Stady On The Clinical Application Of TERC Amplification Of The Exfoliated Cervical Epithelial Cells Detected By FISH

Background and objectivesCervical cancer is the second common gynecological tumor worldwide and more prevalent in developing countries, with an incidence ranging from 10 per 100,000 women in industrialized countries to 60 per 100,000 women in some developing countries. The vast majority of cervical cancer has gradually evolved from cervical intraepithelial neoplasia (CIN). CIN is a series of cervical precancerous lesions which are limited to cervical intraepithelial and have different histological changes. It generally takes about 10 years time for the development from CIN to invasive carcinoma. Cervical lesions can be cure eassily if they can be found and given effective treatments in CIN or early invasive carcinoma stage. Therefore, it is imperative to establish effective cancer screening programs, especially in developing countries, in order to reduce the morbidity and mortality of cervical cancer.Cytopathologic examination and human papillomavirus (HPV) DNA testing are strongly recommended by World Health Organization (WHO) for cervical cancer screening. The conventional Pap smear has been reported to reduce the incidence of cervical cancer by more than 70% in some countries with organized screening program. However, it suffers from relatively low sensitivity and high false negative rate and has variable test results. Compared with conventional Pap smears, the introduction of liquid-based cytology methods has increased the efficacy of population screening but not significantly improved the poor sensitivity. Due to poor sensitivity of cytology, some patients with precancerous lesions can not be detected at early stage and fail to receive effective treatment to progression to invasive cervical cancer.Infection with HPV is a necessary factor of cervical cancer, which can be detected in 99.7% of invasive cervical carcinomas. So far, more than 100 different HPV types have been identified, and approximately 13 HPV (type 16,18,31,33,35, 39,45,51,52,56,58,59,68,73 and 82) are identified as high risk HPV (HR-HPV) that can transform cells in the genital tract and lead to invasive cancer. Among these HR-HPV types, HPV 16 and 18 are the most commonly identified types in cervical cancer worldwide and strongly related to the development of cervical cancer. The causative relationship between HR-HPV infection and the development of cervical cancer provides potential roles for HPV testing in cervical screening. Hybrid captureⅡ(HCⅡ), approved by the US Food and Drug Administration (FDA), can detected 18 types of HPV, containing 13 high risk types and 5 low-risk types (6,11,42,43 and 44), but can not identify which HPV types are present and discriminate between persistent and transient infections. HPV DNA testing is more reliable and more sensitive, but has less specificity and positive predictive value (PPV) than routinely performed cytology for detection of CINⅡor higher grade lesions (≥CINⅡ). This is because most HPV infections are transient infections that can be cleared spontaneously and do not cause≥CINⅡand only persistent infections with HR-HPV, which accounts for 10-20% of HPV infections, is associated with the development of cervical cancer. Interestingly, most HPV infections are transient infections that can be cleared spontaneously and do not cause≥CINⅡand only persistent infection with HR-HPV is associated with the development of cervical cancer. HPV DNA testing is more reliable and more sensitive but has less specificity and PPV than routinely performed cytology for detection of≥CINⅡ. Thus, more specific biomarkers need to be added to HPV DNA testing to increase the specificity for detecting≥CINⅡ. This may significantly reduce the number of women, particularly those with normal cytology, who are subjected to unnecessary follow-up based on a transient HP-HPV infection. This also may highly reduce the number of women who suffer from over-diagnosis and over-treatment.In addition to HR-HPV infection, some chromosomal abnormalities and genomic instabilities are necessary for precancerous lesions to progress to invasive cervical cancer. Among these abnormalities, the most common aberration is gain of 3q26 containing the TERC gene which can be detected in approximately 70% of cervical carcinomas. Telomerase is involved in the maintenance of chromosomes by providing telomere stability and regulating telomere length. Telomerase is composed of two main components, an RNA subunit (hTERC) that serves as a template for telomere addition and a protein subunit (hTERT) that acts to catalyzes telomere synthesis. TERC gene encodes for an RNA unit of telomerase that maintains the length of telomeres through cellular divisions. When this gene is overexpressed, the cells avoid undergoing apoptosis, potentially leading to tumorigenesis. Several recent studies have demonstrated that gain of TERC gene as measured by fluorescence in situ hybridization (FISH) occurs frequently in cervical squamous cell carcinoma, high-grade and light-grade cervical intraepithelial neoplasia and that the frequency of TERC gain increase with the severity of cervical lesions, indicating that TERC might be a useful molecular marker for primary cervical screening. Alameda F found that FISH detection of 3q26 amplification can be used as a persistence-progression indicator in low-grade cervical intraepithelial neoplasia. So, TERC amplification detection of FISH may assist in cervical cancer screening and early