Genomic amplification of the human telomerase gene (hTERC) associated with human papillomavirus is related to the progression of uterine cervical dysplasia to invasive cancer

Cervical cancer (CC) is the second leading cause of cancer deaths in women, with more than 80% of these occurring in developing countries that have limited access to screening programs. It is estimated that 12,170 women will develop cervical cancer and about 4,220 women will die from cervical cancer in the United States during 2012[1]. And in China, the mortality rate of cervical cancer ranged from 2 to 4 per 105 population in urban areas, 0 to 7 per 105 population in rural areas during the period of 1996 to 2005[2]. Remarkably, it always takes about ten years to arise from precancerous lesion to invasive cervical cancer. For this reason, the effective screening of precursor lesion is of great importance, which makes cervical cancer preventable and curable. Cytological examination and HPV test are the most widely applied screening methods for CC and its precursor (cervical intraepithelial neoplasia, CIN) lesion. Although the implementation of the Papanicolaou test has been in routine, the test has limited accuracy not likely to be improved with enhanced collection and screening procedures. Specifically, the inability to distinguish high-grade CIN with potential possibility of progression to invasive cancer from pathologically insignificant or regressing dysplasia contributes to overtreatment, whereas false-negative results could not been eliminated. Furthermore, although 95% of patients with precancerous lesions harbour oncogenic HPV, only a small fraction of these eventually progresses to invasive carcinoma (invCA)[3]. Progression of a high-risk HPV infected CIN lesion to invCA is a result of further specific genetic alterations within the host cell genome. One of the consequences that are crucial for tumor development is an increased activity of telomerase.

Telomerase is a ribonucleoprotein enzyme complex that adds 5^′-TTAGGG-3^′ repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for about 90% of cancers[4]. Telomerase consists of several subunits, including hTERC that serves as a template during telomere elongation and a catalytic subunit, human telomerase reverse transcriptase (hTERT), which has reverse transcriptase activity. The amplifying of hTERC gene can stop cell apoptosis, consequently leading to tumor occurrence. Now we know that the detection of gain of hTERC may make clear distinguish between cytologically low-grade precancerous lesions and high-grade lesions, reaching sensitivity and specificity of over 90%[5, 6].

Tumorigenesis is a complex phenomenon, whose manifestations may appear simultaneously on multiple levels, the genomic, proteomic, and functional alternation. The correlation between telomerase activation during cellular immortalization and in tumor progression suggests that telomerase activation is required for tumor growth. Moreover, telomerase activity is dependent on the expression of 2 main core component genes, hTERT and hTERC. This has prompted a large number of studied addressing telomerase activity using TRAP assay[7, 8], amplification of hTERC using in situ hybridization[5, 6, 9‐12] or expression of hTERT using IHC in CC and CIN lesions[13‐15]. Cluster analysis between methods applied to CIN have shown that each level (phenotype, genotype, progression) should not be studied by itself, as there may be causal or linear relationships between them, which could explain more extensively and accurately the tumorigenesis pathway as a dynamic process[16]. Until now, however, there is no one study that has been designed to detect these three biomarkers in one research in clinical samples from CC and CIN lesions.

In this study, we observed, in a large sample scale, amplification of hTERC for progression from normal cervical tissues to dysplastic lesions and to invasive carcinomas in Chinese women. And we investigated gain of hTERC, expression of hTERT, and telomerase activity, and assessed associations among them. In addition, we assessed gain of hTERC in relation to physical status of HR-HPV infection. The results were analyzed to gain insights into the potential possibility for future clinical applications.

Genomic amplification of the hTERC in cytologic specimens was significantly correlated with the severity of dysplasia. Patients with biopsy diagnoses of CIN or CA had significantly higher percentages of cells with extra copies of hTERC than did patients with a normal biopsy diagnosis. This test may be a complementary to regular screening methods to detect patients with high-grade lesions.

