Telomere length and TERT polymorphisms as biomarkers in asbestos-related diseases

Asbestos consists of mineral fibres with a high malignant potential, listed among carcinogens by the World Health Organisation in 1987.¹ The malignant potential of asbestos fibres lies within their capability to deeply infiltrate the respiratory system and persist there for extended durations.² There is no known safe level of asbestos exposure.³

Inhaled asbestos fibres induce oxidative stress due to the presence of iron as well as frustrated phagocytosis by macrophages, what in turn stimulates Fenton and Haber-Weiss reactions, ultimately resulting in the generation of reactive oxygen species (ROS). Asbestos further elicits the upregulation of the heavy chain of ferritin, consequently leading to an augmented iron burden and increased production of ROS.⁵ Oxidative stress is closely associated with chronic inflamation. The asbestos deposits contribute to the production of iron-rich asbestos bodies, that are accountable for sustaining chronic inflamation.⁴ The pro-inflamatory microenvironment fosters cell survival by inhibiting apoptosis, promoting mesothelial cell proliferation despite DNA damage, activating fibroblasts and inducing immunosupression.^2,5 Furthermore, asbestos fibres also have a detrimental impact on chromosomes. All of these mechanisms eventually contribute to the induction of carcinogenesis.^6,7

Asbestos exposure causes several diseases. In the lung, asbestosis and lung cancer carcinoma affect the lung parenchyma, while pleural plaques (PP) and malignant mesothelioma (MM) affect the pleura, but other serous membranes such as peritoneum may also be affected.⁸

Malignant mesothelioma arises from the malignant transformation of mesothelial cells and is a rare, yet highly aggressive cancer with a poor prognosis. In over 80% of cases, the development of MM is associated with asbestos exposure.⁹ The majority of patients who develop MM have been exposed to asbestos through occupational exposure; however, para-occupational, domestic and environmental exposure have also been associated with MM development.⁸ Due to its long latency period, which can extend up to 40 years or even longer, the MM epidemic continues to rise in Central Europe.^8,10,11 Malignant mesothelioma occurs more often in males, with a median age of 70 years.³ In 2018, the incidence of MM in Slovenia was 3.0/100 000 in males and 1.5/100 000 in females, resulting in a total of 45 new cases that year. At the time of diagnosis, 31.1% of patients had MM in an early localized stage, 55.6% had cancer spread to lymph nodes and in 11.1% it already presented with metastasis.¹² Malignant mesothelioma can be histologically classified into subgroups: epithelioid, biphasic and sarcomatoid subtypes. The epithelioid subtype is the most common and also exhibits the most favourable prognosis, while patients with sarcomatoid MM tend to have the worst prognosis.⁹

The diagnosis of MM is accomplished through clinical examination, thorax CT or MRI, and PET-CT. The prevailing clinical presentation often involves progressive dyspnoea, accompanied by non-pleuritic chest pain. Additional symptoms include cough, fever, asthenia, hypoxia, weight loss, and night sweats. Typically, the disease is detected 3–6 months after the initial clinical presentation.² The therapeutic strategy is tailored based on the tumour resectability and the patient’s performance status. In resectable disease, the treatment involves the combination of surgery, chemotherapy and radiotherapy, while patients with unresectable disease receive systemic treatment. Chemotherapy with pemetrexed/cisplatin doublet has not been changed as a standard treatment since 2004 and immunotherapy with ipilimumab and nivolumab was approved for the first line treatment in late 2020.¹³ Although different chemotherapy (gemcitabine/cisplatin, pemetrexed single, vinorelbine weekly) and immunotherapy (nivolumab/ipilimumab) regimens are used in relapsed MM, there is still no standard of care.^2,13,14

The increasing incidence of MM and the poor prognosis call for the identification of novel noninvasive biomarkers that will enable an earlier diagnosis, will have a prognostic value and/or will predict the response to treatment. Despite the growing numbers of potential biomarkers, there is no reliable diagnostic or prognostic biomarker available yet. Telomerase reactivation may play a crucial role in cancer development and progression, and telomeres could serve both as a potential biomarker and as a therapeutic target in cancer. In somatic cells telomeres shorten with each cell division, ultimately leading to senescence or apoptosis.^15,16 Cancer cells gain the ability to sustain their telomere length by reactivating telomerase, a process typically suppressed under physiological circumstances.^15,16,17,18

The regulation of the expression of the human telomerase reverse transcriptase (hTERT) subunit of telomerase occurs predominantly at the transcriptional level.^19,20,21 Numerous single nucleotide polymorphisms (SNP) in the hTERT gene may also have an impact on telomerase expression levels and activity, and may thus play a role in the risk of carcenogenesis, as well as the prognosis and survival of cancer patients.^18,19,22

Telomere length is influenced by cellular senescence, chronic inflammation and oxidative stress. Telomere shortening itself serves as one of the main markers for senescence, as telomeres typically shorten by 50–200 base pairs (bp) with each cell cycle.²³ Chronic inflammation is associated with elevated hTERT expression, which, in turn, maintains telomere length.⁷ On the other hand, the celullar turnover stimulated by chronic inflamation leads to an increased number of cell divisions, resulting in telomere shortening.¹⁹ Furthermore, oxidative stress causes DNA damage, which subsequently stops telomere elongation.^7,18

Cancer cells exhibit shorter, yet stable, telomeres compared to non-neoplastic cells.^18,20,21 Malignant mesothelioma is considered to be a telomerase dependant cancer.²² The impact of asbestos exposure on telomere length was also established in pleural effusion cells, showing that non-neoplastic cells had longer telomeres than neoplastic MM cells and that telomere shortening and genomic instability play significant roles in MM pathogenesis, and may also serve as biomarkers for disease development, treatment response, and prognosis.²³

To the best of our knowledge, the association between telomere length and hTERT polymorphisms and asbestos-related diseases has not been evaluated yet. Thus, the aim of the present study was to analyse the role of telomere length in leukocytes and hTERT polymorphisms as a biomarker for asbestos-related diseases, in particular MM, its response to treatment and prognosis.

