TERT promoter mutations are rare in parathyroid tumors

in Endocrine-Related Cancer
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  • 1 Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska (CCK), Karolinska University Hospital, SE-171 76, Stockholm, Sweden
  • 2 Yale Endocrine Neoplasia Laboratory, Yale School of Medicine, 333 Cedar Street, FMB130A, Box 208062, New Haven, Connecticut, 06520, USA
  • 3 Department of Surgery, Yale School of Medicine, New Haven, Connecticut, 06520, USA
  • 4 Department of Pathology, Shandong University School of Medicine, Jinan, 250012, People's Republic China
  • 5 Department of Pathology, Yale School of Medicine, New Haven, Connecticut, 06520, USA
  • 6 Department of Medicine, Solna, Division of Haematology, Karolinska Institutet, Karolinska University Hospital, Centre for Molecular Medicine (CMM), SE-171 76, Stockholm, Sweden

Dear Editor,

The majority of parathyroid tumors are benign, and parathyroid carcinomas represent a diagnostic challenge with limited treatment options. Multiple endocrine neoplasia type 1 gene and cell division cycle 73 (CDC73) are major genes in parathyroid adenomas and carcinomas respectively. However, a large group of parathyroid tumors remain without defined genetic background.

Telomeres are chromosome-capping structures designed to protect DNA integrity. During mitosis, the telomere repeats are gradually shortened, which limits the number of possible cell divisions. In order for cancer cells to escape senescence, alterations in telomere maintenance are required. The telomerase reverse transcriptase (TERT) gene encodes the catalytic subunit of the telomerase complex and is largely responsible for maintaining the telomere length. Mutations in the promoter of the TERT gene may cause alterations in transcription factor binding sites, which leads to TERT overexpression and telomerase activation, as was first described for melanoma (Horn et al. 2013).

Recently, the common TERT promoter mutations -146C>T (also called C250T) and -124C>T (C228T) have been implicated in the development of several endocrine tumors. They have been identified in papillary, follicular, and anaplastic carcinomas of the thyroid, adrenocortical carcinomas, and paragangliomas. Interestingly, the mutations were found to be associated with telomerase activation, telomere length, malignant disease, patient age, and adverse prognosis (Liu et al. 2014). In addition to the potential prognostic value, the ongoing development of telomerase inhibitors for clinical use could provide additional treatment options for patients with aggressive endocrine cancer.

Previous studies have identified telomerase activity as well as TERT gene expression in parathyroid carcinomas but not in adenomas or normal parathyroid tissue (Falchetti et al. 1999, Kammori et al. 2003). Additionally, the telomerase inhibitor azidothymidine was able to reduce cell proliferation in parathyroid carcinoma cells in vitro (Falchetti et al. 2005).

In the present study, we sought to investigate the TERT promoter status in parathyroid tumors by direct Sanger sequencing. A total of 120 parathyroid tumors from 119 patients were studied, including 88 adenomas, 22 carcinomas, and ten atypical adenomas (Table 1). The carcinomas were classified according to the World Health Organization 2004 guidelines. The tumors were collected and analyzed at the Karolinska University Hospital, Stockholm, Sweden (n=90) and the Yale University School of Medicine, New Haven, CA, USA (n=30). Samples were obtained with informed patient consent, and the study of the tissue material was approved by the local ethical committees. Subsets of the parathyroid tumors studied at the Karolinska University Hospital have been previously published.

Table 1

TERT promoter sequence and expression in parathyroid tumors

ParameterSeriesTotal
DiagnosisKarolinska (n=90)Yale (n=30)(n=120)
TERT mutation -124C>T or -146C>T
 Adenomas0/620/260/88 (0%)
 Atypical adenomas0/100/10 (0%)
 Carcinomas0/180/40/22 (0%)
TERT variant -145C>T
 Adenomas1/62a0/261/88 (1%)
 Atypical adenomas1/10a1/10 (10%)
 Carcinomas0/180/40/22 (0%)
TERT mRNA expressionb
 Adenomas0/340/34 (0%)
 Atypical adenomas1/61/6 (17%)
 Carcinomas

–, no data; n, number of tumors analysed.