Several authors have shown that the percentage of cells with gain of hTERC copy number increased with the severity of dysplasia[5, 6, 9‐12, 18]. Heselmeyer et al. analyzed 57 thin-layer slides by FISH for the visualization of chromosomal copy number changes and found 63% of the HSIL( CIN2) lesions and 76% of the HSIL(CIN3) lesions showed extra copies of 3q[5]. In another paper, the same group designed a retrospective study set to 59 previously stained Pap smears, and proposed that gains of chromosome arm 3q play a crucial role in the progression of severe dysplasia/carcinoma in situ to invCA[6]. One study else on tissue sections also showed that 3q gain was detected in all of 12 cervical adenocarcinomas[8]. In our study, we analyzed a series of 114 Chinese women. Based on intensive test of exfoliated cell samples, our data show that extra copy numbers of hTERC present in 50% of CIN2, 81.5% of CIN3 and 100% of CA, no matter of what kind of histology type, of cases respectively. This result was similar to other studies in China[18, 19]. The percentage of successfully hybridized samples was 96.49%, considerably higher than in the series of Pap smears[5].

Hongxiu Han et al.[20] suggested helpful biological markers would be in need of distinguishing CIN1 from CIN2/3. The expression of hTERT was evaluated as a potential marker of the high-grade premalignant CIN 2/3 lesions[14]. A recent study showed that overexpression of hTERC and hTERT together in vitro greatly induced telomerase activity and telomere length elongation, while stable overexpression of either hTERC or hTERT alone induced telomerase activity and telomere length elongation to a lesser extent[21]. Consistent with this study, several studies also highlighted the fact that both telomerase components restrict telomerase activity and telomere length in vitro, suggesting that the tumorigenic effects of hTERT overexpression are reliant on hTERC expression and in fact hTERT overexpression in an hTERC deficient background has anti-tumourigenic effects[22‐24]. Our data showed that in cervical intraepithelial lesions, levels of hTERC amplification were correlated with telomerase activity and hTERT expression. Furthermore, comparing with biopsy procedure, exfoliated cells samples used for hTERC testing in our study were easier to get from patients with a concurrent physical examination. From this point of view, we suggest there is the potential value of detecting hTERC gain by FISH method for distinguishing between lesions with and without progression to in situ or invasive malignancy of uterine cervix.

In a previous study by Sokolova I et al.[11], 235 liquid-based preparation from cervical specimens were analyzed for aberrations of 3q26 using a commercially two-color FISH probe. They found in cases diagnosed as LSIL on cytology, the percentage of cells with gain of 3q26 did not differentiate those histologically diagnosed as high-grade dysplasia (CIN2/3) from those diagnosed as low-grade dysplasia (CIN1). Unfortunately, only a few cases had both HPV and FISH testing from one vial in that study. As shown in our study, we not only analyzed abnormal cells on exfoliated cervicovaginal preparations for gain of hTERC using a FISH probe similar to that used by Nancy P et al.[12], but also each case had both HPV testing and concurrent biopsy results. In cases of LSIL, gain of 3q26 differentiated those lesions that had CIN2/3 on biopsy from those that did not.

It is not sufficient for HPV screening assays by itself in predicting the progression from atypical or mild dysplasia to high-grade lesion and subsequently to cancer, because this screening is lack of a high positive predictive value for the presence of dysplasia. Furthermore, a report indicated that some kind of cervical cancer, such as minimal deviation adenocarcinoma, was not associated with high-risk HPV[25]. Qisang et al.[26] reported a significant positive correlation between positive hTERC gain and HR-HPV infection, histopathologic lesions (all P<0.01) in patients with cervical disease. Our study demonstrates that there are 23% HPV-infected positive cases in NNL, and 78.95% in CA group. From the gray-scale graph, we can see the HPV position ratio increase according to the severity of dysplasia. However, the predictive value of hTERC amplification test is much better than HPV test. Only 3.85% hTERC amplification positive ratio was observed in NNL group, and 100% in CA group, including two cases of minimal deviation adenocarcinoma. If combined these two tests together, it seems like much better than any one of them alone. In NNL group, there was not any case with both HPV and hTERC positive. From the clinical perspective, we hypothesize that hTERC gain, combining with testing of HPV infection by HC2, might have a very significant clinical application.

In summary, this study presents gain of hTERC in exfoliated cervicovaginal epithelial specimen and suggests a role of the factor in the biology of cervical cancer. Potential clinical usefulness of the hTERC FISH would be to distinguish patients with clinically significant cervical lesions from those that are insignificant lesions with a high risk of progression from those with a low risk of progression or an adjunct to cytology screening, especially in HPV-infection patients. However, more studies are needed to determine the application of hTERC gain in surveillance and prognosis of invasive cervical cancer.