Telomere length and telomerase activity in non-small cell lung cancer prognosis: clinical usefulness of a specific telomere status

Lung cancer remains the most common cancer in men worldwide [1] and the identification of subjects at higher risk of cancer progression and recurrence, who would benefit from more specific therapies, is necessary [2]. The risk assessment by the Tumour-Nodes-Metastasis (TNM) staging system is relatively inexact as it only considers tumour characteristics at a particular moment (TNM at diagnosis) [3]. Of fundamental concern is the identification and use of new biomarkers, with high specificity and sensitivity, which will allow the prediction of the disease course and identification of patients who would or would not benefit from specific therapy. In this way, telomere function has been recognized as possible biomarker [2]. However, it has not yet been established a consensus for the use of telomere function in cancer clinical practice.

Telomeres are ribonucleoprotein complexes located at the ends of eukaryotic chromosomes which consist of tandem repeats of a DNA sequence (TTAGGG in all vertebrates) [4]. Among its main functions, telomeres mask double strand break DNA damage signals at the extreme of chromosome and prevent chromosomal fusions [5‐8]. In the context of a somatic cell, a significant telomere shortening is monitored by p53 and RB1 and leads to cell death or senescence. However, cells that surpass their normal replicative limit and continue to divide lose all remaining protective telomeric DNA and enter the crisis stage, characterised by massive genomic instability and cell death [9]. At this point, transformed clones emerge and telomerase activation is detectable in the majority of tumour cells and is the main positive power for telomere preservation and elongation [10].

Telomeres and telomerase play a crucial role in human carcinogenesis: the majority of tumours have telomere length alterations which can eventually lead to telomere dysfunction [11]. A disruption of telomere length homeostasis affects telomere structure and leads to genomic instability by generating chromosome end-to end fusions and chromosomal abnormalities [5]. In relation to telomerase, an increase in telomerase activity is often directly correlated with uncontrolled growth of cells, a known hallmark of cancer, and has become a widely acceptable tumour marker and a popular target for anticancer therapeutics [12].

Considering previous data and the need to incorporate new biomarkers for the prognosis of solid tumours into the clinic, our first aim in this work consists of evaluating the potential clinical use of telomeres and telomerase in NSCLC. With this objective, we analyzed both parameters directly implicated in telomere function in a large series of cases of NSCLC. Thus, we identified groups of NSCLC patients with different clinical evolution, independently of the TNM stage tumours, in relation to telomere status (telomere length & T/N ratio).

Malignant cells, in general, have shorter telomeres than their normal counterparts [17] as a reflection of their extended proliferation. In fact, diseases of high cellular turnover are associated with telomere shortening, telomere dysfunction and cancer predisposition [18]. In bronchial carcinogenesis, telomere shortening is an early event [19] and previous results [20] including those of our research group [15] show a significant telomere shortening in NSCLCs in relation to the control tissues. In other tumour types, telomere attrition has been also demonstrated [21]. Therefore, the results shown in this paper, as with the previous data, reflect differences between the mean telomere length in NSCLCs and the non-tumour tissues, which are validated by a T/N ratio <1.

