Clinical Significance Of Fluorescence In Situ Hybridization To Detect HTERC Gene In Early Screening Of Cervical Cancer In Some Areas Of Xinjiang

Background:Cervical disease is one of the most common diseases of women, the prevalence of cervical cancer which accounts for half of the female reproductive system cancer for more than a serious threat to women's health.each year, about 46 million people around the world sick. There are new cases amounting to approximately 13 million in China, accounting for 6% of all the women has been found the tumor, which is the second only to breast cancer-related deaths of women caused by an important reason. However, cervical cancer (SCC) is the only preventable cancer. If that is the precancerous lesions or early diagnosis and timely preventive measures to give positive or appropriate treatment, which can significantly change the prognosis and quality of life. Moreover, early treatment of cervical cancer and five-year survival rate also in the forefront of cancer, this feature makes for cervical cancer screening and the study of the pathogenesis of a more important significance. Over the past decade with the rapid development of molecular cytogenetic techniques, fluorescence in situ hybridization (FISH) came into being, and gradually transformed from basic medical research into clinical testing areas. Chromosome studies have shown that some of the cancer, chromosomal instability usually presents 3q gain and 2,3,4,6 p, 11q's missing, and the abnormal gene region were located in 3 q26.1-q28. In recent years, the U.S. National Institutes of Health (NIH)-led study shows that for cervical cancer, abnormal cervical cells from atypical hyperplasia to cancer almost always associated with changes in the course of the long arm of chromosome 3 amplification, which involves the most important gene to human chromosome telomerase gene may be hTERC, it is expected to be atypical cell cancer genes, the gene amplification can prevent apoptosis, which can lead to tumor occuring. Overseas research shows that by FISH to detect 3q26 hTERC gene, found cervical intraepithelial neoplasia gradeⅡ(CINⅡ), the increase in the number of copies of this gene accounted for 63%, cervical intraepithelial neoplasia gradeⅢ(CINⅢ) accounted for 76%, tetraploid cells with the cellular number and hTERC genes increase the severity of disease, which can be used to predict a high cervical lesions (HSIL) of the independent indicator of specific genome that is abnormal development of precancerous lesions to invasive cervical cancer by the necessary conditions. Determination of the use of FISH in exfoliated cells of cervical lesions hTERC expression of gene amplification, analysis of its relationship with cervical lesions progress to assess the role of FISH technique in screening of cervical lesions in cervical cancer prevention and control will make the prospect of a new work stage of development. There were lots of research reports about the relationship between the development of hTERC with cervical cancer at home and abroad, but the study of the high incidence of cervical cancer in the southern region of Xinjiang Uygur were rarely reported, the differences between ethnic in this grim fact that we have a responsibility to promote. This research work carried out to explore the differences between Uygur and Han nationalities in the pathogenesis of cervical cancer, in order to reduce the incidence of cervical cancer in Xinjiang Uygur, looking for more clinical work to promote the scientific basis. In recent years, the etiology of cervical cancer research has made important progress, which is now a clear evidence for high-risk papilloma virus (human papillomavirus HPV) subtypes infection and multiple types infection can cause cervical intraepithelial neoplasia or cervical cancer. Moreover, there are research suggests that high-risk HPV DNA (HR HPV DNA) integration causes hTERC gene amplification and is closely related to the incidence of cervical cancer. More than 95% of cervical cancer cases occurring related to HPV infection, there are different HPV subtypes in different regions and different ethnic groups, caused by disease development and prognosis are also different. Carcinogenic mechanism of HPV present study shows that high-risk HPV infection lead to the activation of telomerase by the CIN to SCC cervical epithelial transformation process are critical steps. Kanaya and other studies show:high-risk HPV infection and p53 inactivation can activate telomerase, the accelerated proliferation of cervical epithelial cells, causing CIN and SCC. Now that the use of FISH to detect gene amplification in cervical hTERC potentially helpful value in the diagnosis of early cervical cancer. The research aims to analysis the relationship between the women with cervical lesions of all nationalities in Xinjiang of cervical exfoliated cells and cervical hTERC gene expression, the severity of studies to assess the FISH detection hTERC gene in cervical cancer screening in the clinical application and comparative analysis of Xinjiang Uygur and Han Chinese cervical lesions patients infected with high-risk HPV subtypes, whose hTERC gene amplification characteristics and differences, as well as the level of gene amplification in hTERC, discusses the differences characteristics and pathogenesis of cervical cancer between Uygur and Han, in order to seek more efficient and reliable screening, diagnostic methods in cervical lesions and cervical cancer, so as to provide a clinical basis for scientific theory. The past 10 years, due to human papillomavirus infection in a significant increase,10% -15% per year of new cases in domestic, the incidence of CIN has also increased, incidence of cervical cancer seems to be signs of a rebound. Concerning is getting younger and