| BackgroundCervix cancer and breast cancer are frequent gynecological malignant tumor in recent years, the incidences of which are on the top in our country, with the tendency to rise year by year. To the treatment and prevention of tumors, the foremost principle is early diagnosis then early treatment. Therefore, on the process of tumorigenesis and development, the detection to cytogenetic changes, is an important method for early diagnosis and early treatment of tumors, which is of great significance to improve prognosis of patients and raise life quality.At present, the early diagnosis against to cervix cancer, mainly contains cell morphology examine, HPV virus detection, and some other common tumor marker screening. The screen for breast cancer is mainly imageological examine and IHC etc. Nevertheless, the traditional methods can't satisfy the both side of sensitivity and specificity.Fluorescence in situ hybridization (FISH) is a technology conjunct of in situ molecular cyto hybridization and fluorescent technique, compared with the traditional cytogenetical technique, which has the advantages of high sensitivity, strong signal, fast, convenience and accurate allocation etc. we can utilize this to do interphase cytogenetics researches on the solid tumor about chromosomal structural abnormality, chromosomal numerical abnormality, gene allocation etc. Interphase FISH tech associate the cytogenetics changes to the cyto-types, locations, histological structure together, which can be used to study the relationship between genetic materials and the dynamic change of tumors, and conduce to find on early stage, the tiny genetic materials change before tissue pathomorphism change, detecting the low frequency chromosomal abnormality in considerable cells, the sensitivity of which is up to 1/1000, to provide a powerful evidence for the early discovery, early diagnosis and early treatment.Gene amplification is a kind of relative increase of gene copy number in cytes, which is one mechanism of over expression for variety of oncogene in tumor cells. The extent of amplification arranges from one ore two copies to thousands of ones, which is related to selective growth vigor and drug resistance of tumors. Nowadays, various kinds of direct labeling probe systems have been applied to detect different oncogene amplification, and the analytical method of FISH is extremely suitable for study of gene amplification, the sensitivity of which can display the corpuscular heterogeneity well.hTERC(human telomerase RNA complement) gene is on chromosome band 3q26.3. Telomerase, the nuclear ribonuclear protein compound that consists of RNA and protein subunit, synthesizes telomere repeated sequence, using the self RNA complement as template, protein complement as the reverse transcriptase, to maintain the length of telomere, the activity of which is considered to be concerned with malignant cell immortalization. During the normal cell cycle of eucaryon, the terminal end of eucaryon chromosome would shorten with cell division, till the cell senescence to death. The amplification of this gene may prevent cells from apoptosis, thus lead to oncogenesis. hTERC gene unusual amplification may be much probably the early event of neoplasia. The detection of hTERC gene aberration could therefore help to screen and early diagnosis of tumors.Her-2/neu proto-oncogene on the 17q21, belongs to the category of receptor tyrosine kinas (RTK), coding the growth factor receptor C-erb-2 with the activity of RTK. The amplification and overexpression Her-2 gene may lead the signal of growth factor conduction abnormal, disorder of cell cycle control, inhibition of apoptosis, and promote the genesis, development, infiltration and metastasis. According to the information abroad, approximately 1/3 invasive breast cancers have the event of amplification or overexpression, this kind of which is high malignant, early recurrence and metastasis, low DFS and OS, especially insensitive to routine endocrine and chemo treatment program. So, we can take Her-2 as an eligible target for antitumor drugs. Rastuzumab(Herceptin)—the first humanized monoclonal antibody as treatment medicine to be direct against the Her-2 protein on cell surface—is of great significance to antitumor treatment. However, as a target of antitumor treatment and one of the independent markers to prognostic judgment, it is in urgent need to determine the exact state. The positive or not, is the standard to guide clinical medication of Herceptin or not.Our study purposed to set up the standard procedure for FISH detection. The factors influencing the detection quality contain the species of samples, collection methods, the fresh extent of samples, the pretreatment, the hybrid condition and so on. Every factor would affect the final results of FISH detection. Therefore, we should find the optimal experimental condition according to the different states. Discussion how to operate the detection of FISH on the cyto samples get through routine methods, and the possibility