Clinical Characteristics and Prognostic Significance of TERT Promoter Mutations in Cancer: A Cohort Study and a Meta-Analysis

Background The prevalence of telomerase reverse transcriptase (TERT) promoter mutations (pTERTm) in non-small-cell lung cancer (NSCLC) have been investigated, but the results were inconsistent. In addition, several studies have analysed the role of pTERTm in the etiology of various types of cancers, however, the results also remain inconsistent. Methods The genomic DNA sequence of 103 NSCLC samples were analysed to investigate the frequency of pTERTm in these patients and to establish whether these mutations are associated with their clinical data. Furthermore, a meta-analysis based on previously published articles and our cohort study was performed to investigate the association of pTERTm with patient gender, age at diagnosis, metastasis status, tumour stage and cancer prognosis (5-year overall survival rate). Results In the cohort study, 4 patients had C228T and 2 had C250T, with a total mutation frequency up to 5.8%. Significant difference of clinical data between pTERTm carriers and noncarriers was only found in age at diagnosis. In the meta-analysis, We found that pTERTm carriers in cancer patients are older than noncarriers (Mean difference (MD) = 5.24; 95% confidence interval [CI], 2.00 to 8.48), male patients were more likely to harbour pTERTm (odds Ratios (OR) = 1.38; 95% CI, 1.22 to 1.58), and that pTERTm had a significant association with distant metastasis (OR = 3.78; 95% CI, 2.45 to 5.82), a higher tumour grade in patients with glioma (WHO grade III, IV vs. I, II: OR, 2.41; 95% CI, 1.88 to 3.08) and a higher tumour stage in other types of cancer (III, IV vs. I, II: OR, 2.48; 95% CI, 1.48 to 4.15). pTERTm was also significantly associated with a greater risk of death (hazard ratio = 1.71; 95% CI, 1.41 to 2.08). Conclusions pTERTm are a moderately prevalent genetic event in NSCLC. The current meta-analysis indicates that pTERTm is associated with patient age, gender and distant metastasis. It may serves as an adverse prognostic factor in individuals with cancers.


Introduction
The telomerase reverse transcriptase (TERT) gene encodes a highly specific reverse transcriptase that adds repeats to the 3 0 end of chromosomes [1]. The increased telomerase activity allows tumours to avoid the induction of senescence by the preservation of their telomere ends [2,3]. The promoter region of TERT is considered to be the most imperative regulatory element for telomerase expression; it contains several binding sites for factors that regulate gene transcription [4]. Inhibition of telomerase activity for reversion of the immortal phenotype of tumour cells has been one of the most common approaches for cancer therapy [5]. Recent studies have demonstrated that activation of telomerase via transcriptional TERT unregulation can be caused by mutation in the core promoter region of TERT (chr5:1,295,228C>T [C228T], chr5:1,295,250C>T [C250T], et al.) [6,7]. These mutations confer 2-fold to 4-fold increased TERT transcriptional activities by the creation of binding sites for ETS/ternary complex factors (TCF) transcription factors and then upregulate TERT expression, suggesting a potential mechanism for telomerase activation in tumourigenesis [7,8].
The relative characteristics and prognostic effects of TERT promoter mutation (pTERTm) on carriers and noncarriers with cancer are unclear. Statistical difference in gender distribution between pTERTm carriers and noncarriers was found in some studies that male cancer patients are more likely to harbour pTERTm [9,10,11]. Recently, Gandolfi and Wang reported that pTERTm are associated with distant metastases in upper tract urothelial carcinoma and papillary thyroid cancer. Such association of pTERTm may also present in other cancers. In addition, the effects of pTERTm on patient outcome are obscured. Several studies have demonstrated a less favourable prognosis of glioma in pTERTm carriers than in noncarriers [12,13,14,15,16,17], whereas a recent report found a better outcome for pTERTm carriers [18].
