Genetic variability of immune checkpoints in asbestos-related diseases

Occupational or environmental asbestos exposure is the major risk factor for the development of many diseases, including pleural plaques, asbestosis, lung cancer, and malignant mesothelioma (MM), as well as several other types of cancer. Although asbestos has been banned in over 50 countries worldwide, the incidence of asbestos related diseases is still on the rise is some countries due to the long latency period between the exposure and disease occurrence.^1,2

Pleural plaques are nonmalignant thickening on the pleura, often asymmetrical and bilateral and may develop up to 20 to 30 years after asbestos exposure in 50% of asbestos exposed workers. Pleural plaques are often asymptomatic and cause a slight lung function impairment when they grow. In the past, pleural plaques were referred to as a marker of asbestos exposure. However, in the recent years some studies have shown that they can indicate increased risk of asbestosis and MM.^2,3,4

Asbestosis is one of the most common diseases related to asbestos exposure. It is an interstitial lung disease that progresses to diffuse pulmonary fibrosis after a long latency period. The disease is not reversable, and it continues to progress even after even after the end of asbestos exposure. The symptoms of the diseases are often non-specific like dyspnea, dry irritating cough and chest pain. The changes in pulmonary function are initially due to restrictive impairment and later due to obstructive air impairment. Asbestosis is also associated with a higher risk of lung cancer.^1,5

MM is a rare but very aggressive cancer of the serous membranes and the most fatal asbestos-related disease.⁶ MM is found on the mesothelium, most commonly on pleura (65%), peritoneum (30%) or other serosal membranes (1%).² The most common symptoms of pleural MM are dyspnea, chest pain, dry cough, and fatigue.⁷ Due to its non-specific symptoms, MM is often diagnosed in later stages, when the treatment is less effective. MM is treated by surgery in combination with chemotherapy and radiotherapy. In the latest years, the immunotherapy treatment of MM has also been extensively used.^8,9

Studies have proposed that inflammation caused by asbestos is involved in pathogenesis of all asbestos-related diseases.^5,10 After inhalation, asbestos fibers can accumulate in various tissues, including mesothelial cells in the pleural space, but also in other tissues such as the lung parenchyma, pericardium, and lymph nodes. This leads to local inflammatory response and increase in proliferation of mesothelial cells. Due to increase in metabolic activity, mesothelial cells orchestrate the production of local inflammatory mediators such as cytokines and growth factors. That leads to recruitment of leukocytes and enhancing inflam mation.¹¹

The immune response is controlled by immune checkpoints inhibitors that enable effective immune response and immune tolerance. There are two types of immune checkpoints: stimulatory checkpoints that promote activation of naïve T cells and effector, memory and regulatory T cell response and inhibitory checkpoints that limit the threshold of T cell activation and duration of immune response.¹²

Programed death 1 (PD-1) receptor is an inhibitory immune checkpoint. PD-1 is constitutively expressed on T cells in thymus where it is required for their normal selection. PD1 is also found on activated T cells, B cells, monocytes, natural killer cells and dendritic cells. Program death ligand (PD-L1) expressed on cancer cells binds to PD-1 and activates pathways that suppress proliferation and survival of T cells.^13-15

The cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is also an inhibitory immune checkpoint. CTLA-4 is a CD28 homolog. The latter is a costimulatory receptor required for T cell activation. Both CTLA-4 and CD28 bind to the same ligands B7-1 and B7-2, however, CTLA-4 has a much higher binding affinity. As a result, CTLA-4 prevents T cell activation with competitive binding.^15,16 Regulatory T cells constitutionally expressed on regulatory T cells (T_reg) and it is crucial for their immunosuppressive role. T_reg cells are commonly infiltrated in tumor microenvironment and play immunosuppressive role with CTLA-4.¹²

Single nucleotide polymorphisms (SNPs) may have an influence on protein expression and function of proteins. Several studies have evaluated the impact of SNPs in genes coding for immune checkpoints PD-1 (PDCD1), PD-L1 (CD274), and CTLA-4 (CTLA4) and risk of developing different types of cancer.^17-19 However, to the best of our knowledge the role of PDCD1, CD274 and CTLA4 genetic variability in the development of asbestos-related diseases has not been studied so far.

