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Telomerase Reverse Transcriptase Promoter Mutations in Primary Cutaneous Melanoma

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Telomerase Reverse Transcriptase Promoter Mutations in Primary Cutaneous Melanoma ARTICLE Received 13 Nov 2013 | Accepted 6 Feb 2014 | Published 26 Feb 2014 DOI: 10.1038/ncomms4401 Telomerase reverse transcriptase promoter mutations in primary cutaneous melanoma Barbara Heidenreich1,*, Eduardo Nagore2,3,*, P. Sivaramakrishna Rachakonda1, Zaida Garcia-Casado4, Celia Requena2, Victor Traves5,Ju¨rgen Becker6, Nadem Soufir7, Kari Hemminki1,8 & Rajiv Kumar1 We previously reported a disease segregating causal germline mutation in a melanoma family and recurrent somatic mutations in metastasized tumours from unrelated patients in the core promoter region of the telomerase reverse transcriptase (TERT) gene. Here we show that the TERT promoter mutations, besides causing an increased gene expression, associate with increased patient age, increased Breslow thickness and tumour ulceration in 287 primary melanomas. The mutations are more frequent at both intermittently and chronically sun-exposed sites than non-exposed sites and tend to co-occur with BRAF and CDKN2A alterations. The association with parameters generally connected with poor outcome, coupled with high recurrence and mechanistic relevance, raises the possibility of the eventual use of TERT promoter mutations in the disease management. 1 Division of Molecular Genetic Epidemiology, German Cancer Research Center, 69120 Heidelberg, Germany. 2 Department of Dermatology, Instituto Valenciano de Oncologia, Valencia 46009, Spain. 3 Universidad Catolica de Valencia, Valencia 46001, Spain. 4 Laboratory of Molecular Biology, Instituto Valenciano de Oncologia, Valencia 46009, Spain. 5 Department of Pathology, Instituto Valenciano de Oncologia, Valencia 46009, Spain. 6 Department of General Dermatology, Medical University of Graz, Graz 8036, Austria. 7 Departement de Genetique, Hoˆpital Bichat-Claude Bernard, APHP, Paris 75877, France. 8 Center for Primary Health Care Research, Lund University, Malmo¨, Lund 221 00, Sweden. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to R.K. (email: [email protected]). NATURE COMMUNICATIONS | 5:3401 | DOI: 10.1038/ncomms4401 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4401 utaneous melanoma, one of the most aggressive form of À 124C4T and À 146C4T accounted for 91 of 109 (83.5%) skin cancers owing to its high propensity to meta- mutations with the most common being the À 146C4T (51 of Cstasize and intrinsic drug resistance, accounts for the 109; 46.8%). The position at À 124 bp was altered in 36.7% (40/ majority of skin-cancer-related deaths1. Sun exposure coupled 109) of tumours by the C4T transitions and one tumour carried with characteristic phenotypes define the most important risk aC4A transversion. Four tumours (3.7%) carried CC4TT factors for developing melanoma2. Sequencing data have shown tandem mutation at À 124/ À 125 bp and seven tumours (6.4%) that of all cancer types, melanoma genomes are marked by the had a similar tandem mutation at the positions À 138/ À 139 bp. highest prevalence of somatic mutations and mutational pattern A previously described causal A4C alteration at À 57 bp in a depict a characteristic ‘ultraviolet-signature’, indicated by a high melanoma family was present as a somatic mutation in 3 of 109 rate of transitions at dipyrimidinic sites3–5. Frequently mutated (2.8%) tumours with mutations (Fig. 1). In addition, three genes are involved in various signalling cascades and cell cycle tumours with the À 146C4T mutation also carried a C4T base regulation. Besides the recurrent alterations in BRAF/NRAS, change at À 149 bp position. One tumour with the À 124C4T CDKN2A, PTEN and others, various sequencing projects have and another with the À 138/ À 139CC4TT mutations also car- also identified mutations in a number of additional genes ried the À 101C4T alteration. In total 15 tumours with muta- including GRIN2A, RAC1, BCL2L12, PPP6C and STK19 (refs 4–8). tions at À 146, À 124 and À 138/ À 139 bp also carried We previously described a highly penetrant, disease segregating additional alterations (Table 1). In addition, eight melanomas causal germline mutation in a melanoma family and recurrent carried exclusive mutations at various other positions within the somatic mutations in tumours from unrelated patients within the core TERT promoter, which included À 46C4T, À 58C4T, core promoter region of the telomerase reverse transcriptase À 144C4T, À 154CT, À 156C4T, À 176C4T, À 187C4T (TERT)gene9. The somatic mutations in the TERT promoter and À 242C4T. The observed alterations lead to putative simultaneously discovered in another study have been shown to be changes in transcription factor-binding sites including creation of frequent in a wide range of cancer types10–15. The familial a Ets/TCF-binding motif by À 156C4T (Table 1)22. From six germline A4C mutation at À 57 bp (from ATG start site; Chr patients, besides primary tumours, additionally, metastases were 5:1,295,161 hg19 co-ordinate) and