The prevalence and association of pTERTms with non-small-cell-lung-cancer (NSCLC) patients have been studied but showed different results. Ma and colleagues found a proportion of 2.67% NSCLC patients in their cohort had pTERTm [19], whereas other studies failed to detect pTERTm [20,21,22]. By conducting a cohort study in NSCLC patients and a meta-analysis, we have attempted to further strengthen the prevalence of pTERTm in NSCLC and to provide definitive evidence of the relative effectiveness and characteristics of pTERTm in cancer patients. This is the first meta-analysis to evaluate the association of pTERTm with cancer. The results could provide insight into the biology of pTERTm, to understand the clinical prognosis of these mutation carriers and to offer implications for the design of clinical trials, particularly those of anticancer agents that target the TERT.
informed consent from each patient. Each sample was placed in liquid nitrogen immediately after resected and stored in -80°C refrigerator. Patient clinical data were collected and their information was anonymized and de-identified prior to this analysis. This cohort study was conducted under the approval of the Ethics Committees of the First affiliated Hospital of Zhejiang University DNA extraction and mutation analysis. DNA extraction and polymerase chain reaction amplification for sequencing of the TERT promoter were performed in all cases by standard protocols. The genomic DNA of tumour tissue was extracted with a QiAamp DNA Mini Kit (Qiagen, Hilden, Germany) and purified with an EZNA MicroElute DNA Clean-Up kit (OMEGA). Polymerase chain reaction (PCR) amplification of the TERT promoter region covering the mutations (from -27 to -286) was performed using primers: 5 0 CCC ACG TGC GCA GCA GGA C3 0 (forward) and 5 0 CTC CCA GTG GAT TCG CGG GC3 0 (reverse), With 3 minutes at 95°C; 35 cycles at 95°C 15 seconds, 63°C 15 seconds, 72°C 1 minute, followed by a final step at 72°C for 5 minutes. After gel electrophoresis to confirm the quality of the PCR products, sequencing PCR was performed using a Big Dye terminator version 3.1 cycle sequencing ready reaction kit (Applied Biosystems), and DNA sequence was analysed on an ABI PRISM 3730 automated genetic analyser (Applied Biosystems), All samples were checked in forward and reverse directions.
Statistical method of cohort study. Statistical analyses were carried out using the SPSS16.0 software package. Associations between pTERTm and the patients' categorical variables were analysed with a chi-square test, Continuous data were summarised as the mean ± SD and analysed with the Mann-Whitney Wilcoxon test. Values of p less than 0.05 were considered significant.

Meta-analysis
Literature search. We searched PubMed and Web of Science for articles published before March 2015, using the systemic literature search terms "telomerase reverse transcriptase", "promoter", and "mutation". The reference lists of the articles retrieved were further screened for other potential studies. We made every attempt to obtain the necessary information from the first and corresponding authors by e-mail if insufficient data were reported in the article (i.e., missing data, missing Kaplan-Meier curves or any other uncertainties).
Inclusion and exclusion criteria. All of the studies included in this meta-analysis met the following criteria: (a) articles about the pTERTm and human cancer that were published in English. (b) availability of detailed genotype data or frequencies that could be calculated from the article text; (c) sufficient data to calculate an odds ratio (OR) or hazard ratio (HR, for prognosis analysis) with a 95% confidence interval (CI); (d) if survival data is not available for calculating HR, survival curves for pTERTm carriers and noncarriers is necessary. The exclusion criteria were: (a) published as an abstract, case report, comment letter, review or editorial; (b) non-human studies; (c) duplicate studies, in which case the latest or largest study were included.
Data extraction. Two reviewers independently assessed all of the potentially relevant studies and reached a consensus on all of the items. Any disagreements were reconciled by discussion and consensus. The following data were collected from each study: first author, year of publication, type of cancer, population, sequencing method and the number of carriers and noncarriers.
Quality assessment. The quality of the studies included was evaluated according to the Newcastle-Ottawa scale (NOS) quality assessment, which is available at http://www.ohri.ca/ programs/clinical_epidemiology/oxford.asp. This evaluation system focuses on three aspects of a study (selection of patients, comparability of baseline characteristics and outcome assessment). The quality of the study was denoted by a numerical score from 0 to 9, with 9 representing the highest quality. Quality assessment was conducted by two independent reviewers. The original papers were scanned when disagreements occurred. Unsettled disagreements were referred to a third researcher for a final decision.
Statistical method of meta-analyses. The meta-analyses, subgroup analyses and sensitivity analyses were performed with Review Manager (revman) version 5.1 software. The metaregression, Begg's and Egger's test were performed with STATA software (version 12.0 Stata Corp LP, College Station, Texas).
