Telomerase Reactivation Is an Early Event in Laryngeal Carcinogenesis

Abstract

The exact role and timing of reactivation of telomerase, a key enzyme implicated in cellular immortalization and transformation in the multistep process of laryngeal carcinogenesis, is still unknown. We attempted to (1) determine that quantitative differences exist in the levels of telomerase catalytic subunit (hTERT) mRNA expression among different grades of laryngeal epithelial abnormalities classified according to the Ljubljana classification; (2) determine that telomerase reactivation is an important, most probably early event in laryngeal carcinogenesis; and (3) analyze whether the relative quantity of hTERT mRNA can be used as a molecular biomarker in the early detection of precancerous lesions. The relative quantity of hTERT mRNA, expressed as an hTERT index, was analyzed in 140 frozen laryngeal tissue specimens representing different morphological stages of laryngeal carcinogenesis by using a commercially available LightCycler Telo TAGGG hTERT Quantification kit. The presence and relative quantity of hTERT mRNA in laryngeal epithelium increases progressively with the degree of epithelial abnormalities. hTERT mRNA was detectable in 1/15 normal laryngeal epithelia (7%, mean hTERT index 0.02), 3/15 simple hyperplasias (20%, mean hTERT index 0.09), 10/27 abnormal hyperplasias (37%, mean hTERT index 0.18), 9/12 atypical hyperplasias (75%, mean hTERT index 0.74), 8/9 intraepithelial carcinomas (89%, mean hTERT index 1.82), and 53/62 invasive laryngeal squamous cell carcinomas (85%, mean hTERT index 2.51). Statistical analysis revealed two groups of laryngeal epithelial changes with significant differences in the levels of hTERT mRNA expression (P < .0033): (1) normal and reactive hyperplastic laryngeal epithelium (simple and abnormal hyperplasia) and (2) atypical hyperplasia (precancerous lesion), intraepithelial and invasive laryngeal squamous cell carcinoma. The results of the present study suggest that telomerase reactivation is an early event in laryngeal carcinogenesis, detectable already at the stage of precancerous laryngeal epithelial changes. Nevertheless, other genetic abnormalities appear to be necessary for progression of these epithelial abnormalities toward invasive laryngeal squamous cell carcinoma.

Telomerase catalytic subunit in laryngeal carcinogenesis—an immunohistochemical study

“…15,16,22 We have previously shown by RT-PCR that telomerase reactivation, measured indirectly by the levels of hTERT messenger RNA (mRNA), is an early event in laryngeal carcinogenesis, detectable already at the stage of precancerous lesions. 12 Although the RT-PCR is sensitive for detecting even small quantities of hTERT mRNA, its main drawback is that the tissue morphology is destroyed, thereby preventing the detection of particular cells with telomerase activity. It is well known that, in addition to neoplastic cells, activated lymphocytes do possess low levels of telomerase activity and may influence RT-PCR results.…”

“…Interestingly, although the relative quantities of hTERT mRNA detected by RT-PCR in squamous and basal/ parabasal cell hyperplasia were higher than in normal laryngeal epithelia, the relative quantities were still low and the differences were not statistically significant. 12 Morphologically, only nuclei of basal and parabasal cells showed hTERT protein immunoreactivity in squamous and basal/parabasal cell hyperplasia, mostly as one single small dot, while the nuclei in the spinous (prickle cell) layer were negative. These data suggest that hTERT protein becomes undetectable with maturation or differentiation of laryngeal epithelial cells, reflecting inhibition of hTERT gene trascription and the disappearance of telomerase activity.…”

“…These data support the results of our previous study, in which we detected significantly higher relative quantities of hTERT mRNA in atypical hyperplasia, intraepithelial carcinoma and invasive laryngeal squamous cell carcinoma than in reactive hyperplastic laryngeal epithelia. 12 We believe that significantly higher hTERT protein signals per nucleus in precancerous lesions (atypical hyperplasia), intraepithelial and laryngeal squamous cell carcinomas are the consequence of hTERT gene re-expression as a part of the multistep process of laryngeal carcinogenesis. Since no statistically significant differences exist in the numbers of hTERT protein signals per nucleus between atypical hyperplasia, intraepithelial and invasive squamous cell carcinoma of the larynx, other genetic abnormalities than telomerase reactivation must be responsible for their development and/or progression.…”

“…24 In agreement with the low number of hTERT protein signals per nucleus in normal laryngeal epithelium, as shown by immunohistochemistry, we previously detected low relative quantities of hTERT mRNA in just 7% of normal laryngeal epithelia. 12 hTERT Protein in Regenerative (Squamous and Basal/ Parabasal Hyperplasia) Laryngeal Epithelium…”

“…11 We have recently demonstrated that telomerase catalytic subunit (hTERT) mRNA relative quantities increase progressively with the degree of laryngeal epithelial abnormalities by using RT-PCR, and hypothesised that hTERT gene re-expression represents an early event in the multistep process of laryngeal carcinogenesis. 12,13 The aim of the present study was, therefore, to analyse whether hTERT protein immunohistochemistry parallels hTERT mRNA relative quantities, for example, reflects hTERT gene expression, in different morphological grades of laryngeal epithelial abnormalities and squamous cell carcinoma of the larynx.…”