IJC International Journal of Cancer

TERT promoter mutations in clear cell renal cell carcinoma €rje Ljungberg2, Ismail Hosen1, P. Sivaramakrishna Rachakonda1, Barbara Heidenreich1, Raviprakash T. Sitaram2, Bo 3 1,4 1 €ran Roos , Kari Hemminki and Rajiv Kumar Go 1

Division of Molecular Genetic Epidemiology, German Cancer Research Center, Heidelberg, Germany Department of Surgical and Perioperative Sciences, Urology and Andrology, Umea˚ University, Umea˚, Sweden 3 Department of Medical Biosciences, Umea˚ University, Umea˚, Sweden 4 €, Sweden Center for Primary Health Care Research, Lund University, Malmo 2

The human telomerase reverse transcriptase (TERT) gene encodes the catalytic subunit of telomerase, a ribonucleoprotein complex that maintains genomic integrity through de novo synthesis of telomere repeat units at chromosomal ends.1 Activation of telomerase is dependent on a number of factors in which the transcriptional regulation of the TERT gene constitutes a rate limiting step. The TERT promoter harbors binding sites for a number of transcriptional activators and repressors and is considered to be the most important regulatory element for telomerase expression.2 Since initial discovery, activating somatic mutations in the promoter region in the TERT gene have been reported in many cancers.3, 4 The promoter mutations result in creation of

Key words: TERT promoter mutations, telomere length, mutations, survival Abbreviations: ccRCC: clear cell renal cell carcinoma; ETS: E-twenty Six; HR: hazard ratio; OR: odds ratio; RTL: relative telomere length; TERT: telomerase reverse transcriptase Additional Supporting Information may be found in the online version of this article. DOI: 10.1002/ijc.29279 History: Received 1 Sep 2014; Accepted 7 Oct 2014; Online 21 Oct 2014 Correspondence to: Rajiv Kumar, Division of Molecular Genetic Epidemiology, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany, Tel.: 149-62-21-42-18-06, Fax: 149-62-21–42-18-10, E-mail: [email protected]

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binding sites for the E-twenty Six (ETS)/ternary complex factors (TCFs) transcription factors located mainly at two residues at 2124C > T (G > A) and 2146C > T (G > A) from the ATG start site in the TERT promoter. The mutations in the TERT promoter result in increased TERT expression and have been shown to be associated with more advanced forms of malignant diseases. In primary cutaneous melanoma the mutations were associated with increased patient age, increased tumor thickness and tumor ulceration.5 In bladder cancer and gliomas, the mutations were associated with risk of tumor recurrence and poor survival.6,7 In thyroid cancer, the TERT promoter mutations were more frequent in advanced tumors.4 In follicular thyroid cell-derived carcinomas, the TERT promoter mutations have been shown to be associated with increased age and decreased telomere length.8 Renal cell carcinoma (RCC) includes different distinct sub-types that differ in genetic aberrations, histology, clinical course and responses to therapy.9 Clear cell renal carcinoma (ccRCC) is the most frequent renal tumor subtype, comprising around 80% of the RCCs. While an earlier study based on a small number of different RCCs found no TERT promoter mutations, a subsequent communication reported TERT promoter mutations at a low frequency.10,11 In order to establish the frequency of TERT promoter mutations, we screened DNA from 188 patients with tumors of different stages and grades, measured relative telomere length (RTL) and analyzed data for the effect of mutations on diseasespecific patient survival.

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We screened promoter region of the telomerase reverse transcriptase (TERT) for activating somatic mutations in 188 tumors from patients with clear cell renal cell carcinoma (ccRCC). Twelve tumors (6.4%) carried a mutation within the core promoter region of the gene. The mutations were less frequent in high grade tumors compared to low grade tumors [odds ratio (OR) 5 0.15, 95% confidence interval (CI) 5 0.03–0.72, p 5 0.02]. Multivariate analysis for cause specific survival showed statistically significant poor outcome in patients with TERT promoter mutations [hazard ratio (HR) 5 2.90, 95% CI 5 1.13–7.39, p 5 0.03]. A common polymorphism (rs2853669) within the locus seemed to act as a modifier of the effect of the mutations on patient survival as the noncarriers of the variant allele with the TERT promoter mutations showed worst survival (HR 5 3.34, 95% CI 5 1.24–8.98, p 5 0.02). We also measured relative telomere length (RTL) in tumors and difference between tumors with and without the TERT promoter mutations was not statistically significant. Similarly, no difference in patient survival based on RTL in tumors was observed. Our study showed a relatively low frequency of TERT promoter mutations in ccRCC. Nevertheless, patients with the mutations, particularly in the absence of the rs2853669 variant showed the worst disease-specific survival. Thus, it is possible that the TERT promoter mutations define a small subset of tumors with an aggressive behavior.