For dichotomous outcomes, Odds Ratio (OR) with 95% confidence intervals was calculated by using a fixed effect model (Mantel-Haenszel method) [23] for P Heterogeneity > 0.05, or random effect model (DerSimonian and Laird method) [24] for P Heterogeneity < 0.05. Such as the assessment of association between pTERTm and gender (male vs. female), lymphatic metastasis (positive vs. negative), distant metastasis (positive vs. negative), tumour stage (III/IV vs. I/II), and Glioma WHO grade (III/IV vs. I/II). The dependent variables in these studies are the frequencies of event versus non-events. The significance of the combined OR was determined with a Z test, in which p < 0.05 was considered statistically significant. For continuous outcomes, the mean difference (MD) was calculated based on the mean and standard deviation given in the included studies. So the association between pTERTm and patient age at diagnosis was evaluated by mean age difference (carriers vs. noncarriers) combined with the corresponding 95% CIs. Pooled HR with a 95% confidence interval was calculated for the association between 5-year overall survival and pTERTm status (carriers vs. noncarriers). HR < 1 means that the prognosis of patients of pTERTm carrier is worse than non-carriers, while HR > 1 means the opposite. If a direct report of survival were not available, then the survival data read from Kaplan-Meier curves were read by Engauge Digitizer version 4.1 (http://digitizer. sourceforge.net/). Population data sets were categorized as Asian and non-Asian. Stratified analyses were performed by cancer type (If a cancer type contained only one data source, it was combined into the "other cancers" group.). The evaluation of the meta-analysis results included an examination of the heterogeneity, an analysis of the sensitivity, meta-regression and an examination for publication bias.
The heterogeneity between studies was evaluated using a chi-square-based Q test and a p value of less than 0.05 was considered statistically significant. The Higgins I 2 was calculated to quantitatively estimate the heterogeneity, with I 2 < 25%, I 2 = 25-75% and I 2 > 75% representing low, moderate and high heterogeneity, respectively. Subgroup and meta-regression were conducted to delineate the major sources of heterogeneity. Sensitivity analyses were performed to assess the stability of the results and to identify the individual potential influences on the OR or HR. Funnel plots and Egger's test were used for the diagnosis of potential publication bias, An asymmetric plot suggests a possible publication bias and the P value of Egger's test being considered representative of significant publication bias if it was less than 0.05.

Results of the cohort study
The study included 103 surgical specimens from patients with NSCLC. The results of the cohort study are shown in Table 1. We identified six mutations (5.8%) in the TERT promoter region (four C228Ts and two C250Ts) ( Table 2). The associations of the patient characteristics and clinical features with pTERTm status amongst our patients showed a statistically significant difference only for age. The pTERTm carriers tended to be older at the time of diagnosis than the noncarriers (p = 0.031). No significant differences were found in the distributions of

Results of the meta-analysis
Characteristics of the identified studies. The detailed selection process is demonstrated in Fig 1. In the initial search, 245 studies were found in PubMed, 193 studies were found in Web of science. A total of 388 studies remained after the initial elimination for duplication. 341 studies were excluded after the titles and abstracts were examined. Following a full text review and detailed evaluation, 35 articles were included in our analyses (Table 3). Each study was published between 2013 and 2015 by authors from China, Korea, Japan, Austria, The United States, Germany, Italy, France, Sweden and Portugal. Among the 35 studies, Nine studies assessed glioma [12,13,18,25,26,27,28,29,30], seven studies assessed thyroid cancer [9,14,31,32,33,34,35], five studies assessed melanoma [10,15,16,36,37], two studies each assessed bladder cancer [38,39], renal cell carcinoma [40,41] gynecologic cancer [42,43], hepatocellular carcinoma [11,44] and urothelial carcinoma [17,45]. One study each assessed lung cancer [19], adrenal cancer [46] laryngeal cancer [47] and meningioma [48]. The results of our cohort study (Yuan P) are also included in this meta-analysis. Thus, 36 studies with 3001 carriers and 8384 noncarriers were analysed. In addition, in that some independent variables are not available in certain articles, the numbers of studies in different analyses are varied.
Association of pTERTm with Patient age, gender, metastasis status and tumour stage. The overall results show that pTERTm carriers were older than noncarriers (MD = 5.24; p < 0.001) from a random model. Stratification analysis decreased heterogeneity and identified increased MD in subgroup of glioma and lung cancer, whereas melanoma displayed a reversed pattern (MD = -5.74; p = 0.02). No significant difference was found in other cancers. (Table 4, S1 Fig) We also found that male cancer patients were more likely to harbour pTERTm (OR = 1.38, p < 0.0001). But non-significant risk was found in glioma, lung cancer and renal cell carcinoma (  Fig and S4 Fig). Stratified analyses of distant metastasis and stage performed on cancer types revealed that the significant risk was only observed in thyroid cancer. In addition, an analysis of tumour stage was not available for glioma, but glioma patients with pTERTm were more likely to have a higher WHO grade (III/IV vs. I/II): OR, 2.41; p < 0.0001) ( Table 4).