Both tumors with -145C>T were from the same patient.

relative to HeLa-cells.

Genomic DNA isolated from fresh frozen or paraffin-embedded tumor samples was used for amplification of the TERT promoter and sequencing as previously described (Liu et al. 2014). Sanger sequencing was carried out at the KIGene core facility at Karolinska Institutet for 90 cases and the Keck DNA Sequencing Facility, Yale University School of Medicine, for 30 cases. All samples displaying TERT promoter alterations or with ambiguous chromatograms were reanalyzed in the Clinical Molecular Pathology division at Karolinska University Hospital.

All 120 tumors exhibited the WT sequences at positions -124C and -146C (Table 1). A TERT promoter variant was identified in an adenoma (T1) and an atypical adenoma (T2) from a single patient. This heterozygous variant -145C>T was also detected in constitutional DNA from the patient (Fig. 1A). It has previously been reported in a pleomorphic xanthoastrocytoma (Koelsche et al. 2013). The patient, a 16-year-old male with unknown family history of the disease and lost to follow-up, carries a constitutional W45X mutation of CDC73 together with a second W30X mutation of CDC73 in T1 (Sulaiman et al. 2012).

Figure 1
Figure 1

Analysis of the TERT promoter. (A) Chromatograms showing the WT sequence and the heterozygous -145C>T variant. (B) Predicted transcription factor binding sites for -146C>T and -145C>T. The -146C>T mutation is predicted to lose the MAZ and gain the STAT4, c-ETS1, and Elk-1 motifs. The -145C>T variant is predicted to lose the MAZ and TFII-I motifs.

Citation: Endocrine-Related Cancer 22, 3; 10.1530/ERC-15-0121

The potential effect of the -145C>T variant on transcription factor motifs in the promoter was analyzed in silico using PROMO (Transfac database v8.3, dissimilarity margin ≤15%) together with the -146C>T mutation as a control. As a validation of the method, PROMO identified the putative Ets-1 and Elk-1 binding sites induced by the -146C>T mutation (Horn et al. 2013). Furthermore, PROMO predicted loss of the MAZ and TFII-I binding sites (Fig. 1B) for the -145C>T variant. This specific MAZ binding site has previously been identified in the TERT promoter, and the MAZ transcription factor has been suggested to suppress TERT expression (Su et al. 2007).

TERT expression analysis was undertaken using previously described methodology based on quantitative real-time PCR (Liu et al. 2014). In short, sample cDNA were run in quadruplicate for the Taqman gene expression assays for TERT (Hs00972656_m1, catalogue no. 4331182) and the endogenous control RPLP0 (Hs99999902_m1, catalogue no. 4331182) A HeLa cell line was used as a positive control and an arbitrary relative reference. Reactions with TERT Ct values of <35 in all replicates were classified as having detectable expression (as per the manufacturer's recommendations).

TERT expression was only identified in one of the six atypical adenomas and in none of the 34 adenomas (Table 1). In the TERT positive atypical adenoma (T2 from the patient carrying -145C>T), the TERT expression level was 220-fold higher than that in HeLa cells. The adenoma (T1) from the same patient showed no detectable expression.

The present study suggests that TERT promoter mutations are rare or absent in parathyroid tumors. The role of -145C>T in telomerase activation is ambiguous, and it is uncertain whether this represents an unusual single nucleotide variant or a mutation.

Previous studies have reported telomerase activity using telomeric repeat amplification protocol (TRAP) analysis in all analyzed parathyroid carcinomas (6/6) as well as in TERT mRNA expression (4/4) by RNA in situ (Falchetti et al. 1999, Kammori et al. 2003, Onoda et al. 2004). By contrast, telomerase activity was virtually absent in adenomas (0/47), hyperplasias (0/11), and normal parathyroid tissues (0/23) (Falchetti et al. 1999, Velin et al. 2001, Kammori et al. 2003, Onoda et al. 2004). These observations suggest that telomerase activation is necessary for parathyroid malignancy but not for the proliferation of benign lesions.