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TERT promoter mutations

What’s new? The human telomerase reverse transcriptase (TERT) gene encodes the catalytic subunit of telomerase, a ribonucleoprotein complex that maintains genomic integrity. Activating somatic mutations in the promoter region of the TERT gene have been reported in many cancers. Here, the authors describe new TERT promoter mutations in clear cell renal cell carcinoma. Although present only in a proportion of the tumors, the TERT promoter mutations were independently associated with poor patient survival. The effect was enhanced by a common polymorphism within the core TERT promoter. The TERT promoter mutations may thus define a small subset of tumors with an aggressive behavior.

Material and Methods Study population

A total of 188 patients, 100 men and 88 women, mean age 65 (range 32–87 years), with different grades and stages of ccRCC were enrolled in this study and DNA was extracted from the tumor tissues using a standard protocol. There were 99 patients with Stage I and II tumors, 39 with Stage III and 50 patients with Stage IV tumors. Mutation detection

The core TERT promoter region (from the position 165 to 2278 bp from ATG start site) was amplified by PCR and mutations were screened by Sanger sequencing. Each PCR was carried out with 10 ng DNA as a template in a 10-lL volume containing 0.15 lM each primer (Supporting Information Table S1 for primer sequences), 0.11 mM dNTPs, 2.0 mM MgCl2, 50 mM KCl and 10% (vol/vol) glycerol. The PCR product was purified using ExoSapIT (Amersham Biosciences, Uppsala, Sweden) and sequencing reaction was carried out in a 10-lL volume using forward and reverse primers separately using a dideoxy terminator kit (Big Dye; Applied Biosystems, Foster City, CA, USA). Purified sequencing products were electrophored in a capillary sequencer (ABI prism 3130XL Genetic Analyzer; Applied Biosystems, Foster City, CA, USA). The sequences generated were analyzed in Geneious (www.geneious.com).

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Telomere length measurement

RTL was measured using a previously reported multiplex quantitative PCR assay.12 Briefly, the reactions were performed in triplicates in an optical 384-well reaction plate in a 10-mL reaction volume using 2 mL of 53 HOT FIREPol Probe qPCR Mix Plus with ROX (Solis BioDyne, Tartu, Estonia), 1.5 mM of Syto 9 (Invitrogen, Carlsbad, CA, USA) and 5–10 ng of genomic DNA. Reactions without any genomic DNA were included as negative controls. Four primers (Supporting Information Table S1) were used in each reaction to amplify telomere sequences (telg at 200 nM and telc at 400 nM) and albumin gene (albugcr2 at 200 nM and albdgcr2 at 400 nM). The real-time PCR experiments were performed on the sequence detection system (Applied Biosystems Viia-7) using two simultaneous programs to acquire the respective Ct values for telomere sequences and albumin (control) gene. The

conditions for telomere sequences were 95 C/15 min, 2 cycles of 95 C/20 s and 49 C/1 min, followed by 25 cycles of 85 C/ 20 s with signal acquisition at 59 C/30 s. Thermal conditions for albumin gene were 35 cycles of 95 C/15 s, 85 C/30 s, with signal acquisition at 84 C/30 s. The specificity of all amplifications was determined by melting curve analysis done at default settings (95 C/15 s, 60 C/1 min with continuous signal acquisition at 0.05 C/s ramping, 95 C/15 s). Seven concentrations of a reference DNA sample (genomic DNA pooled from ten healthy individuals) were included in triplicates in a twofold serial dilution (from 20 ng to 0.3 ng) to generate standard curves for telomere (T) and albumin (S) PCR products, respectively. The quality control was performed using the Applied Biosystems Viia-7 software. The standard curve was used to quantify the telomere and albumin sequences based on the respective Ct values and the obtained triplicate values were averaged. The RTLs was expressed as the ratio between T/S. Inter-assay variation and intra-assay variation was determined by duplicating the reference DNA for all the dilutions in all the assays performed.

