The Value Of HTERC Gene Detection By FISH In Predicting Progression And Regression Of Cervical Intraepithelial Neoplasia Grade â… 

Worldwide, incidence of cervical cancer ranks the fourth in female malignant tumor, approximately 530,000 new cases each year, including 266,000 death. In China, there are 62,000 new cases approximately every year, accounting for 30% of the world, next to breast cancer. It usually takes 10 to 20 years for the occurrence and development of cervical cancer from HPV infection to cervical cancer. Cervical cancer is the only preventable disease due to the long process. It is vital important for timely detection of precancerous lesions and effective intervention to block the occurrence of cervical cancer and to reduce the morbidity and mortality of cervical cancer.Currently, The three-step screening is widespread used in the world. Firstly, using cytology and HPV testing to screen, and then using colposcopy to check patients with abnormal results, histology examination is used to confirm lastly. Through the screening model, most of the patients with cervical lesion can be detected and received timely diagnosis and treatment. The morbidity and mortality of cervical cancer has been controlled significantly. The TCT has disadvantages such as low sensitivity, high proportion of atypical squamous cells of undetermined significance influenced by materials, human factors. Although HPV testing is high sensitivity, but low specificity,and most women are mostly transient infections, especially among young sexually active women. Above two methods can not be used to predict cervical lesions. Therefore it is essential to develop a new clinical indicator to predict the occurrence and development of cervical cancer and assist the above checks.The molecular biology and genetics show that most human malignancies is associated with chromosome instability. Many studies have found that gain of chromosome 3q defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix, which is located on chromosome 3q26 (human telomerase RNA component, hTERC). The hTERC gene prevents apoptosis, resulting in tumorigenesis. The hTERC gene amplification is the early events in the pathogenesis of uterine cervical carcinoma, maybe it is the pathogenesis of cervical cancer. To investigate the expression and clinical significance of hTERC gene amplification may provide a another way for early screening of cervical cancer and also can assist clinical diagnosis and prognosis of cervical cancer.Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s and is used to detect and localize the presence or absence of specific DNA sequences on chromosomes. Currently FISH has been widely used in cytogenetics, pathogenic microorganisms, tumor biology and prenatal diagnosis, especially in cancer diagnosis, such as breast cancer, bladder cancer, bile duct cancer, ovarian cancer detection. The research of National Institutes of Health shows that FISH technique can also be applied to detect cervical cancer which has important clinical significance. FISH can detect hTERC gene amplification of precancerous cervical lesions in varying degrees from the perspective of genomics. The amplification of hTERC gene in cervical exfoliated cells and paraffin-embedded tissue has a higher sensitivity, specificity and positive predictive value by FISH.As we all known, there are three development tendency of cervical intraepithelial neoplasia:lesions progressed, lesions regressed and lesions persisted. In fact, most CIN can regress, of all factors the regression rate depends on the level of CIN and age. There are about 60% regressed,30% persisted and 10% progressed of CIN I,40% regressed,40% persisted and 20～30% progressed of CIN II, less 30% regressed,10～50% progressed of CINIII. Depending on the age, women under the age of 30 are more likely tend to regress than women over 35 years. In general,1/3 early lesions can regress,1/3 can persist and 1/3 can progress. Aggressive treatment be taken for CIN Ⅱ/Ⅲ, but treatment of CIN I is still confused. Due to the small proportion progress of CIN I, it is more likely to lead to over-treatment. But there are still a little part of the progression, if it is not be treated, which may lead to disease progression. So now it is vital important to develop a clinical indicator to predict the occurrence and development of CIN IFor this reason, based on study of Cancer Screening Project of Zhongshan city (a large-scale screening of cervical cancer and breast cancer for women was conducted from Mar 2012 to Mar 2013). To investigate its clinical application in predicting progression and regression of cervical intraepithelial neoplasia grade I and provide original data, a restrospective study of hTERC gene in paraffin-embedded cervical tissue specimens were detected by FISH.Objective:1. To detect hTERC gene in paraffin-embedded cervical tissue specimens for women with CIN I of "Cancer Screening Project" by FISH.2. To investigate the value of hTERC gene detection in predicting progression