the recurrent mutually exclusive also available. Out of those, five metastases were from five C4T somatic mutations at À 124 (1,295,228) bp and À 146 patients, whereas from one patient two metastases were available. (1,295,250) bp resulted in creation of E-twenty six/ternary complex Sequencing showed that primary and corresponding metastatic factor (Ets/TCF)-binding motifs with consequent tumour-specific (four locoregional cutaneous and one locoregional lymph node) increased TERT expression as shown by gene reporter assays9,10. tumours from five patients did not carry mutations in the TERT The frequency of the alterations in the TERT promoter exceeded promoter. In contrast, all three tumours from one patient, one that of any known gene mutation in melanoma. primary and two metastases, showed the À 124C4T and The widespread occurrence of the TERT promoter mutations in additional À 91C4T mutations. Within the primary tumour many cancers has now been confirmed. The mutations have been and one metastasis (locoregional cutaneous), the TERT promoter associated with an increased gene expression, decreased telomere mutations were heterozygous; in the second metastasis (soft tissue length and adverse forms of the disease and poor outcome11,16–21. from maxillary mucosa) both mutations were in the homozygous Here we screen 287 primary cutaneous melanomas to validate state indicating loss of the wild-type allele (Supplementary Fig. 1). the presence of mutations in primary melanoma and to find association, if any, with different parameters. We analyse the data for association with different phenotypic, epidemiologic and clinical features. The results show that activating TERT promoter mutations, which result in an increased gene expression, associate Table 1 | Types of TERT promoter mutations in primary with increased Breslow thickness and tumour ulceration. The melanomas. mutations are more frequent at sun-exposed than at non-exposed sites and tend to co-occur with BRAF and CDKN2A alterations. Position* Numberw % Effectz The findings, if confirmed, coupled with high recurrence and y mechanistic relevance, raise the possibility of the eventual use of À 146C4T5146.8 Ets/TCF 124C4T35|| 31.2 Ets/TCF TERT promoter mutations in disease management. À À 124C4A 1 0.92 Ets/TCF z À 124_125CC4TT 4 3.7 Ets/TCF Results À 138_139CC4TT 7# 6.4 Ets/TCF À 57A4C 3 2.8 Ets/TCF Tumour information and sequencing. In this study we screened ** primary tumours from 287 melanoma patients. Of 287 tumours, Others 8 7.3 À 46C4T BRCA1 227 were formalin-fixed paraffin-embedded (FFPE) tumour tis- ww À 58C4T ELK1 or AP sues for which corresponding mutational data for the BRAF and À 144C4T PLAG1 NRAS genes were available. Additional 60 tumours were available À 154C4T— as fresh frozen tissues, which were, besides TERT promoter À 156C4T Ets/TCF mutations, also sequenced for BRAF and NRAS. In addition, in À 176C4T E2F1 those tumours alterations at the CDKN2A locus were determined À 187C4T BRCA1 using methylation-sensitive multiplex ligation-dependent probe À 242C4T SP1 amplification (MS-MLPA). Detailed epidemiological data, phe- *Position refers to distance to ATG start site. w notype information about the patients and the available tumour Number of melanomas with mutations (n ¼ 109) in a set of primary melanomas (n ¼ 287). zPutative creation of a transcription factor binding site. y characteristics were available and are given in Supplementary Number of tumours that carried additional mutations: À 149C 4 T3Â ; À 126 C 4 T2Â ; Table 1 and Supplementary Data 1. À 120G 4 A and À 156C 4 T1Â ; À 156C 4 T1Â ; À 89_90CG 4 AA 1 Â . ||Number of tumours that carried additional mutations: À 91C 4 T1Â ; À 101C 4 T1Â ; À 105_106CC 4 TT 1 Â ; À 126 C 4 T1Â . z Number of tumours that carried additional mutations: À 149C 4 T1Â ; À 161C 4 T1Â . Mutations in the TERT promoter. Mutations in the TERT #One tumour carried an additional mutation: À 101C 4 T. promoter region were detected in 109 of 287 (37.9%) melanomas **These tumours carried only the enlisted mutations. wwDisruption of transcription binding site. (Table 1). The two mutually exclusive somatic changes 2 NATURE COMMUNICATIONS | 5:3401 | DOI: 10.1038/ncomms4401 | www.nature.com/naturecommunications & 2014 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4401 ARTICLE a Melanoma samples Wild-type TERT TERT promoter mutation BRAF BRAF mutation NRAS NRAS mutation No data b Ets/TCF Ets/TCF Ets/TCF Ets/TCF –146C>T –138_139CC>TT –124C>T –57A>C Figure 1 | Distribution of mutations in primary melanomas and representative sequences showing somatic mutations in the TERT promoter. (a) The distribution of mutations in the TERT promoter, BRAF and NRAS genes in 287 melanomas are indicated by different colours. (b) Representative sequences of the TERT promoter from different tumours with mutations; À 146C4T mutation shown in the left most panel followed by the À 138/ À 139CC4TT tandem mutation, À 124C4T and À 57A4C. The wild-type and mutated sequences are shown on the top, and the Ets/TCF consensus motifs created by the mutations are marked.
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