For the overall comparisons, significant heterogeneity was observed except for gender analysis. However, most of the heterogeneity decreased markedly or disappeared after stratification, excepted for "other cancer" in age analysis, renal cell carcinoma in distant metastasis and melanoma in stage analysis (I 2 > 75). Sensitivity analysis with one study omitted each time showed that the significance of the result was not affected by any single study (S1-S4 Tables) pTERTm and prognostic significance. The HRs for 5-year overall survival were available from 25 studies. All of the studies were published between 2013 and 2015 and were carried out in China, Japan, Austria, the United States, Germany, France, Spain and Portugal. We found a significant increased risk of death for the pTERTm carriers (HR = 1.71; p <0.0001) (Tables 4  and 5). Stratification analysis identified significant risk in subgroups of glioma (HR = 1.52; p = 0.004), thyroid cancer (HR = 2.73; p = 0.002), gynecologic cancer (HR = 2.08; p = 0.006) and "other cancer" (HR = 1.45; p = 0.0005) (Fig 2, Table 4). All the results of the meta-analyses are showed in a simplified table (Table 5).
We preformed meta-regression analyses by covariates including population, sample size, age, treatment, HR estimation and NOS score. No significant alteration was found in the HR by these covariates, and the results showed that the differences between the subgroups did not reach statistical significance (Table 6). No evidence was found to demonstrate that any of these covariates could explain the heterogeneity. In addition, sensitivity analyses omitting one study each time showed that the study of Chen, A K (glioma), Liu, T (Thyroid cancer) and Egberts, F (Melanoma) had the largest influence on the result; The heterogeneity become non-significant when they are omitted. And the summary HR of melanoma became significant and heterogeneity disappeared when the study of Egberts, F was omitted (HR = 2.04; 95% CI = 1.41 to 2.95) (S5 Table). Publication bias. Begg's funnel plot and Egger's test were both performed to evaluate the publication bias of the studies. The shapes of the funnel plots did not show any evidence of an obvious asymmetry in any comparison model. As shown in S5 Fig The p value of Egger's regression tests further provided evidence of funnel plot symmetry. (Table 7).

Discussion
The maintenance of telomere length is of ultimate importance to normal self-renewing stem cells and cancer cells for preventing senescence induction. It has been suggested that tumour cells rely on epigenetic mechanisms or alterations that maintain telomerase activity to retain their immortality [49,50,51]. The recurrent pTERTm creates a putative binding site for ETS/ TCF binding motifs, thereby facilitating the transcription of TERT [7,8]. pTERTm have recently been shown as a novel genetic mechanism underlying telomerase activation and present in diverse human tumours with a large range of prevalence. It was first reported in the melanoma, and then the prevalence of pTERTm was reported in 43-51% of cancers of central nervous system, 59-66% of bladder, 59% of hepatocellular, 10% of thyroid cancer, and 29-73% of skin cancers. Nonetheless, pTERTm was found absent in breast carcinoma, low in cancers of digestive system organs, haematopoietic system and certain reproductive system (serous carcinoma) [52].
The prevalence of pTERTm in small-cell lung cancer (SCLC) and NSCLC have been investigated. Zheng et al [22] failed to detect presence of pTERTm in SCLC. Chen et al [21] and Li et al [20] tried to identify pTERTm in NSCLC but no positive result was found. However, in the present studies, we identified a low frequency of pTERTm (5.8%) in NSCLCs and the mutation was significantly associated with older patients, similar to the result of Ma and his colleagues [19]. They detected 8 adenocarcinomas, 3 squamous carcinoma and 1 other histologic type of 467 NSCLC patients are pTERTm carriers. we tried to further investigate the association of pTERTm with tumour size, differentiation level and distant metastasis, but no significant association was found.
In the current meta-analysis, a borderline significant association between pTERTm and relevant clinical data was observed in overall analysis except for lymphatic analysis. The obvious between-study heterogeneity in each analysis decreased markedly in stratification analyses by tumour types, suggesting that different tumour types might be a potential source of heterogeneity. Interestingly, we observed a significant association of pTERTm with a higher age at diagnosis in patients with glioma and thyroid cancer, whereas patients with melanoma displayed an opposite pattern. This is probably because genetic factors and environmental factors contribute equally to the development of melanoma. Recent studies suggested that melanoma is found more frequently in skin with intermittent sun-exposure than in skin that is not exposed or chronically exposed [53,54].