Progressive telomere erosion is countered with telomerase, which is expressed in the vast majority of human cancers [12]. Our data indicated more than 80 % of NSCLCs analysed expressed telomerase activity, in agreement with previously published works [15], without significant differences in the mean telomere length values between telomerase positive and negative tumours. Although telomerase activation can be an early event in cancer, it is not necessary for cancer initiation. However, telomerase can stimulate tumour progression by ensuring maintenance of telomeres above a critically short length, thus preventing induction of cellular senescence or apoptosis [22]. Appart from telomerase, possible implication of Alternative Lengthening of Telomeres (ALT) mechanisms in telomere elongation of telomerase-negative tumours should be considered. However, the ALT phenotype was not investigated in our research work considering the limited role of this mechanism in lung cancer. In fact, the ALT phenotype is common in tumours such as osteosarcomas, undifferentiated pleomorphic sarcomas, leiomyosarcomas, astrocytic tumours grades 2 and 3, and pancreatic neuroendocrine tumours [11, 23]. However, ALT is a very infrequent mechanism in the most common cancer types, carcinomas, which are derived from epithelia [24]. Heaphy et al. assessed the ALT phenotype in 6110 primary tumours from 94 different cancer subtypes and this mechanism was not detected in most lung carcinoma subtypes, only individual cases of small cell (2 %) and large cell lung carcinomas (3 %) were observed. ALT-positive cells are characterized by striking telomere length heterogeneity [11]. In the present work, it is noteworthy that the maximum value in telomere length for telomerase positive and negative lung tumours is close to that defined in the normal human population: the length of telomeres is heterogeneous, ranging between 5 and 15 Kb [25]. Therefore, very long telomeres, other feature of ALT, were not detected.

The role of telomeres in the initiation and progression of carcinogenesis has been widely recognized [11]; thus, its relation with clinical variables, in conjunction with others molecular markers, could represent a therapeutic opportunity for cancer patients. Currently, except for recent developments in relation to the involvement of EGFR mutational status [26], limited advances in the detection of molecular targets in lung cancer were obtained. For NSCLCs, the statistical association between the tumour size (T descriptor) and telomere status is in line with previously published data [15]: the highest degree of telomere shortening is detected for tumours that grow into the area of mediastinum or cancers in which a malignant pleural effusion is reached (T4).

An altered telomere length in cancer cells could give the ability to metastasize and cause recurrent disease and, in consequence, be a predictor of clinical outcome. Previous works concluded that careful assessment of telomere length or its proxies, such as DNA content, will be part of novel risk assessment and prognostic modalities for patients [2]. More recently, in glioblastoma multiform tumours, telomeres were always shorter when compared with normal brain tissue, and together with telomerase activity seem to be associated with malignancy and poor outcome [27]. In lung cancer, previous studies have evaluated whether telomere length could represent a risk factor or a prognostic marker, however most of them are contradictory: for NSCLCs patients both shorter and longer telomeres has been associated with decreased overall survival [28]. Results obtained in the present work indicate that the worst prognosis is seen in patients whose mean tumour telomere length is lower than 7.29 Kb or when almost any degree of telomere shortening is reached in tumours cells. Both parameters related to telomere status proved to be independent of other known prognostic factors. However, we also demonstrated in colorectal cancer that telomere attrition conferred good clinical evolution. Therefore, cancer prognosis associated to telomere status could be dependent on the tumour type [14]. A deficiency in the function of senescence and cell death pathways [29], or other molecules related to telomere maintenance and genome instability [30] could explain the unfavorable prognosis for post-surgical patients with NSCLCs and short telomeres. Genetic context underlying telomere status, which confers the different clinical outcome, must be carefully considered.

Recently, two works have demonstrated that leukocyte relative telomere length in peripheral blood is an independent prognostic marker in glioma and gastric cancer patients, being shorter telomeres associated with the worst clinical evolution of patients [31, 32]. Telomere analyses in leukocytes from peripheral blood could be a useful biomarker to improve the prognosis prediction in cancer patients. However, previously it is necessary corroborate and validate correlations between telomere status in blood cells and tumours through prospective studies. This is a field that needs to be investigated in lung cancer.

Finally, the presence of telomerase activity in NSCLCs conferred the worst outcome, confirming previous results [15]. Telomerase activity or hTERT expression has proved to be a marker of malignancy. Furthermore, its diagnostic utility is being demonstrated [11].

The novel finding of this study is the independent prognosis role of a specific telomere status in NSCLC patients. Moreover, telomerase activity is confirmed as a prognostic marker in NSCLC. According to results from the present work, telomere function may emerge as a useful molecular tool that allow to select groups of NSCLC patients with different clinical evolution, in order to establish personalized therapy protocols.