The aim of our study was to investigate the associations of PDCD1, CD274 and CTLA4 polymorphisms with the risk of developing asbestos-related diseases.

Our study assessed the association between genetic variability in three immune checkpoints: PD-1, PD-L1 and CTLA-4 and the risk of developing asbestos related diseases (pleural plaques, asbestosis and MM). The key finding in our study was the association of polymorphic CTLA4 rs4553808 G allele with decreased risk of developing asbestos-related diseases of pleura, in particular MM and pleural plaques. However, carriers of polymorphic CTLA4 rs231775 GG genotype had a higher risk of developing MM than the group of subjects with pleural plaques. None of investigated polymorphisms were associated with the risk of asbestosis.

Our study showed that the CTLA4 rs4553808 G polymorphism had a protective role in development of pleural plaques and MM. This could be perhaps attributed to the fact that rs4553808 is located in the upstream regulatory region of CTLA4 and may influence the binding site for TFs, but the consequences of such change in the binding side are still controversial. In silico, it was shown that change of A to G in position-1665 could create a new binding site for C/EBPβ TF.²⁵ However, another study reported that the A to G substitution could lead to a loss of binding site for C/EBPβ TF.^26,27 Therefore, the role of this SNP is still inconclusive and must be experimentally validated.

To the best of our knowledge there is no study that has evaluated associations between CTLA4 rs4553808 and MM. However, it was widely studied in other types of cancer. Nevertheless, the results differ among studies. In a meta-analysis from 2020, it was shown that polymorphic CTLA4 rs4553808 G allele significantly increased the risk of cancer in Asian population, but there were no significant associations observed in Caucasians.²⁸ Therefore, the results of studies on Asian population are in agreement with hypothesis that A to G substitution leads to higher expression of CTLA4. However, another study reported that carriers of at least one A allele have a higher risk for development of gastric cancer.²⁹ Consequently, that would be in agreement with hypothesis that rs4553808 downregulates CTLA4 expression. Therefore, further studies are needed to evaluate the influence of rs4553808 on CTLA4 expression.

Our study also showed the association of CTLA4 rs231775 GA and GG genotype with increased risk of MM development when compared to pleural plaques. CTLA4 rs231775 is a non-synonymous polymorphism which causes amino acid change from threonine (Thr) to alanine (Ala) in position 17 (p.Thr17Ala). It was reported that p.17Thr was associated with lower levels of T cell activation and proliferation.²⁸ Moreover, lower levels of expression of CTLA4 with p.17Ala were observed on the surface of T-cells. Therefore, CTLA4 rs231775 AA genotype may be considered as a risk factor for cancer development.^28,30 Indeed, CTLA4 rs231775 was widely studied in different types of cancers, however, its influence on cancer risk is still controversial. It was shown that CTLA4 rs231775 A allele increased the overall risk of cancer in Asian but not in Caucasian population.^30,31 Two meta-analyses reported an increased risk of colorectal cancer in carriers of CTLA4 rs231775 G allele.^32,33 However, another meta-analysis showed that allele G plays a protective role in cancer development.²⁹ A metaanalysis of several cancer types showed no evidence that CTLA4 rs231775 would have an impact on lung cancer, and MM was not studied.³⁴ Our results therefor warrant further research of the role of CTLA4 rs231775 in the development of asbestos related diseases.