Statistical analysis

The associations between the TERT promoter mutations and other parameters were determined by chi-square test. Student’s t-test was used to analyze difference in patient age at diagnosis between the patients with and without the TERT promoter mutations. A difference was considered statistically significant if p value was  0.05. Odds ratios (ORs) were calculated to assess the size of the effect of different parameters on the occurrence of the TERT promoter mutations. The associations between the age at diagnosis, sex, tumor stage, tumor grade, lymph node, metastasis, TERT promoter mutations and cause specific survival were determined separately using the log rank test. The cumulative curves for the survival outcome in patients with and without the TERT promoter mutations were drawn using the Kaplan-Meier method. The differences between the curves were analyzed with the log rank test. A Cox proportional hazard regression model was used to determine the association between the TERT promoter mutations and cause-specific patient survival. The model included the age at diagnosis, tumor stage, tumor grade, lymph node and metastasis. Box-plots were drawn to show distribution of RTL in tumors with and without mutations and differences in length between the two groups were C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

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Table 1. TERT promoter mutations in patients with ccRCC in relation to different subcategories TERT promoter mutation status Characteristics

Mutated

Wild type

Number of tumor analyzed

12

176

OR

95% CI

Age at diagnosis (median age and range in years)

66.5 (51–79)

66 (32–87)

Women

6

82

1.00

Reference

Men

6

94

0.87

0.27- 2.81

Low grade (1 1 2)

9

71

1.00

Reference

High grade (3 1 4)

2

105

0.15

0.03–0.72

T1 (T1a 1 T1b; tumor size < 7 cm)

6

67

1.00

Reference

(T2 1 T3 1 T4; tumor size > 7 cm)

6

109

0.61

0.19–1.98

Nonmetastatic (M0)

8

129

1.00

Reference

Metastatic (M1)

4

45

1.43

0.41–4.99

Stages I 1 II

7

92

1.00

Reference

Stages III 1 IV

5

84

0.78

0.24–2.56

Alive (or dead of other disease)

6

104

1.00

Reference

Dead of disease

6

72

1.44

0.45–4.66

Carrier

2

79

1.00

Reference

Noncarrier

10

97

4.07

0.87–19.13

p-value 0.911

Gender

0.82

Tumor grade

0.02

Tumor stage (T)

0.42

Tumor metastasis (M)

0.57

Group stage (based on TNM status)

0.68

Death

0.54

rs2853669 carrier status

0.08

analyzed by t-test. The association with patient survival was determined by Cox regression model using RTL as a continuous variable and the model included age at diagnosis, tumor stage, tumor grade, lymph node, metastasis and the TERT promoter mutations.

Results The study included tumors from 188 patients with ccRCC with different clinical characteristics (Supporting Information Table S2). Twelve of 188 (6.4%) tumors carried mutations within the core promoter region of the TERT gene. Nine tumors carried the 2124 C > T mutation, one tumor each carried 2124 C > A, 2146 C > T and 257A > C mutations (Supporting Information Table S3). The screened TERT promoter region included the rs2853669 polymorphism at 2245 bp position. The minor allele frequency for the polymorphism in this set of patients was 0.25. The mutations in the TERT promoter were not associated with the patient age at diagnosis or sex (Table 1). The frequency of mutations was significantly lower in tumors with Grades 3 and 4 than Grades 1 and 2. A total of 9 of 12 tumors that carried mutations had histological Grades 1 and C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

2 compared to two tumors carrying TERT mutations, which were Grades 3 and 4 [OR 5 0.15, 95% confidence interval (CI) 5 0.03–0.72, p 5 0.02]. The difference between low and high stage tumors was not statistically significant or between the tumors that did or did not metastasize. The TERT mutations were evenly distributed in patients within different TNM based stages. Of the 99 patients in the lower stage (Stages I and II), 7 carried TERT mutations, while 5 of 89 patients in the more aggressive stages (Stages III and IV) had mutations (OR 5 0.78, 95% CI 5 0.24–2.56, p 5 0.68). The survival of the patients was independently associated with age at diagnosis, tumor grade, tumor stage, lymph node status and presence of metastasis (data not shown). The median survival for the patients with TERT mutations was 14.3 years compared to 18.6 years for the patients without TERT mutations; the difference, however, was not statistically significant (p 5 0.78; Supporting Information Fig. S1). A Cox proportional model, which included age at diagnosis, tumor grade, tumor stage, lymph node and metastasis, showed that patients with TERT promoter mutations were at risk of poor survival (HR 5 2.90, 95% CI 5 1.13–7.39, p 5 0.03). Stratified analysis based on the carrier status of the rs2853669

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All other p-values were determined using the chi-square test and considered statistically significant if < 0.05. Bold type OR is statistically significant. 1 p values were determined using the t-test and considered statistically significant if < 0.05.

