loss of heterozygosity both at the SDHAF2 and SDHD loci and for a microsatellite marker telomeric to SDHB gene.

Role of TERT promoter mutation on gene expression

To determine if this mutation resulted in increased TERT expression, quantitative RT-PCR was performed on a single TERT promoter-mutated ACC for which frozen material was available. Significant TERT expression was detected in the promoter- mutated ACC, while the non-mutated ACCs demonstrated very low to negligible TERT expression similar to that detected in normal adrenocortical tissue as shown in

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Figure 2. TERT expression in the TERT promoter-mutated ACC was approximately half that of the control HEK and SW13 cell lines.

Discussion

Telomerase reverse transcriptase promoter mutations have recently been shown as a novel genetic mechanism underlying telomerase activation and present in diverse human tumors with the highest frequencies in bladder cancer, CNS tumors, melanomas, hepatocellular carcinomas and myxoid liposarcomas (Allory et al. 2013, Arita et al. 2013, Horn et al. 2013, Huang et al. 2013, Hurst et al. 2013, Killela et al. 2013, Koelsche et al. 2014, Landa et al. 2013, Liu et al. 2013, Nault et al. 2013, Papadopoulos et al. 2013, Tallet et al. 2013, Vinagre et al. 2013). In this study, we expand the spectrum of TERT promoter mutated tumors to ACC, extra-adrenal PGL and GIST, while adding TERT promoter mutations to other mechanisms of TERT mRNA upregulation in adrenocortical tumorigenesis (Else 2009, Else et al. 2008, Liu et al. 2014) consistent with previously reported associations in other tumor types (Arita et al. 2013, Nault et al. 2013, Tallet et al. 2013, Vinagre et al. 2013).

Interestingly, we found that two extra-adrenal PGLs of urinary bladder harboring TERT promoter mutations were SDH-deficient tumors. Other tumors that have been linked to SDH-deficiency are GISTs (Barletta & Hornick 2012). To further explore a potential association between the presence of these mutations and SDH-deficiency, a series of 36 GISTs was examined and subsequently revealed one SDHD-mutated GIST containing a TERT promoter mutation. This prompted us to examine the SDH- status of the TERT promoter-mutated ACCs. Despite the fact that this latter tumor type has never been associated with SDH-deficiency, we showed loss of SDHB

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expression in one of the aforementioned ACCs, but without any germline SDH-x pathogenic mutations or gross deletions detected. This finding further extends the spectrum of tumors displaying loss of SDHB and/or SDHA expression in the absence of causative SDH-x mutations, including a clinico-pathologically and biologically distinctive subset of KIT/PDGFRA wild-type GISTs (Barletta & Hornick 2012, Nannini et al. 2013), poorly and/or un-differentiated neuroblastomas (Feichtinger et al. 2013) and a clear cell RCC with sarcomatous dedifferentiation (Papathomas et al. 2013).

Although only a small subset of SDH-deficient extra-adrenal PGLs and GISTs harbored a TERT promoter mutation, the latter did occur exclusively in the SDH- deficient setting. As all SDH-deficient TERT promoter-mutated tumors were clinically aggressive, these observations may reflect that TERT promoter mutations can co- operate in SDH-deficient cells to support an enhanced tumor progression. Whether the latter could be attributed to telomerase-mediated extension of telomeres extending the lifespan of mutated clones, conferring them infinite proliferation potential as well as enabling the accumulation of additional genetic alterations and/or to other non- canonical functions interfering with extra-telomeric tumor promoting pathways 342 remains to be determined (Cao et al. 2002, Choi et al. 2008, Greider et al. 1985, Liu et al. 2013, Martinez & Blasco 2011, Mukherjee et al. 2011, Park et al. 2009, Parkinson et al. 2008, Stewart et al. 2002).

Likewise, a selective combinatorial genetic alteration pattern has been highlighted in various tumor types (Arita et al. 2013, Heidenreich et al. 2014, Horn et al. 2013, Killela et al. 2014, Killela et al. 2013, Landa et al. 2013, Liu et al. 2013, Pópulo et al. 2014, Tallet et al. 2013, Vinagre et al. 2013, Wu et al. 2013). In CNS tumors, TERT promoter mutations mostly occur in (i) tumors with EGFR amplification; (ii)

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IDH wild-type tumors; (iii) almost all tumors with concurrent total chromosome 1p and 19q loss and IDH1/2 mutations and (iv) IDH1/2-mutated tumors exhibiting oligodendroglial morphologies (Arita et al. 2013, Killela et al. 2014, Killela et al. 2013). Similar to the previously reported coexistence with BRAF activating mutations or with concomitant BRAF and CDKN2A alterations in melanomas (Heidenreich et al. 2014, Horn et al. 2013, Pópulo et al. 2014), two independent groups displayed a preferential occurrence of TERT promoter mutations in BRAF V600E mutation- positive papillary thyroid carcinomas (Liu et al. 2013, Vinagre et al. 2013), while Landa et al. (Landa et al. 2013) observed a significant co-occurrence of TERT mutations with BRAF and RAS mutations in poorly differentiated thyroid carcinomas and anaplastic thyroid carcinomas. In bladder cancer and mesotheliomas, TERT promoter mutations were frequently associated with inactivating mutations in the TP53/RB1 signaling pathway (Wu et al. 2013) and tumor suppressor CDKN2A gene inactivation respectively (Tallet et al. 2013), while a significant co-occurence with CTNNB1 activating mutations has been reported in hepatocellular carcinomas and adenomas with malignant transformation (Nault et al. 2013, Pilati et al. 2014).

