• UIP
• TERT promoter mutations
• NSCLC

Background. The mutations detected in the promoter of the telomerase reverse transcriptase (TERT) gene, namely C228T and C250T, were first identified in sporadic and familial melanoma and subsequently in several cancer models, notably in glioma, thyroid cancer and bladder cancer. Recent findings demonstrate that mutation of the TERT promoter may be one of the most common genetic mechanisms contributing to telomerase activation in malignant cells. Mutation of the TERT promoter seems to be an indicator of worse outcome and is correlated with tumor aggressiveness and patient survival. The mutation frequency varies considerably among cancer types, although to date, the mutation frequency of the TERT promoter in lung cancer remains to be addressed.
Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause associated with the histopathologic and/or radiologic pattern of usual interstitial pneumonia (UIP). IPF is the most common manifestation of telomere-mediated disorders. Germline mutations in the essential telomerase genes, hTERT and hTR (human telomerase RNA), are the most commonly identified genetic risk factor in IPF. Recent reports have documentated that genetic variants within hTERT or hTR are associated with familial pulmonary fibrosis and are present in some patients with sporadic IPF.
Goal. We would like to investigate the presence of TERT promoter mutations in NSCLC, in UIP and in diffuse pulmonary fibrosis. In patients with two synchronous lesions we studied the mutational status of TERT promoter gene both in the neoplastic lesion and in the surrounding parenchyma (with fibrosis or UIP).
Materials and methods. In this study, polymerase chain reaction and direct sequencing analysis of a TERT promoter region using formalin-fixed and paraffin-embedded tissues were performed to investigate the presence of TERT promoter mutations in a series of 95 early-stage (T1N0M0) non-small cell lung cancer (NSCLC) samples, including 48 adenocarcinomas (ADCs) and 47 squamous cell carcinomas (SCCs). In the NSCLC samples that harbored the mutations we also analysed the surrounding parenchyma. Histological analysis of the parenchyma of mutated cases revealed diffuse fibrosis in 4 cases, emphysema in 5 cases and organizing pneumonia in 1 case. Then immunohistochemical (IHC) staining was performed to detect the expression o f TERT.
Moreover, with the same methodology, we examined TERT promoter gene mutations in 24 cases of UIP. In two cases UIP was associated with adenocarcinoma, in these cases our analysis was extended to the neoplastic tissue.
Results. Sequencing analysis was performed in the TERT promoter region containing the two previously reported hot spot mutations. Our results revealed that TERT promoter mutation occurred in 10.52% of early-stage (T1N0M0) NSCLC patients, with higher frequency observed in ADCs (14.58%) compared to SCCs (6.38%). This result is of particular interest, although it does not reach statistical significance. Sequence analysis revealed that these mutations affected only one of the alleles of the TERT gene locus; their somatic nature was confirmed by their absence in normal tissues collected at a distance from the tumor in the 10 patients harboring TERT mutations. In eight mutated cases out of ten the DNA of the surrounding parenchyma was analyzable: none of them showed the hot spot mutations. Three samples carried mutations at other positions within the TERT promoter core, different from the hot spots (C206T/C205T, C212T/C211T and C205T).
Immunohistochemically TERT was expressed in 55 samples out of 93 analyzed (59.1%): 33 adenocarcinomas and 22 squamous cell carcinomas. The adenocarcinomas seem to express it in a higher percentage of cases (71.7% vs 46.8%). Although no correlations between TERT mutations, TERT immunohistochemical expression, clinicopathological parameters and survival were found in our series, this study represents a starting point for further research to better understand the molecular mechanisms involved in the transcriptional regulation of TERT and its role in lung cancer progression.
For what concerns the UIP samples 20 of them were amplifiable: none harbored the hot spot mutations. Nine samples out of 20 showed the presence of mutations at other positions within the TERT promoter core, different from the hot spots (C209T in two cases, C211T in two cases, C217T in two cases, C231T, C236T, C237T/C240T).
Finally, the two adenocarcinomas samples associated with UIP showed a wild-type genotype of TERT promoter gene both in the neoplastic tissue and in the surrounding parenchyma with UIP.
Conclusions. In our study we showed that TERT promoter mutations are present even in non-small cell lung cancer, with particular incidence in adenocarcinomas. More detailed studies in larger cohorts of patients will be necessary to clarify the role of these mutations in NSCLC cancer progression with specific reference to the regulation of transcriptional mechanisms of TERT gene.
To the best of our knowledge this is the first study that investigate on the presence of TERT promoter gene mutations in UIP and in non small cell lung carcinomas associated with UIP.
None of the mutations found at other positions within the TERT promoter core generate de novo consensus binding sites for transcription factors. The molecular significance of these mutations, different from the hot spots, remain to be addressed.
Although hot spot mutations seem to be absent in UIP and in adenocarcinomas associated with UIP, further investigations are necessary to confirm these data and to understand the pathogenesis of UIP and the cancerogenesis in lung tissue with UIP.