Given the absence of -124C>T and -146C>T, TERT promoter mutations should be considered a rare event in parathyroid tumors. The previously reported telomerase activation is therefore expected to be caused by other mechanisms, which could include copy number alterations, DNA methylation, or other genetic and regulatory events.

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 work was supported by the Swedish Cancer Foundation, the Swedish Research Council, the Cancer Research Foundations of Radiumhemmet, Karolinska Institutet, and Stockholm County Council.

References

  • Falchetti A, Becherini L, Martineti V, Morelli A, Benvenuti S, Picariello L, Gennari L, Lampugnani R, Bordi C & Brandi ML 1999 Telomerase repeat amplification protocol (TRAP): a new molecular marker for parathyroid carcinoma. Biochemical and Biophysical Research Communications 265 252255. (doi:10.1006/bbrc.1999.1320).

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  • Falchetti A, Franchi A, Bordi C, Mavilia C, Masi L, Cioppi F, Recenti R, Picariello L, Marini F & Del Monte F et al. 2005 Azidothymidine induces apoptosis and inhibits cell growth and telomerase activity of human parathyroid cancer cells in culture. Journal of Bone and Mineral Research 20 410418. (doi:10.1359/JBMR.041123).

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  • Horn S, Figl A, Rachakonda PS, Fischer C, Sucker A, Gast A, Kadel S, Moll I, Nagore E & Hemminki K et al. 2013 TERT promoter mutations in familial and sporadic melanoma. Science 339 959961. (doi:10.1126/science.1230062).

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  • Kammori M, Nakamura K, Ogawa T, Mafune K, Tatutomi Y, Obara T, Onoda N, Fujiwara M, Izumiyama-Shimomura N & Mori M et al. 2003 Demonstration of human telomerase reverse transcriptase (hTERT) in human parathyroid tumours by in situ hybridization with a new oligonucleotide probe. Clinical Endocrinology 58 4348. (doi:10.1046/j.1365-2265.2003.01642.x).

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    • Export Citation
  • Koelsche C, Sahm F, Capper D, Reuss D, Sturm D, Jones DT, Kool M, Northcott PA, Wiestler B & Bohmer K et al. 2013 Distribution of TERT promoter mutations in pediatric and adult tumors of the nervous system. Acta Neuropathologica 126 907915. (doi:10.1007/s00401-013-1195-5).

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  • Liu T, Wang N, Cao J, Sofiadis A, Dinets A, Zedenius J, Larsson C & Xu D 2014 The age- and shorter telomere-dependent TERT promoter mutation in follicular thyroid cell-derived carcinomas. Oncogene 33 49784984. (doi:10.1038/onc.2013.446).

    • Search Google Scholar
    • Export Citation
  • Onoda N, Ogisawa K, Ishikawa T, Takenaka C, Tahara H, Inaba M, Takashima T & Hirakawa K 2004 Telomerase activation and expression of its catalytic subunits in benign and malignant tumors of the parathyroid. Surgery Today 34 389393. (doi:10.1007/s00595-003-2729-6).

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    • Export Citation
  • Su JM, Lai XM, Lan KH, Li CP, Chao Y, Yen SH, Chang FY, Lee SD & Lee WP 2007 X protein of hepatitis B virus functions as a transcriptional corepressor on the human telomerase promoter. Hepatology 46 402413. (doi:10.1002/hep.21675).

    • Search Google Scholar
    • Export Citation
  • Sulaiman L, Haglund F, Hashemi J, Obara T, Nordenström J, Larsson C & Juhlin CC 2012 Genome-wide and locus specific alterations in CDC73/HRPT2-mutated parathyroid tumors. PLoS ONE 7 e46325. (doi:10.1371/journal.pone.0046325).

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    • Export Citation
  • Velin AK, Herder A, Johansson KJ, Trulsson LM & Smeds S 2001 Telomerase is not activated in human hyperplastic and adenomatous parathyroid tissue. European Journal of Endocrinology 145 161164. (doi:10.1530/eje.0.1450161).