In the present study, all TERT promoter-mutated tumors except one appeared to be metastatic (Table 1) being in accordance with previous studies demonstrating that these mutations are more highly prevalent in advanced forms of particular malignancies, including follicular cell-derived thyroid cancer, melanoma, and primary glioblastoma (Horn et al. 2013, Killela et al. 2013, Landa et al. 2013, Liu et al. 2013, Vinagre et al. 2013). By contrast, TERT promoter mutations occur as an early genetic event in bladder tumorigenesis (Allory et al. 2013, Hurst et al. 2013, Papadopoulos et al. 2013), meningiomas prone to malignant progression (Goutagny et al. 2013), while in CTNNB1-mutated hepatocellular adenomas associated with the

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last step of the adenoma-carcinoma transition (Nault et al. 2013, Pilati et al. 2014). In this context, BRAF V600E mutated papillary thyroid carcinomas, which are more aggressive than their BRAF wild-type counterparts (Liu et al. 2013), are preferentially enriched for TERT promoter mutations (Liu et al. 2013, Vinagre et al. 2013).

TERT promoter mutations seem to be present in neuroblastomas at low frequencies (~9%; 2 out of 22) (Killela et al. 2013). Neuroblastomas are characterised by high expression and/or amplification of NMYC, the neuronal equivalent of c-myc. A direct binding of NMYC to the TERT promoter has not been established. In the present study, TERT promoter mutations were not detected in any peripheral neuroblastic tumor being consistent with data stemming from a recent whole genome sequencing project for neuroblastomas (Molenaar et al. 2012) and similar observations concerning other pediatric embryonal tumors, such as a clinically distinct molecular subtype of medulloblastoma (Killela et al. 2013, Koelsche et al. 2013, Remke et al. 2013). Nevertheless, three human NBL cell lines harboured TERT promoter mutations indicating that lack in tumor samples could be attributed either to decreased sensitivity of the technique owing to presence of normal cells or to inclusion of other peripheral neuroblastic tumor types, such as ganglioneuroblastomas and/or ganglioneuromas.

In summary, this study demonstrates that TERT promoter mutations occur, albeit rarely, in tumors originating from the adrenal cortex (ACCs) and extra-adrenal paraganglia of urinary bladder. Their absence in PCCs and HN PGLs indicates that these seem unlikely to be critical genetic events in their development and/or progression. In addition, it provides preliminary evidence of a potential association with the acquisition of TERT promoter mutations in a subset of aggressive SDH- deficient tumors. Further studies are warranted to elucidate this connection and

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provide mechanistic insights into the effects of these gain-of-function mutations at the TERT promoter on SDH-x related tumorigenesis as well as their prognostic relevance in SDH-related tumors types.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was supported by the Seventh Framework Programme (FP7/2007-2013) under grant agreement nº 259735 (ENS@T-Cancer).

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Figure legends

Figure 1. Somatic TERT promoter mutations in ACC2 and G104 as detected by Sanger sequencing (upper panel) and confirmed by a SNAPshot assay (lower panel). Arrows in the upper panel indicate the C228T and C250T mutations as displayed in the sequencing chromatograms (from left to right), while arrows in the lower panel indicate the same mutations in the SNAPshot electropherograms.

Figure 2. Quantitative real-time TERT expression analysis in human HEK and SW13 cell lines, normal adrenocortical tissues (NATs), adrenocortical adenoma (ACA), and adrenocortical carcinomas (ACCs) with or without TERT promoter mutations.TERT expression was measured relative to the housekeeping HPRT gene with fold changes normalized to expression in human adrenocortical tissue for all samples.

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CC CC/T T CC

hTERT 1295228 C > T

G104

CCTCTCCG

hTERT 1295250 C > T

228

242

250

228

242

250

200

Relative TERT expression

100

王

0

HEK

SW13

TERT mutated ACC

TERT WT ACC

TERT

ACA

NAT

NAT

WT ACC

Table 1. Clinicopathological and genetic data of patients with TERT promoter-mutated tumors

Case No	Tumor Type	Anatomic Site	Sex	Age	TERT promoter mutation	SDH- deficient	Weiss score	Metastatic Disease/ Site	Follow-up/ Status
1	ACC	Adrenal gland	F	50	C228T	No	5	Yes/ liver	9 mo/ DOD
2	ACC	Adrenal gland	M	51	C228T	No	6	Yes/ liver, lung, bone	12 mo/ DOD
3	ACC	Adrenal gland	M	42	C228T	Yes	8	Yes/ liver, lung, LNs	2 mo/ DOD
4	ACC	Adrenal gland	F	58	C228T	No	7	None	105 mo/ AWED
5	ea PGL	Urinary bladder	M	46	C228T	Yes *	-	Yes/ LNs	NA
6	ea PGL	Urinary bladder	M	61	C228T	Yes **	-	Yes/ LNs	226 mo/AWED
7	GIST	Stomach	F	57	C250T	Yes ***	-	Yes/ liver	33 mo/DOD

* SDHB IHC -/ SDHA IHC - as previously published in Korpershoek et al. J Clin Endocrinol Metab 96 E1472-1476 (non-informative on mutational analysis due to poor DNA quality)

** SDHB IHC -/ SDHA + (SDHB c.292T>C p. Cys98Arg)

*** SDHB IHC -/ SDHA IHC + (SDHD c.416T>C p.Leu139Pro)

Abbreviations: ACC, adrenocortical carcinoma; AWED, Alive without evidence of disease; DOD, Dead of disease; ea PGL, extra-adrenal paraganglioma; GIST, gastrointestinal stromal tumor; LN, lymph nodes; NA, not available