| Ovarian cancer has the highest mortality in gynecological malignant diseases. Its treatment strategies are mainly consisted of de-bulking surgery combined with platinum-based chemotherapy. Although the survival rate has increased in recent years, about 80-85% of ovarian cancer patients will result in relapse, and chemotherapy drugs such as cisplatin tolerance. The five-year survival rate fluctuates at 30-40%. With the deepening of cytogenetics and cancer etiology understanding, a variety of studies have shown that chromosomal instability, aneuploidy and chromosome structural rearrangements(deletions, gene amplification and translocation) are closely related to tumor formation and development. However, research on multi-gene copy number changes in ovarian cancer is still in its infancy. Quantitative Multi-gene Fluorescence in situ Hybridization, referred to QM-FISH, can achieve 5-10 fold increase in Signal/Noise Ratio compared to the traditional FISH technique. This approach can quantitatively analyze more than one gene(currently more than 20 genes) simultaneously within one single tumor cell nucleus at the same time, and the target gene can be freely chosen and combined. The technique is an extremely effective tool to find and confirm the tumor specific allelic imbalance in clinical specimens that can be used for large-scale clinical studies. Furthermore, QM-FISH also has a significant role in determining resistant subpopulation in tumor cells, clonal evolution during disease progression and to identify cancer stem cells. This study is the first to apply QM-FISH technique to ovarian cancer, finding advanced epithelial ovarian cancer multi-gene copy number variations.Studies on genomic abnormalities in ovarian cancer, including comparative genomic hybridization in ovarian cancer reveals a series of gene plays an important role in the development of ovarian cancer, including MYC, RB1, CHEK2, TP53 and BRCA1. So we designed a set of probe in this study, including the combination of the five-gene probes mentioned above. The BAC clones used for the synthesis of probes were identified, and peripheral blood mononuclear cells were used in probes verification. The probes we designed and made were applied to ovarian cancer specimens and found that the image was clear, it is possible to obtain satisfactory signal intensity, the fluorescence signal / background signal ratio and signal splitting rate. Due to the various types of clinical specimens we may obtain, this study compared four kinds of samples in QM-FISH, including paraffin sections, frozen sections, tissue imprint and cytospin slides, and also developed appropriate sample selection strategy. In the case of access to a variety of specimens, the investigator should prefer frozen section, followed by the choice of paraffin. Tissue sample imprints are favorable in patients given surgical treatment. In advanced ovarian cancer patients with ascite, no matter whether or not performed the de-bulking surgery, it is always beneficial to make cytospin samples and perform QM-FISH.The second part of the application used fluorescence labeled probes same as the first part to test the five-gene copy number abnormalities in 10 patients. The results showed that 70% of the cases appeared MYC amplification, RB1 deletion was found in 40% of patients. To further study MYC amplification and RB1 deletion, study was extended to 58 epithelial ovarian cancer cases, and nearly one-third of the patients and both these two chromosomal abnormalities. Paraffin immunohistochemistry and Western blot performed in ascites cells confirmed increased MYC amplification resulting in higher protein expression levels. Combined with clinical data and postoperative follow-up, statistics showed that RB1 gene deletion is correlated with FIGO stage and histological grade. A significant increase of RB1 deletion was found in FIGO stage III-IV epithelial ovarian cancer. RB1 deletion rate is higher in poorly differentiated and undifferentiated group than in differentiated group. The study also found that the survival of patients with RB1 deletion is significantly shortened. In one specific epithelial ovarian cancer case, patient was examined both when newly diagnosed and relapsed using QM-FISH, clonal evolution was also observed. After relapse, the proportion of the subclone "4MYC1RB" rose from 11.00% to 27.59%, accompanied by two novel subclones. The study suggests RB1 as one of the indicator for ovarian cancer staging and prediction of disease prognosis.A patent was granted based on the first two parts of the work. The patent provides a method for detecting epithelial ovarian cancer gene variations. Probe compositions include MYC gene probe, RB1 gene probe, CHEK2 gene probe, TP53 gene probe and BRCA1 gene probe. With this probe set we can simultaneously detect 5 gene copy number variations for the same sample at the single epithelial ovarian cancer cell level. The kit can determine molecular pathology of epithelial ovarian cancer, indicate prognosis and guide personalized therapy, assess clinical outcomes, and monitor tumor relapse and metastasis.Ovarian cancer stem cells are closely related to the tumorigenesis, progression, relapse, drug resistance and metastasis. Therefore, the third part of this study used flow cytometry to sort ascites tumor cells and enrich the ALDH-positive subpopulation of ovarian cancer stem cells. And then the ALDH-positive cells were cultured in ovarian cancer stem cell medium in ultra-low attachment plates. After 2 weeks sphere were obtained. To verify that the cultured sphere-forming cells are cancer stem cells, both flow cytometry and immunofluorescence staining was performed. QM-FISH on tumor cells and cancer stem cells indicates the presence of less number of sub clones, and low chromosome variability. MYC appears to have increasing copy number. In one patient, BRCA1, TP53, CHEK2, RB1 were missing in a variety of combinations in different subclones.Because MYC and RB1 chromosomal abnormalities were confirmed in ovarian cancer stem cells, the fourth part of this study was designed for further extension. We analyzed published microarray data, and screened for differentially expressed genes between tumor cells and epithelial ovarian cancer stem cells. Pathway clustering of the differentially expressed genes indicate ErbB pathway are often affected in ovarian cancer stem cells. Based on abnormal expression of these genes, 18 known small molecule drugs were selected in CMAP database. Some of these small molecules have been demonstrated effective in the treatment of other cancers, some can overcome the resistance of tumor cells, and these small molecules are expected to play a role in the treatment of ovarian cancer. |
PDF Full Text Request