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*

(F Haglund and C C Juhlin contributed equally to this work)

 

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    Analysis of the TERT promoter. (A) Chromatograms showing the WT sequence and the heterozygous -145C>T variant. (B) Predicted transcription factor binding sites for -146C>T and -145C>T. The -146C>T mutation is predicted to lose the MAZ and gain the STAT4, c-ETS1, and Elk-1 motifs. The -145C>T variant is predicted to lose the MAZ and TFII-I motifs.

  • Falchetti A, Becherini L, Martineti V, Morelli A, Benvenuti S, Picariello L, Gennari L, Lampugnani R, Bordi C & Brandi ML 1999 Telomerase repeat amplification protocol (TRAP): a new molecular marker for parathyroid carcinoma. Biochemical and Biophysical Research Communications 265 252255. (doi:10.1006/bbrc.1999.1320).

    • Search Google Scholar
    • Export Citation
  • Falchetti A, Franchi A, Bordi C, Mavilia C, Masi L, Cioppi F, Recenti R, Picariello L, Marini F & Del Monte F et al. 2005 Azidothymidine induces apoptosis and inhibits cell growth and telomerase activity of human parathyroid cancer cells in culture. Journal of Bone and Mineral Research 20 410418. (doi:10.1359/JBMR.041123).

    • Search Google Scholar
    • Export Citation
  • Horn S, Figl A, Rachakonda PS, Fischer C, Sucker A, Gast A, Kadel S, Moll I, Nagore E & Hemminki K et al. 2013 TERT promoter mutations in familial and sporadic melanoma. Science 339 959961. (doi:10.1126/science.1230062).

    • Search Google Scholar
    • Export Citation
  • Kammori M, Nakamura K, Ogawa T, Mafune K, Tatutomi Y, Obara T, Onoda N, Fujiwara M, Izumiyama-Shimomura N & Mori M et al. 2003 Demonstration of human telomerase reverse transcriptase (hTERT) in human parathyroid tumours by in situ hybridization with a new oligonucleotide probe. Clinical Endocrinology 58 4348. (doi:10.1046/j.1365-2265.2003.01642.x).

    • Search Google Scholar
    • Export Citation
  • Koelsche C, Sahm F, Capper D, Reuss D, Sturm D, Jones DT, Kool M, Northcott PA, Wiestler B & Bohmer K et al. 2013 Distribution of TERT promoter mutations in pediatric and adult tumors of the nervous system. Acta Neuropathologica 126 907915. (doi:10.1007/s00401-013-1195-5).

    • Search Google Scholar
    • Export Citation
  • Liu T, Wang N, Cao J, Sofiadis A, Dinets A, Zedenius J, Larsson C & Xu D 2014 The age- and shorter telomere-dependent TERT promoter mutation in follicular thyroid cell-derived carcinomas. Oncogene 33 49784984. (doi:10.1038/onc.2013.446).

    • Search Google Scholar
    • Export Citation
  • Onoda N, Ogisawa K, Ishikawa T, Takenaka C, Tahara H, Inaba M, Takashima T & Hirakawa K 2004 Telomerase activation and expression of its catalytic subunits in benign and malignant tumors of the parathyroid. Surgery Today 34 389393. (doi:10.1007/s00595-003-2729-6).

    • Search Google Scholar
    • Export Citation
  • Su JM, Lai XM, Lan KH, Li CP, Chao Y, Yen SH, Chang FY, Lee SD & Lee WP 2007 X protein of hepatitis B virus functions as a transcriptional corepressor on the human telomerase promoter. Hepatology 46 402413. (doi:10.1002/hep.21675).

    • Search Google Scholar
    • Export Citation
  • Sulaiman L, Haglund F, Hashemi J, Obara T, Nordenström J, Larsson C & Juhlin CC 2012 Genome-wide and locus specific alterations in CDC73/HRPT2-mutated parathyroid tumors. PLoS ONE 7 e46325. (doi:10.1371/journal.pone.0046325).

    • Search Google Scholar
    • Export Citation
  • Velin AK, Herder A, Johansson KJ, Trulsson LM & Smeds S 2001 Telomerase is not activated in human hyperplastic and adenomatous parathyroid tissue. European Journal of Endocrinology 145 161164. (doi:10.1530/eje.0.1450161).

    • Search Google Scholar
    • Export Citation