In addition, we found that thyroid cancer patients with pTERTm have a higher risk of distant metastasis that is four times greater than that of patients without pTERTm (OR = 4.01, 95% CI = 3.15 to 5.10), in line with the study done by Gandofi et al. They found that pTERTm are strongly associated with tumour progression and development of distant metastasis in papillary thyroid cancer [31]. Similarly, landa et al demonstrated that pTERTm are highly prevalent in advanced thyroid cancers (51%) compared to well-differentiated tumours (22%) [55]. Taken together, these data indicate that pTERTm is probably a genetic event during the acquisition of metastatic potential. The mechanism of pTERTm in cancer progression is still unclear. It has been reported that pTERTm is able to increase the transcriptional activity of TERT promoter in tumours and express higher level of TERT mRNA compared with wild type-tumours [7,8,11,33,39,56]. In this regard, it is conceivable that the acquisition of pTERTm leading to TERT activation is an important event during cancer progression, as it allows tumour cells to avoid proliferation limitation and to acquire immortalization [37]. Another study done by Papathomas et al reported that pTERTm occur preferentially in succinate dehydrogenase (SDH)-deficient tumours, and this genetic alteration might cooperate with pTERTm to extend the lifespan of mutated clones, so as to render them infinite proliferation potential and accumulation of additional genetic alterations [57]. However, such association was not found in melanoma, renal cell carcinoma and "other cancer". Whether this effect may be cancer-type specific and play a different role in the etiology of other cancer are still unclear, thus the results should be interpreted with caution. The 5-year overall survival data from 25 studies indicated that patients with pTERTm had a 70% greater risk of death than those without pTERTm. Since pTERTm results in the creation of binding sites for ETS/TCF transcription factors, which are downstream targets of RAS-RAF-MAPK pathways. pTERTm are suggested to have synergistic effects to promoter tumour cell proliferation with activating BRAF or NRAS mutations, which have been proposed to be driver mutations in the development of cutaneous melanocytic neoplasms. It is likely that these mutations turn the pTERTm into a target of ETS-domain transcription factors. Thus additional studies could further investigate whether pTERTm are of therapeutic significance, either in terms of influencing the efficacy of established therapies (ie, BRAF/NRAS inhibitors or immunotherapies) or whether they might even prove to be directly valuable to therapeutic targets [6,58,59]. The association between pTERTm and cancer prognosis was carefully investigated. We attempted to trace the origin of the substantial heterogeneity by performing subgroup and meta-regression analyses. Prognosis analyses in gynecologic cancer, bladder cancer and "other cancer" filed to exhibit significant heterogeneity when stratified by cancer types without changing the HR materially. Further Meta regression analysis by prespecified factors such as population, sample size, age, treatment, method of HR estimation and NOS score did not change the HR as well, and provide no evidence to account for the heterogeneity. In addition, the heterogeneity became non-significant in glioma, thyroid cancer and melanoma by sensitivity analysis. The funnel plots and Egger's test did not identify any publication bias. However, some limitations should be addressed in the interpretation of the results of our cohort study and metaanalysis. First, the sample size of our cohort study was relatively small. Well-designed population-based studies with large sample sizes and detailed exposure information are needed to further confirm our findings. Second, subgroup meta-analysis stratified by cancer type, such as hepatocellular carcinoma, bladder cancer and laryngeal cancer, might contain insufficient data to enforce statistical power to check for an association, despite our efforts to contact the authors for data. We were unable to include more articles because the authors of a few studies with incomplete data failed to reply to our requests. Hence, more individual study would be required to draw a more precise conclusion In conclusion, we found that pTERTm is present in a small fraction of NSCLCs and are significantly associated with older patients. The meta-analyses suggested that pTERTm carriers were older than noncarriers in glioma, thyroid cancer and lung cancer, with melanoma demonstrate a reserved pattern. Male cancer patients exhibited a significantly elevated risk of having pTERTm in thyroid cancer, melanoma and hepatocellular carcinoma. Apart from other cancers, we also identified thyroid cancer patients with hTERTm are more likely to have distant metastasis and higher tumour stages. In addition, pTERTm carriers had a higher risk of death in our prognosis analysis in giloma, thyroid cancer, gynecologic cancer and "other cancers". All in all, the detection of pTERTm appears to be a promising prognostic indicator in patients with cancer and may have potential as a biomarker for treatment stratification. More welldesigned prospective studies are needed to validate our findings.