Our study observed no significant association between CTLA4 rs5742909 and the risk of developing any asbestos disease. As this polymorphism is located in the promotor region, the T allele could have an impact on transcription factor LEF1 binding. As higher CTLA4 mRNA expression levels were observed in T allele carriers, some studies concluded that this allele could be a risk factor for cancer.^28,35 A meta-analysis showed association of rs5742909 T allele with increased risk of overall cancer susceptibility in both Asian and European population.¹⁹ However, CTLA4 rs5742909 was not associated with risk for lung cancer.^36,37

Our study did not show any statistically significant associations between PDCD1 SNPs and susceptibility for developing any of investigated asbestos related diseases. PDCD1 rs2227982 (c.644C>T, p.Ala215Val) is located in exon 5 and as it causes a nonsynonymous amino acid substitution in the extracellular domain of the PD-1, it could lead to a change in its structure and function.^38,39 A meta-analysis performed in 2019 did not show a significant association between PDCD1 rs2227982 and overall risk of cancer, however MM was not investigated.¹⁸ There was also no significant association between PDCD1 rs2227982 and non-small cell lung cancer.⁴⁰ Another investigated PDCD1 polymorphism, rs2227981 (c.804T>C. p.Ala268=) in exon 5 leads to a synonymous amino acid substitution in PD-1. However, it was shown that PD-1 expression was lower in individuals with a more common rs2227981 genotype. Therefore, it was speculated that rs2227981 may be in linkage disequilibrium with another polymorphism influencing expression levels.^28,41 Previous studies have reported that PDCD1 rs2227981 TT genotype decreases the overall risk of cancer.^18,28 However, no significant association were observed between PDCD1 rs2227981 and many different types of cancer such as non-small cell lung cancer (NSCLC), colorectal cancer and breast cancer.³⁹ The third investigated PDCD1 polymorphism, rs10204525 (c.*889G>A) is located in a miRNA binding site in the 3’ untranslated region (3’UTR). According to one study, miRNA-4717 significantly decreased expression of the PD-1 in subjects with GG genotype, therefore it could have a protective role in cancer development.^28,42 However, our results are in concordance with a meta-analysis did not find a significant association between PDCD1 rs10204525 and the overall risk of cancer.¹⁸

Our study also did not show any significant association between any of the three investigated CD274 SNPs: rs2297136, rs4143815, and rs4742098 and asbestos-related diseases. All of them are in 3’UTR region that it is crucial for post-translational modification.²⁸ In silico prediction has shown that CD274 rs2297136 (c.*93G>A) could affect the binding side for miRNA-296-5p and miRNA-324-5p.⁴³ It was shown that expression of PD-L1 was inhibited in carriers of G allele. Therefore, G allele could play a protective role in cancer development.^28,43 There have been many studies that investigate SNP CD274 rs2297136 as risk factor for developing cancer. A previous study has shown that carriers of SNP CD274 rs2297136 A allele had increased risk for developing NSCLC.⁴³ Polymorphism CD274 rs4143815 (c.*395G>C) is located in the binding site for miR-7-1*, miR-495, miR-298 and miR-570. As allele G changes binding site for miRNA-570, it could be cancer risk factor.^28,43 According to the meta-analysis, CD274 rs4143815 was associated with increased overall risk of cancer.⁴⁴ There was no association between CD274 rs4143815 and risk of developing NSCLC.⁴⁴ Polymorphism CD274 rs4742098 (c.*2635A>G) is located in the binding site for miRNA-138. Allele A increases the capability of binding miRNA-138 and could therefore be protective for cancer development.^28,43 Carriers of CD274 rs4742098 G allele had significantly higher risk of developing NSCLC, according to one study.⁴³

The limitation of our study is a small control group. However, the strength of our study is its relatively large and well-defined sample. All subjects were from genetically homogenous Central Europe Caucasian populations, therefore there are not a lot of other variables that could influence our analysis. Another limitation of our study was that the group of MM patients was significantly older than other groups. This finding is in agreement with some other studies, probably due to the long latency period between asbestos-exposure and MM development.^2,45 Therefore, we performed multivariable analysis with adjustment for age.

In conclusion, our data indicate that CTLA4 polymorphisms may have an impact on the risk of asbestos-diseases of the pleura such as pleural plaques and MM. Therefore, future studies should investigate if these polymorphisms may also play a role in the outcome of MM treatment with immunotherapy.