younger cervical cancer, cervical cancer incidence of young women by 2%-3% annual growth rate, according to the ststistics of Chinese Academy of Medical Sciences Cancer Hospital, which show that cervical cancer for women under 35 years of age accounted for about the same period the proportion of the total number of cervical cancer were treated from 1.22% in 70s to 9.88% in 90s. Each year about 30,000 women were died of cervical cancer, however, cervical cancer is a threat to women's health and of the first line killer. Early diagnosis of cervical cancer and precancerous cervical lesions is to improve the cure rate, survival rate,and also the key to reducing the incidence of cervical cancer. The current general methods for the screening of cervical lesions is liquid-based smears method combined with high risk human papillomavirus detection methods. Although the high specificity of cervical cytology, simple, low cost, large-scale screening for cervical cancer, but sensitivity is not high, it will lead to a greater degree of false negatives, and also can not be carried out quantitative analysis of the cellular level. Abnormal cytology results, the consistency between the observers is not ideal, poor reproducibility. Although HPV detection sensitivity is higher but the false positive rate is high. Positive cervical HPV infection only show that but does not mean that pathological changes in cervical cytology, which can not make accurate judgments about the pathological type and duration to development of cervical cancer, therefore, the HPV test can not replace cytology. FISH, due to its high specificity, sensitivity, fast, stable and non-invasive characteristics, to some extent make up for the existing limitations of screening methods in cervical cancer and precancerous lesions, which can be used as a clinical screening for cervical disease of important adjuvant examinations. Objectives:1) Through the detection of the amplification of human telomerase RNA gene in chromosome of women with cervical lesions of all ethnic groups in Xinjiang, to study the relationship between hTERC gene expression and HPV infection, understanding the biological mechanisms of cervical cancer, and providing a scientific basis for genetic hTERC detection in the clinical value of cervical cancer screening for early diagnosis of cervical cancer.2) Human exfoliated cells of cervical lesions telomerase RNA (hTERC) differences in gene expression of Xinjiang Uygur and Han women, as well as high-risk subtypes of HPV infection, evaluation clinical value of hTERC genetic testing in the Uygur, Han cervical cancer screening.3) Through comparative analysis of cervical exfoliated cells hTERC gene amplification, high-risk HPV DNA infection and TCT test results, to be on the FISH method for detection of hTERC gene amplification in cervical lesions early screening programs, to evaluate the application of the cervical cancer screening programs to find the best available scientific evidence. Methods:1) Select by liquid-based cervical cytology (TCT) is normal-cervical carcinoma (SCC) of the 149 cases of cervical lesions of women of all ethnic groups in Xinjiang as the research object, the use of human papillomavirus (HPV) HybrMax to detect high-risk type HPV (HR-HPV) infection, pathological examination of their lesions, fluorescence in situ hybridization (FISH) technique to detect the abnormal gene amplification of hTERC. Final pathology results to be the gold standard for the level of the research group, results were compared among of hTERC genetic test, liquid-based cervical cytology and HPV test.2) Using fluorescence in situ hybridization, comparative study of hTERC gene amplification in 50 cases of Uygur patients and 52 cases of Han patients with cervical lesions, while high-risk HPV DNA infection were detected, the pathology results as the gold standard, two groups of patients Uygur, Han hTERC genetic test results were compared with HPV test results.3) The first two parts of 156 cases of cervical lesions in cervical exfoliated cells hTERC gene amplification and high-risk HPV DNA test results and the TCT test results were compared, colposcopic cervical biopsy histopathologic diagnosis of cervix as final diagnosis gold standard, the three detection methods were calculated in sensitivity, specificity, positive predictive value, negative predictive value and Youden index,which were compared screening accuracy of cervical lesions of 3 methods, and evaluated prospects for clinical application in FISH detection of hTERC gene amplification. Results:1)①Through hTERC genetic testing cervical exfoliated cells in 129 cases of cervical lesions and 20 normal women, indicating the group of liquid-based cervical cytology is normal (NILM), atypical squamous cells (ASCUS), low grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesion (HSIL) and invasive cervical cancer (SCC) cases of abnormal cervical cytology hTERC gene amplification rate was 5.00%, 17.24%,29.73%, 80.48% and 81.82% respectively, study of hTERC gene amplification ratio of ASCUS group, LSIL group, HSIL group and SCC group was significantly higher than the normal control group, the difference was statistically significant (P<0.05); levels of lesions in the ASCUS group, LSIL group were lower than HSIL group (P<0.05), but the ASCUS and LSIL was no significant difference between groups (P>0.05).②Pathology group at all levels in cervical lesions, genetic abnormalities in patients with CINⅠ, CINⅡ, CINⅢhTERC and SCC were amplified by 21.95%, 34.48%, 85.71%. 