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TERT promoter mutations

Table 2. Effect of the TERT promoter mutations on patient survival by Cox regression analysis Survival analysis

Number of patients

Dead

Hazard ratio (HR)

95% CI

Without mutation

176

72

1.00

Reference

With mutation

12

6

2.90

1.13–7.39

p value

1

All patients

0.03

Stratification by rs2853669 Status1 No mutations and noncarriers

97

38

1.00

Reference

TERT mutation and noncarriers

10

6

3.34

1.24–8.98

0.02

TERT mutation and carriers

2

0







No mutations and carriers

79

34

1.17

0.69–1.98

0.57

188

78

0.67

0.39–1.16

2

Telomere Length

1

0.15 2

Bold HR values are statistically significant. HR adjusted for age at diagnosis, tumor grade, tumor stage, lymph node and metastasis. Telomere length was considered as a continuous variable and HR adjusted for TERT mutations, age at diagnosis, tumor grade, tumor stage, lymph node and metastasis.

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Figure 1. Association of TERT promoter mutations with telomere length. Wt: wild type; Mut: mutation.

polymorphism showed that the median survival in the noncarrier patients with TERT mutations was 4.1 years compared to the 18.6 years for the patients without mutations and carried variant allele of the polymorphism. The effect, however, was not statistically significant (log rank test p 5 0.50; Supporting Information Fig. S2). On the other hand, a multivariate analysis showed that those patients were at statistically significant increased risk of death with HR of 3.34 (95% CI 5 1.24–8.98, p 5 0.02, Table 2). The median RTL in tumors with and without TERT promoter mutations was 0.74 (range 5 0.48–1.61) and 0.78 (range 5 0.09–2.33), respectively (Fig. 1; p 5 0.75). Association between RTL and cause specific patient survival was also not statistically significant (HR 5 0.67, 95% CI 5 0.39–1.16, p 5 0.15).

Discussion Inherited forms of RCCs are associated with a number of genes that include von Hippel-Lindau (VHL), mesenchymalepithelial transition factor (MET) gene, folliculin (FLCN)

gene, fumarate hydratase gene and others.13–15 Somatic alterations in the VHL gene alterations in kidney cancer have a potential for use as prognostic and predictive markers.16 Other somatic mutations in kidney cancers have been documented in genes involved in histone modification, including SET domain containing 2 (SETD2), Jumonji/ARID domaincontaining protein 1C (JARID1C), as well as mutations in the histone H3 lysine 27 demethylase, and SWI/SNF chromatin remodeling complex gene, PBRM1.17,18 In this study on ccRCC tumors, we observed that though present in tumors from only a proportion of patients, the multivariate analysis indicated that the TERT promoter mutations, in agreement with previous observations in different cancer types, can define poor cause-specific patient survival. Like most cancers, RCCs exhibit widespread telomerase activation, and a close correlation between TERT expression and telomerase activity is well documented.19 In RCCs increased telomerase activity have been correlated with the expression of full length TERT compared to the alternatively spliced variants of TERT in normal tissues.19 In this study we found the TERT promoter mutations in relatively low proportion of ccRCC tumors, mainly at 2124 bp from ATG start site, which generate binding motifs for the Ets transcription factors. In ccRCC inactivation of VHL has been reported to be associated with accumulation of hypoxic inducible factors (HIFs); increased level of HIFs enhances Ets expression.20 ETS overexpression along with TERT promoter mutations are expected to exert positive effect on the malignant tissue specific telomerase activation in ccRCCs. Recently, a study has reported significantly higher levels of TERT mRNA in TERT promoter mutation-carrying ccRCC tumors than in those lacking the mutation.11 In this study, we observed that the TERT promoter mutations in ccRCC were associated with statistically significant poor disease-specific survival in a multivariate model. That finding is in line with earlier reports on bladder cancer, gliomas and thyroid cancer where the mutations associate with poor outcome.4,6 Earlier, we showed a modification of the C 2014 UICC Int. J. Cancer: 00, 00–00 (2014) V

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effect of the TERT promoter mutations on patient survival and disease recurrence in bladder cancer by a common polymorphism within the sequence.6 Even within a limited number of tumors with mutations, we observed that patient without variant allele of the polymorphism and the mutations in tumor showed worst survival. Experimental data based on promoter assays and gene expression supported the observed effects.6 While somatic mutations result in creation Ets/TCF binding sites, the variant allele of the rs2853669 polymorphism results in disruption of a preexisting noncanonical Ets2 binding site adjacent to an E-box, in the proximal region of the TERT promoter. This functional polymorphism has been shown to modulate the risk of cancer by regulating telomerase activity and telomere length in a tissue specific manner.21 We did not find any statistical significant association between RTL in tumors and patient survival. And in contrast to an earlier report on thyroid cancer, we did not observe any difference in RTL based on the TERT promoter mutational status.8 Presumed acquisition of the somatic TERT promoter mutations at the point of telomere crisis in the course of cellular transformation has been cited as the reason for the association with telomere length. It is thus possible in this study relatively low number of cases precluded any such observation.

