In the present study, we investigated the relation between the hTERC amplification and laryngeal lesions using fluorescent in situ hybridization (FISH) in Chinese Han population. We found that the percentage of hTERC amplification in patients with moderate dysplasia (MD), severe dysplasia (SD), carcinoma in situ (CIS), and invasive carcinoma (IC) was significantly higher than those with normal epithelium (NE) or mild dysplasia (Md). The amplification of hTERC was significantly associated with the histopathologic diagnosis.
Emerging evidence suggest that transition from normal epithelium to dysplasia and squamous cell carcinoma is related to the progressive accumulation of genetic changes leading to a clonal population of transformed epithelial cells . Despite extensive research into these genetic changes in laryngeal carcinogenesis, reliable genetic markers with diagnostic and prognostic value are still lacking.
Telomerase is a ribonucleoprotein enzyme that adds the TTAGGG repeats to the chromosomal ends and maintains chromosome integrity during DNA replication. Activation of telomerase can lead to cellular immortalization, prevent from apoptosis and potentially promote tumorigenesis . The human telomerase RNA component (hTERC) gene, localized on chromosome 3q26, encodes the RNA component of human telomerase, constituting one of two major subunits, and acts as a template for the addition of the repeat sequence . When hTERC is overexpressed, the cells with critically short telomeres avoid undergoing apoptosis, potentially leading to tumorigenesis .
In our study, we detected the amplification of hTERC by FISH in all cases of invasive carcinoma of larynx. This result indicates demonstrated that amplification of the hTERC gene is one of the most frequent amplifications described in LSCC, and it was increased with increasing stage of dysplasia and had a high incidence in invasive carcinoma. These results support a previous study by Soder et al, which detected more than two copies of hTERC per cell in 97% (29/30) of head and neck squamous cell carcinoma (HNSCC) by FISH . While studies employing comparative genomic hybridization (CGH) have reported lower frequency for 3q26 gains (between 50-82%) in HNSCC [23–26], this difference is likely attributable to the different sensitivity of the respective techniques. Detection of trisomy using CGH requires this numerical aberration to be present in at least 40% of the cells. In contrast, FISH detects changes on a single-cell basis and is therefore not sensitive to dilution. Furthermore, small, regional, low-copy number amplification may escape detection by CGH .
Interestingly, the amplification of hTERC was observed at low frequency in normal epithelium and mild dysplasia, compared with a significantly higher frequency in cases of the moderate dysplasia, severe dysplasia, carcinoma in situ and invasive carcinoma. This finding supports that the amplification of hTERC may be a transition event in the progression to invasive squamous cell carcinoma of larynx. It is in agreement with previous studies, the gain of chromosome 3q was reported as the most common site of genetic overrepresentation in mucosal squamous cell carcinomas and has suggested it is a pivotal transition event in cancer pathogenesis [10, 11, 28]. The finding also supports the possibility for hTERC amplification as a clinically useful genetic marker assisting histopathologic analysis for the differential diagnosis of low-grade (< mild dysplasia) versus high-grade (> moderate dysplasia) of laryngeal dysplasia. The finding was consistent with results reported by other investigators. Luzar et al demonstrated that the presence and relative quantity of Human telomerase reverse transcriptase (hTERT) mRNA increases progressively with the degree of laryngeal epithelial abnormalities. Statistical analysis revealed two groups of laryngeal epithelial changes, with significant differences in the levels of hTERT mRNA expression (P < 0.0033): (i) normal and reactive hyperplastic laryngeal epithelium (squamous and basal-parabasal hyperplasia), and (ii) atypical hyperplasia (potentially malignant lesion), CIS and invasive laryngeal SCC . They also found the telomerase catalytic protein immunohistochemistry parallels well with hTERT mRNA relative quantities in laryngeal carcinogenesis. The results indicate that telomerase reactivation is an early event in laryngeal carcinogenesis, already detectable at the stage of precancerous laryngeal epithelial changes .
Gene mutations and chromosomal abnormalities are commonly present in tumor cells. Chromosome aneuploidy and structural anomalies lead to genomic instability, resulting in tumorigenesis. In the present study, the percentages of cells with hTERC amplification increased with increasing severity of disease, and the amplification patterns got more diverse and complex as well. Notably, more complicated patterns of hTERC amplification other than 2:3 were found only in the high-grade lesions. The study demonstrated that chromosome aneuploidy was a common event in laryngeal lesions. Whether amplification patterns can be used as diagnostic or prognostic markers warrants further investigation.
In conclusion, We have analyzed the amplification of hTERC in different stages of laryngeal carcinogenesis. The present study suggests that FISH analysis can be performed on histological specimens in order to detect the amplification of hTERC for the diagnosis of laryngeal lesions. The hTERC amplification assay may be an adjunct to histopathologic screening and may be useful in assessing the potential of individual lesions to progress.