to take the hTERC gene as a marker of early diagnosis of cervix cancer. By now, the research on the relationship between hTERC, Her-2 gene and cervix cancer, breast cancer respectively, are both from reports abroad. In order to spread the FISH technique on clinic in our country, to establish the detection threshold of Chinese, to analyze the genetic changes of organism, it is necessary not only to early diagnosis, but also the research on medication of targeted therapy for cancers.Methods1. on the foundation of early researches and experiments set up the complete technology for FISH to detect cervical epithelial cells and breast glandular epithelium cells. Standardize the method of collection and pretreatment for both samples of cervical epithelial cells drop slides and mammary gland histological section. Cervical epithelial cells are collected with thin-layer cervical cytology test (TCT) and normal sodium respectively, while breast glandular epithelium cells are divided to paraffin section and frozen section, the procedures of which are standardized and improved to the optimal hybrid condition, to ensure the stability of results.2. Establish the detection threshold of samples. Collect 10 cervical samples of health adults under the standard method from early experiment to have FISH detect, every piece analyzed 100 cells at least, and then calculate the percentage of cell numbers with hTERC signals above 2. Threshold=Mean(M)+3×Standard deviation(SD). The threshold of breast glandular epithelium cells is refer to the guide instituted combined with American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP), related to Her-2 of breast cancers in clinical detection.3. Take the 95 cervical samples as detection object, comparing the different amplification states in four different extents of neoplasia; refer to the pathologic diagnosis, detecting the states of high risk HPV infection at the mean while; Take the 40 breast cancers as detection object, detecting the amplification states of Her-2 gene, to make sure that the proportion with amplification in breast cancer patients, and compare FISH with IHC, whether has a coincident result or not. Evaluate and compare the two methods to detect gene amplification with FISH, and determine the standard detection procedure for clinical samples.ResultsWe complete the technology of FISH, which can be used to detect the different origins and types of samples, and the hybrid signals are clear, has a better stability and reproducibility.2. The threshold of hTERC gene in cervical epithelial cells is not below 1.7%.3. The results of amplification for hTERC gene detected in cervical samples showed: the positive percentage in high-grade CIN(Ⅱ/Ⅲ) is 65.8%(25/38), and the one in cervix cancer is 93.3%(14/15), are both significantly higher(P<0.05) than that of inflammation/CIN group 3.13%(1/32); the positive percentage in CINⅡand CINⅢis 60%(6/10) and 72.2%(13/18) respectively, which are both significantly higher (P<0.05)than that of inflammation/CIN group 3.13%(1/32). The positive percentage of CINⅢgroup and cervix cancer group has no significance(P>0.05). The positive percentage of hTERC gene amplification increased as the advancement of pathological grade. The sensitivity(Se) of the two methods of FISH to detect hTERC gene and HC-2 to detect HR-HPV infection were 39/53(73.6%),37/53(69.8%); the specificity(Sp) were 31/32(96.9%),17/32(53.1%); the positive prediction value(PPV) were 39/40(97.5%),37/52(71.2%); the negative prediction value(NPV) were 31/45(68.9%),17/33(51.5%).4. The results of amplification for Her-2 gene detected in breast cancer samples showed: 37.5%(15/40)of the samples are positive for Her-2 gene amplification. 8 cases which had the positive result for FISH, and the IHC were all 3+; 9 cases had the result of 2+ for IHC, while 6 in 9 had a positive result for FISH; 23 cases had the result of 0/1+ for IHC, only 1 in 23 had a positive result for FISH. The two methods to detect had a concordance on the statistic analysis of their results. Conclusion1. hTERC gene took a part in the carcinogenesis and development of cervix cancers, could be taken as one of the markers of CIN and cervix cancers. The detection to hTERC gene with FISH could be more reliable to identify the benign from the malignant neoplasia, connected to the detection of HR-HPV infection would provide a more effective, more reasonable program to improve the early diagnosis percentage of cervix cancers.2. The methods of FISH and IHC to detect Her-2 gene amplification or overexpression respectively had concordance in statistics; however the combination CEN probe and GLP probe, not only can observe morphology and detect amplification, but also correct the false positive caused by the aneuploid phenomenon which the method of IHC can't do.3. The technique of FISH could detect the aberration on chromosome or certain gene in some tumors, and be operated on the cyto drop slides, tissue paraffin sections and frozen sections. |
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