and regression of CIN I by observing the amplification of three groups.3. To explore the relationship between hTERC gene hybridization signal ratio patterns and CIN I by comparing hTERC gene hybridization signal ratio patterns in three groups.Methods:1. The study group consisted of paraffin-embedded cervical tissue specimens from 55 patients with CIN I,13 patients whose lesions progressed from CIN I to CINII/Ⅲ or cervical cancer (The progression), in 19 of the 55 patients whose lesions persisted with CIN I (The persistence) and 23 patients whose lesions regressed (The regression). Fluorescence in situ hybridization was performed to detect the hTERC gene.2. Selecting 20 cases normal, chronic cervicitis as a control group at the same time, and establish the threshold.3. Calculating hTERC gene amplification positive rate in three groups, and use chi-square test between groups.4. Counting cells of hTERC gene amplification hybridization signal ratio patterns in three groups, and calculate the proportion of cells in all abnormal signal cells, and use chi-square test between groups.Results:1. The study group consisted of paraffin-embedded cervical tissue specimens from 55 patients with CIN I, in which 1 specimen is too small to abandon. Finally, a total of 54 cases were enrolled in the study,12 patients whose lesions progressed from CIN Ⅰ to CINII/Ⅲ or cervical cancer, in 19 of the 54 patients whose lesions persisted with CIN I and 23 patients whose lesions regressed. There were 50 cases hybrided successful at the first time,4 cases hybrided successful at the second time. The rate of hybridization success rate was 54/54 (100%).2. The threshold was established using the mean and 3 times standard deviation of percentages of the cells with abnormal FISH signal patterns based on 20 normal or chronic cervicitis as controls. The comparative threshold is 8.5%. Samples were considered positive for hTERC amplification if nuclei with abnormal FISH signal patterns were observed at values greater than 9.3. Paraffin-embedded cervical tissue specimens from 54 patients with CIN I were enrolled in this study. Among the specimens,22 samples were positive of hTERC gene amplification,12 samples whose lesions progressed,9 samples whose lesions persisted with CIN I and in 1 of the 23 patients whose lesions regressed spontaneously. The positive rates of hTERC gene amplification in patients who progressed, persisted and regressed were 12/12(100%),9/19(47.37%) and 1/23(4.35%) respectively. The difference of hTERC genomic amplification in three groups was statistically significant(P<0.001). Pairwise differences among the three groups were statistically significant by Fisher’s exact test. The hTERC positive rate of the group of progression was significantly higher than the group of regression.4. Each case counted 100 cells, the group of progression counted 1200 cells, abnormal gene amplification signal cells were 573, the rate of abnormal cell signal was 47.75%(573/1200); the group of persistence counted 1900 cells, abnormal gene amplification signal cells were 456, the rate of abnormal cell signal was 24.00% (456/1900). The group of regression counted 2300 cells, abnormal gene amplification signal cells were 92, the rate of abnormal cell signal was 4.00%(92/2300). The difference of hTERC gene hybridization signal ratio in three groups was statistically significant(P< 0.001).5. The number of cells with signal ratio pattern of 2:3,2:4,4:4 and N:≥5 in the group of progression were 8.55%(49/573),2.27%(13/573),32.46%(186/573),2.44% (14/573),34.90%(200/573),19.37%(111/573) respectively. The number of cells in the group of persistence were 23.02%(105/456),7.24%(33/456),28.51%(130/456)、 3.07%(14/456),21.27%(97/456),16.89%(77/456). The number of cells in the group of regression were 41.30%(38/92).30.43%(28/92),21.74%(20/92),3.26%(3/92), 3.26%(3/92),0.00%(0/92). Among the abnormal cells, the percentage of cells with a 2:3n 2:、4:4 and N:>5 signal ratio pattern were statistically significant (P< 0.001).The percentage of cells with a 2:3 and 2:4 signal ratio pattern in group regression was higher in patients with progression, while the percentage of cells with a 4:4 and N:>5 signal ratio pattern was higher in patients with regression. None of the patients with a 4:4 signal ratio pattern regressed spontaneously.Conclusion:1. The genomic amplification of hTERC in Paraffin-embedded tissues by FISH has the advantages of Simple, reproducible. From the perspective of genetics, using FISH can detect the amplification of hTERC cervical lesions of varying degrees.2. The detection of amplification of hTERC in paraffin-embedded cervical tissue specimens by FISH has the advantages of objectivity high successful rate, satisfied fluorescence signal.3. The detection of amplification of hTERC in paraffin-embedded cervical tissue specimens by FISH could predict the progression and regression of cervical intraepithelial neoplasia grade 1.4. The 4:4 or N:>5 signal ratio pattern may indicate the progression of CIN I.