Study Of Microsatellite Instability And The Expression Mismatch Repair Gene In Epithelial Ovarian Cancer

Ovarian cancer represents the most lethal malignancy among gynecological tumours in China, in which 90% are epithelial ovarian tumors. Etiology is yet unknown. Multistep process, with accumul ation of genetic alterations concerning factors with key role in cell regulation -oncogenes, tumor-supressor genes and mismatch-repair genes is supposed. This study was conducted to evaluate the frequency of MSI and the expression of the mismatch repair gene in epithelial ovarian tumors and their relationship with clinicopathologic features, in order to know whether there is genomic instability and losing expression of mismatch repair gene. In this study we have explored the mechanism of hMLH1 gene silencing by analyzing the aberrant methylation of CpG islands in its promoter. It helps us to understand the molecular events involved in the development of ovarian tumors.Part I: Study of microsatellite instability and its relationship withclinicopathologic features in epithelial ovarian tumorsObjective: This study was conducted to evaluate the frequency of MSI inepithelial ovarian tumors and its relationship with clinicopathologicfeatures.Methods: ninety fresh specimens of epithelial ovarian tumors (primary 74,secondary 16) and twenty fresh specimens of ovarian cyst as control werecollected from The Obstetrics and Gynecology Hospital of Medical Centerof Fudan University from 2004 to 2005. Microsatellite analysis was carriedout using 5 mono- and dinucleotide markers from the National CancerInstitute Consensus Panel by fluorescence-labeled polymerase chainreaction. The results were auto-analyzed by software of GeneScanAnalysis3. 7 and Genotyper.Results: 1): Of the 20 ovarian cysts analyzed, all were demonstrated MSS.Of the 90 epithelial ovarian tumors analyzed, 18 were demonstrated a highlevel of MSI (MSI-H), 30 were demonstrated a low level of MSI (MSI-L),and the remaining 42 exhibited microsatellite stability (MSS).2): Frequency of MSI at loci BAT-25 was higher than that at any other loci. No correlation was found between MSI level and patient age, tumor type, tumor differentiation (P >0.05).3): The microsatellite instability-high phenotype correlate with clinical stage, it tended to occur more frequently in early-stage tumors (P=0.03).Conclusions: Our results indicate that there are frequent MSI in epithelial ovarian tumors. It is an early event and it is involved in the development of epithelial ovarian tumors.Part II: Study of expression of mismatch repair genes and its relationship with clinicopathologic features in epithelial ovarian tumors Objective: This study was conducted to evaluate the expression of mismatch repair genes in epithelial ovarian tumors and its relationship with clinicopathologic features. Investigate the correlation between the MSI and the expression of mismatch repair genes.Methods: Ninety fresh specimens of epithelial ovarian tumors (primary 74, secondary 16) and twenty fresh specimens of ovarian cyst as control were collected from The Obstetrics and Gynecology Hospital of Medical Center of Fudan University from 2004 to 2005. Comparatively quantitative analysis of expression of MMR genes (hMLH1, hMSH2, hMSH3, hMSH6, hPMS1, hPMS2) were conducted by real-time polymerase chain reaction. Results:1): The quantity of expression of mRNA of the hMLH1 gene in the ovarian cyst is significantly higher than that in the epithelial ovarian tumors (P<0.05) . The quantity of expression of mRNA of the hMLHl gene in high grade tumor is significantly higher than that in the low grade tumor (P<0.05) .There is a significant correlation between the quantity of expression of mRNA of the hMLHl gene and the status of the microsatellite(P<0.05). No correlation was found between the quantity of expression of mRNA of the hMLHl gene and tumor type, tumor stage (P>0.05). 2) : The quantity of expression of mRNA of the hMSH2 gene in the ovarian cyst is not significantly higher than that in the epithelial ovarian tumors (P>0.05) . No correlation was found between the quantity of expression of mRNA of the hMSH2 gene and tumor type, tumor stage, tumordifferentiation and the status of the microsatellite. (P>0.05).3): The quantity of expression of mRNA of the hMSH3, hMSH6, hPMS1, hPMS2 gene in the ovarian cyst is significantly higher than that in the epithelial ovarian tumors (P<0.05) . No correlation was found between the quantity of expression of mRNA of the hMSH3, hMSH6, hPMS1, hPMS2 gene and tumor type, tumor stage, tumor differentiation and the status of the microsatellite (P>0.05).Conclusions: The losing expression of MMR genes, especially hMLH1 gene have a key effect in the tumorgenesis of the epithelial ovarian tumors, which can result in MSI in the tumor.Part III: Study of aberrant methylation of promoter CpG islands of hMLHlgene in epithelial ovarian tumorsObjective: To analyze the aberrant methylation of CpG islands in hMLHlgene promoter by using MSP (methylation specific polymerase chainreaction), and then to investigate its relationship withclinicopathologic features, the expression of hMLHl gene and the statusof the microsatellite.Results: 1): The frequency of the methylation of CpG island in the hMLHlgene in control group (0%) were significantly lower than that inepithelial ovarian tumors (16.6%) (P<0.05).2): No correlation was found between the frequency of the methylation of CpG island in the hMLHl gene and tumor type, tumor stage, tumor differentiation (P>0. 05) .3) : There was a highly significant negative correlation among methylation of CpG island in hMLHl gene, mRNA expression of hMLHl gene and the status of the microsatellite(P>0.05).Conclusions: Methylation of CpG island in hMLHl is the main cause of its loss expression. It may be also the key mechanism which induce the MSI-H in the epithelial ovarian tumors.