| Background:Ovarian cancer is a very serious threat to the lives of all women.Platinum-based chemotherapy is the main adjuvant therapy for advanced ovarian cancer.However,primary or secondary drug resistance to this therapy is a major obstacle for the treatment of ovarian cancer.By using a high-throughput sequencing technique,we sought to delineate genetic alterations in recurrent ovarian cancer patients and further com-pare genetic changes in drug-resistant and-sensitive recurrent ovarian cancer patients.We also sought to study the specificity,sensitivity,and consistency of DNA biomarkers in liquid biopsy specimens and ovarian cancer tissue DNA.and aims to investigate the mechanism of the BRCA2 N372H mutation in the regulation of platinum resistance in ovarian cancer.Methods:Tumor tissue specimens and blood samples were obtained from pathologically proven recurrent ovarian cancer patients.Genomic DNA was extracted from tumor tissues,blood cells,ascites,and urine samples.The DNA Library was constructed and we use the Illumina HiSeq 4000 high-throughput sequencing platform.Bioinformatic analysis was done using the Tor-rent Suite software.(?)The BRCA2 N372H 1342A>C mutant gene was synthesized and sequenced.Plasmids were constructed and used to transfect the OVCAR-3 cell strain after lentiviral packaging.qPCR was used to detect BRCA2 N372H mRNA.The negative control for viral transfections was constructed using the same method,and OVCAR-3 empty cells were also used as a control.Western blotting was used to compare the expression of BRCA2 protein in between the mutant,transfection control,and empty cell types.Immunofluorescence was used to detect the binding of the three types of cells to Rad51 protein.A CCK8 assay was used to detect the sensitivity of the cells to cisplatin treatment Results:Ten patients with pathologically proven drug-resistant recurrent ovarian cancer and 11 patients with sensitive recurrent ovarian cancer were included.The 5-year OS for drug-resistant recurrent ovarian cancer patients(44.0±11.07months,95%CI:231.24-53.66 months)was significantly lower than that of drug-sensitive recurrent ovarian cancer patients(58±3.97months;95%CI:50.05-65.59 months;p=0.024)TP53 was the most frequently mutated gene in both drug-resistant(9/10,90%)and drug-sensitive recurrent ovarian cancers(10/11,91%).MYC and RB1 had the highest frequency of copy number variations(6/21,29%)in recurrent ovarian cancers,followed by PIK3CA(3/21,14%).BRCA2 N372H polymorphism was found in 40%(4/10)of drug-resistant recurrent ovarian cancer patients.The specificity,sensitivity,and consistency of TP53 and BRCA1 in circulating tumor-free DNA and tumor tissue DNA were 100%,73.7%,76.2%and 100%,75%,95.24%,respectively.(?)BRCA2 N372H 1342A>C mutation regulates the sensitivity of ovarian cancer cells to platinum-based drugs.Our results suggest that the BRCA2 N372H 1342A>C mutation increases the expression of BRCA2 protein in ovarian cancer cells,thus increasing the binding of BRCA2 and Rad51 proteins.This interaction enhances the DNA homologous recombination repair function of ovarian cancer cells,resulting in the development of resistance to platinum-based drugs in ovarian cancer cells.Conclusion:We uncovered extensive genetic alterations in recurrent ovarian can-cer and drug-resistant recurrent ovarian cancer exhibited unique genetic changes compared with recurrent ovarian cancer and drug-sensitive recurrent ovarian can-cer.We further showed that high-throughput sequencing using liquid biopsy spec-imens could provide an effective,specific,and sensitive approach for detecting genetic alterations in ovarian cancer.The heterozygous variation of BRCA2 N372H may be an effective screening tool to predict the recurrence of ovarian cancer due to platinum resistance. |
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