83.33%, low grade lesions (CINⅠ) cells group abnormal hTERC gene amplification was significantly lower than the high-level lesion group (CINⅡ～SCC).2)①Number of hTERC gene amplification rate of 50 Uygur patients with cervical lesions is 76.46%, 52 cases of cervical cancer Han number of hTERC gene amplification rate is 80.91%. As the lesions progress, Uygur, Han hTERC patients with cervical lesions significantly increased the number of gene amplification. Compared with the control group, the difference was statistically significant (P<0.05); Uygur, Han cervical lesions hTERC gene amplification rate between the two groups, the difference also statistically significant (P<0.05).②In the Uygur, Han patients in two groups of multiple HPV infection and single infection group differences were statistically significant (P<0.05); HPV single infections Uygur, Han hTERC gene amplification rate between the two groups showed no statistically significant (P>0.05), but multiple HPV infection Uygur, Han hTERC gene amplification rate between the two groups was significantly (P<0.05). Uygur, Han hTERC average gene amplification factor of cervical cancer, the infection of HPV subtypes in the difference between the groups was not significant (P>0.05), total cervical lesions hTERC gene amplification factor difference between the Uygur and Han was statistical significance, may be related to Uygur multiple infections in patients with cervical lesions than the rate of cervical lesions related to the Han. Uygur, Han groups in HR-HPV DAN infection hTERC positive and negative groups were significantly different rate of gene amplification, but the Uygur, Han was no significant difference between the two groups (P>0.05).3)①156 cases of cervical cytology specimens of cervical lesions detected by FISH applications hTERC gene amplification, exfoliated cells detection rate of abnormal hTERC gene amplification in the CINⅠgroup was significantly lower than the CINⅢand SCC group, the difference was statistically significant (P<0.05); between CINⅠgroup and CINⅡgroup, CINⅢand SCC groups, hTERC gene amplification was no significant difference (P>0.05). As the lesions from low grade to high-level development, hTERC gene significantly increased the positive detection rate of amplification, and disease severity were positively correlated.②HPV infection detected the most common subtype were HPV 16, HPV58, and HPV 18, followed by HPV52, HPV31, HPV68, HPV56, HPV39, HPV33, HPV45 and HPV51,83 cases were infected with high-risk HPV, the positive rate was 53.20%, of which HPV 16 positive rate of 57.83%, HPV58-positive rate of 18.07%, HPV18-positive rate of 9.64%. (3) Classification of cervical cytology, LSIL is generally consistent with histopathologic grade in CINI, HSIL corresponding CINII and CINIII. TCT for CINI's diagnostic accuracy was 34.78%, CINII, CINIII the diagnostic accuracy was 37.83%, SCC of the diagnostic accuracy was 58.82%, the total detection rate is 43.82%.④3 ways to compare the efficiency of screening for cervical lesions, for the low-lesion group (CINI), three detection methods in terms of positive rate, sensitivity and specificity, the differences were not statistically significant (P>0.05). For a high degree of lesion group (CINII-SCC), FISH detection of abnormal gene amplification for the hTERC positive in 65 cases (59.1%), liquid-based cytology in HSIL was only 19 cases (17.3%), HR-HPV DNA-positive infections 66 cases (60.0%), pairwise comparison, only liquid-based cytology group's positve detection rate was significantly lower than FISH testing group and HPV testing group (P<0.05), three kinds of detection sensitivity and specificity were: FISH detection of group 82.7% and 73.6%, liquid-based cytology group 48.4% and 66.8%, HR-HPV DNA testing group to 86.5% and 46.5%, pairwise comparison the sensitivity of TCT group is lower than HPV group and the FISH group, specificity of HPV group is lower than TCT group and FISH testing group, the difference was statistically significant (P<0.05). Negative predictive value of the TCT group of the lowest in the three groups, HPV positive predictive value of the lowest group in the three groups, Youden index of FISH detection methods is higher than the other two groups, TCT was the lowest, the differences were statistically significant (P<0.05). Conclusion: l)In liquid-based cervical cytology and histological examination, hTERC gene amplification rate with the increase of the severity, especially in low-grade cervical lesions hTERC and high lesion group were significantly different gene expression, which can be applied FISH to detect abnormal hTERC gene amplification distinguish low-grade cervical lesions with high degree of lesions.2) hTERC gene amplification in the Uygur and Han nationalities in both groups increased with the severity of progress, amplification of hTERC gene and the signal ratio of polyploid types of high grade cervical lesions and cervical cancer in a high proportion, difference between low grade lesions and HSIL was significantly (P<0.05), hTERC gene amplification in high-risk HPV infection group was significantly higher than that of HPV-negative group, suggesting that high-risk HPV was associated with hTERC gene amplification, which can be as a progressive monitoring predictor of cervical precancerous lesions. Uygur patients with multiple HPV cervical lesions infected with a higher proportion of high-risk subtypes, leading to hTERC gene amplification obvious, may be one of the reasons the high incidence of cervical cancer in the Uygur than Han's.3) FISH technology because of its high specificity, sensitivity and speed, good stability, non-invasive and so on, to some extent make up for the current screening of cervical cancer and precancerous lesions of the limitations, it can be used as clinical cervical disease an important auxilury screening examinations. High risk of cervical cancer groups can be concentrated by genetic hTERC testing, which can increase the cervical cytology screening efficiency of cervical lesions, predict the development trend of cervical lesions.