diagnosis. At present, fluorescence in situ hybridization (FISH) technology is considered as the standard method of detection of TERC gene amplification. FISH technique is simple, reproducible and has high stability and better sensitivity and specificity, so it is suitable for clinical testing. FISH has been widely used in the areas of cellular genetics, tumor biology, gene mapping, gene amplification and prenatal diagnosis.In the present study, we detected TERC gene amplification of cervical exfoliated epithelial cells by fluorescence in situ hybridization, and compared these results with cytology, HPV results and histopathology results. Our purpose was to assess the clinical significance of FISH detection of TERC amplification in cervical cancer screening and CIN progression prediction. It may provide a new approach for cervical cancer screening, early diagnosis and progression prediction in Chinese women.Methods/MaterialsA total of 120 patients with informed consent were recruited from Nafang Hospital affiliated to Southern Medical University. All of them were the inpatients or outpatients of the hospital from March 2008 to December 2008 and excluded acute pelvic infection, pregnancy, hysterectomy, pelvic radiotherapy and chemotherapy. Each of them was subject to FISH analysis with the TERC-specific probe, HPV DNA testing, liquid-based cytology and histopathological examination in a blind fashion. Cervical cells collected from squamocolumnar junction of the cervix were stored in liquid-based cytology storage solution used for liquid-based cytology and FISH detection and HPV-DNA test dedicated storage solution used for HPV DNA testing, respectively. The biopsy specimens were also taken from the squamocolumnar junction of the cervix under colposcopy-directed. Either cytological or histopathological examination was confirmed by the experienced pathologists. The cytological diagnoses were categorized as follows:normal (21 cases), atypical squamous cells of undetermined significance (ASCUS,8 cases), atypical squamous cells-cannot exclude a high-grade lesion (ASCH,5 cases), low-grade squamous intraepithelial lesion (LSIL,22 cases), high-grade squamous intraepithelial lesion (HSIL,52 cases), and squamous cervical cancer (SCC,12 cases). The histologic diagnoses were categorized as follows:20 cases of normal,14 cases of CINI,35 cases of CINⅡ,36 cases of CINIⅡ, and 15 cases of SCC.ResultsTERC amplification was observed in 0 of 20 (0.00%) of normal and inflammatory cases, in 1 of 14 (7.14%) of CINⅠcases, in 23 of 35 (65.71%) of CINⅡcases, in 35 of 36 (97.22%) of CINⅢcases and in 15 of 15 (100.00%) of SCC cases, respectively. TERC amplification was significantly associated with cervical lesions according to cytological and pathological evaluation (P<0.001), and the positive rate of TERC amplification increased with the increasing severity of cervical lesions. Except of normal and CINⅠ, CINⅡ/Ⅲand SCC, the positive rate of TERC amplification were significantly different between other groups (P<0.005). In normal and inflammatory, CINⅠ, CINⅡ, CINⅢand SCC, the mean of abnormal cells number with TERC amplification was 0.70,3.57,10.97,17.86 and 35.87, respectively. The mean of abnormal cells number with TERC amplification was significantly different among different cervical lesions group classified by histopathological evaluation (P=0.000). Comparison between two groups, the mean of abnormal cells number with TERC amplification was significantly different among any two different cervical lesions groups (P=0.000).000). The complexity of abnormal signal pattern of TERC gene amplification increased as the severity of cervical lesions. In normal control group, the abnormal signal pattern was only 2:3. In CINⅠgroup, the abnormal signal pattern were 2:3 and 2:4, respectively accounting for 82% and 18% of the total abnormal signal pattern. In CINⅡor higher lesions group,2:5,4:4,5:5,6:6 and more complex signal types were found and the proportion of complex signal types increased. In SCC group, the abnormal signal pattern appeared more complex types such as 2:5,5:5,6:6, in which the signal type of 5:5 accounted for 22.3% of the total abnormal signal pattern. With FISH positive results as the standard, the sensitivity and specificity of detection of≥CINⅡlesions were 84.9% and 97.1%, respectively. With HPV DNA positive results as the standard, the sensitivity and specificity of detection of≥CINⅡlesions were 96.5% and52.9%, respectively. The specificity of FISH for detecting CINⅡor more severe cervical lesions (≥CINⅡ) were obviously higher than those of HPV DNA testing.ConclusionsTERC gene amplification play an important role in the development of cervical cancer. With high sensitivity and specificity, detection of TERC gene amplification using FISH has important clinical value in the screening and early diagnosis of cervical cancer and precancerous lesions. In addition to cytology and HPV testing, detection of TERC gene amplification using FISH method, significantly improve the specificity of screening for cervical cancer without significantly reducing its sensitivity. It may be serve as an important biological marker of screening for cervical cancer and precancerous lesions and a progression indicator of CIN and may provide more evidence for the clinical diagnosis and treatment of cervical cancer. In addition, as FISH detection of TERC gene can effectively distinguish between different levels of CIN, it can be used as a reliable measure to assist in diagnosis of CIN grade and guide the rational treatment of CIN.