In our study, we evaluated whether telomere length or their dynamics and hTERT polymorphisms could serve as a biomarker for the risk of developing asbestos-related diseases, chemotherapy response, and progression in MM patients. Consistent with previous studies, we observed that patients with MM had shorter telomeres compared to cases with PP. Previous studies stated that the telomeres in cancer patients are shorter but stable when compared to healthy individuals.^18,20,21 Also, a study analysing telomere length in pleural effusion cells reported shorter telomeres in 12 MM patients compared to 35 cases with non-neoplastic disease.²³

Interestingly, older patients with MM had longer telomeres than younger patients with MM. According to our knowledge, telomere shortening is one of the most important markers of ageing.^19,33,34 Furthermore, cancer cells have typically short telomeres^18,20,21, as also shown for MM.²³ However, the presence of telomerase reactivation in MM²² allows for an unlimited cell division potential and telomere length maintenance, which, on the contrary, does not occur in non-neoplastic cells,³⁵ potentially contributing to the observed results.

In the first part of the study, we evaluated the association of TERT SNPs with a risk for MM. We observed rather consistent associations of the polymorphic rs2736098 T allele with an increased risk of MM in the additive or in the dominant genetic model. Although no specific studies on this association with MM have been conducted, recent studies have shown associations between rs2736098 and lung cancers^36,37 as well as an increased risk for bladder cancer, while the risk was decreased for breast and colon cancers.³⁶

Another important finding of this study was the decreased risk of MM associated with homozygosity for polymorphic rs2736100 A allele after adjustment for age. This observation emphasizes the importance of age as a contributing factor to carcinogenesis, although further investigation is needed to determine whether this association is coincidental. Our results are in agreement with previous studies indicating that carriers of two reference rs2736100 C alleles generally have a higher risk of developing idiopathic lung fibrosis, chronical obstructive pulmonary disease (COPD) and laryngeal cancer.^18,39,40

Furthermore, we observed a significantly higher risk for MM in carriers of two polymorphic rs10069690 T alleles. To our knowledge, there are no other studies investigating this association in MM; however, rs10069690 has been linked to a higher overall cancer risk, specifically in breast, ovarian, lung and thyroid cancers.⁴¹

In the second part of the study, we evaluated the association of telomere length and TERT SNPs with a treatment outcome in MM. We did not find any associations between telomere length and MM chemotherapy response. While studies on breast cancer reported that chemotherapy can lead to telomere shortening in the short term, telomere length was shown to return to its pre-treatment level after two years.⁴² Given that our findings are based on a limited number of participants and that studies in MM patients are lacking, further analyses and investigations are necessary to gain a deeper understanding of the relationship between telomere length and a chemotherapy response in MM.

In our study, polymorphic rs10069690 T allele was associated with a good chemotherapy response. Moreover, this association became even more statistically significant after adjustment for age. Interestingly, our findings differ from a previous study in breast cancer, which is also telomerase-dependent cancer, where rs10069690 was associated with poor chemotherapy outcome.⁴³ As there are currently no studies specifically exploring this relationship in MM, further studies are required to fully understand the impact of rs10069690 on a chemotherapy response in MM.

No significant associations were identified between telomere length and PFS in patients with MM. To our knowledge, there are no other studies that investigated the association between telomere length and survival in MM, and the existing survival analyses conducted for other cancer types yield contradictory results. An extensive study examining the effect of telomere length on survival in various benign and malignant diseases found no influence on cancer patients’ survival.⁴⁴ Conversely, an American study on pancreatic cancer observed that shorter telomeres were linked to poorer OS, while hTERT polymorphisms had no statistically significant impact on OS.⁴⁵ Similarly, our study showed no significant associations between the investigated hTERT polymorphisms and overall survival of MM patients. Due to the inconsistent knowledge in this field, further studies should be performed to better define the factors influencing the outcome of MM.

Finally, our study has shown that carriers of two polymorphic rs2736100 A alleles had a lower risk for MM progression. However, this area of research is still limited, and our finding contrasts with a kidney cancer study that identified rs2736100 as an independent factor associated with a poor prognosis.⁴⁶ Similarly, an Indian study reported that rs2736100 contributes to a poorer prognosis of glioma patients.⁴⁷ On the other hand, a Chinese study did not seem to validate the relationship between this polymorphism and a poor prognosis in papillary thyroid carcinoma.⁴⁸ It is essential to consider that the mentioned studies did not focus on MM and were not conducted on Caucasians, thus caution should be applied when interpreting the data. Therefore, future studies investigating these associations with a specific focus on MM are required.

In conclusion, our results suggest that telomere length and genetic polymorphisms in the hTERT gene have a limited role as a biomarker for the risk of developing asbestos-related diseases. Collectively, our study did not demonstrate the role of telomere length as a biomarker for a MM chemotherapy response; however, with a cautious interpretation, hTERT polymorphisms may represent a biomarker for the chemotherapy outcome in MM. Similarly, telomere length does not seem to impact PFS, while hTERT polymorphisms may be used as a biomarker for the risk of MM progression. So far, our findings have been encouraging, yet further studies are necessary to validate these associations in independent patient cohorts and elucidate the role of telomere length and genetic variants of the hTERT gene in MM.