Overall, our result shows that the TERT promoter mutations do occur in ccRCC but are less frequent than in many other cancer types. The possible explanation of the less frequent TERT promoter mutation in ccRCC could be that the activation of telomerase in those tumors could follow a route independent on the TERT promoter mutations. Several factors including, oncogenes like Myc and Wnt, alternatively spliced variants of TERT, posttranslational regulation via reversible phosphorylation of TERT catalytic subunit at specific serine or threonine residues, interaction with telomeric proteins and others have been reported to activate telomerase.22,23 Nevertheless, the consistent association of the TERT promoter mutations with poor patient survival augments the relevance of those mutations in cellular transformation and beyond. As the mutations impart effect through increased gene expression, the observed poor outcomes are in concordance with experimental data that have shown telomerase reactivation in tumors with telomere dysfunction reportedly lead to malignant progression of prostate cancer in a mouse model.24 And suppression of telomerase activity reduced tumor invasion and metastatic potential in a melanoma mouse model.25 It is possible the TERT promoter mutation confer a stochastic level of telomerase to derive tumor cells to adversity. Thus, even in ccRCC, with relatively low mutation frequency, the TERT mutations define subset of tumors that require aggressive treatment.

1.

Mocellin S, Pooley KA, Nitti D. Telomerase and the search for the end of cancer. Trends Mol Med 2013;19:125–33.

2.

Kyo S, Takakura M, Fujiwara T, et al. Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers. Cancer Sci 2008;99:1528–38.

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Horn S, Figl A, Rachakonda PS, et al. TERT promoter mutations in familial and sporadic melanoma. Science 2013;339:959–61.

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Heidenreich B, Rachakonda PS, Hemminki K, et al. TERT promoter mutations in cancer development. Curr Opin Genetics Dev 2014;24:30–7.

5.

Heidenreich B, Nagore E, Rachakonda PS, et al. Telomerase reverse transcriptase promoter mutations in primary cutaneous melanoma. Nature communications 2014;5:3401.

13.

6.

Rachakonda PS, Hosen I, de Verdier PJ, et al. TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism. Proc Natl Acad Sci USA 2013;110:17426– 31.

14.

7.

Simon M, Hosen I, Gousias K, et al. TERT promoter mutations: A novel independent prognostic factor in primary glioblastomas. Neuro Oncol 2014;doi:10.1093/neuonc/nou158.

8.

Liu T, Wang N, Cao J, et al. The age- and shorter telomere-dependent TERT promoter mutation in follicular thyroid cell-derived carcinomas. Oncogene 2014;33:4978–84.

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SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 2011;469:539–42. Dalgliesh GL, Furge K, Greenman C, et al. Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes. Nature 2010;463:360–3. Fan Y, Liu Z, Fang X, et al. Differential expression of full-length telomerase reverse transcriptase mRNA and telomerase activity between normal and malignant renal tissues. Clin Cancer Res 2005;11:4331–7. Arjumand W, Sultana S. Role of VHL gene mutation in human renal cell carcinoma. Tumour Biol 2012;33:9–16. Xu D, Dwyer J, Li H, et al. Ets2 maintains hTERT gene expression and breast cancer cell proliferation by interacting with c-Myc. J Biol Chem 2008;283:23567–80. Bollmann FM. Physiological and pathological significance of human telomerase reverse transcriptase splice variants. Biochimie 2013;95:1965–70. Greider CW. Molecular biology. Wnt regulates TERT—Putting the horse before the cart. Science 2012;336:1519–20. Ding Z, Wu CJ, Jaskelioff M, et al. Telomerase reactivation following telomere dysfunction yields murine prostate tumors with bone metastases. Cell 2012;148:896–907. Bagheri S, Nosrati M, Li S, et al. Genes and pathways downstream of telomerase in melanoma metastasis. Proc Natl Acad Sci USA 2006;103: 11306–11.

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References

TERT promoter mutations in clear cell renal cell carcinoma.

We screened promoter region of the telomerase reverse transcriptase (TERT) for activating somatic mutations in 188 tumors from patients with clear cel...
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