| Objective:To detect the protein expression and gene mutation of P53,Vascular endothelial growth factor(VEGF)and Poly-ADP ribose polymerase-1(PARP-1)in the Umbrella of normal fallopian(UNF),Ovarian Serous borderline tumor(OSBT)and High-grade serous ovarian cancer(HGSOC)and explore the role of the three molecules in the occurrence and development of HGSOC and their relationship with clinicopathological features and prognosis in this study.Methods:1.Specimens were collected from 114 cases of ovarian tumors surgically resected from January 2008 to December 2019 in the Department of Pathology of Affiliated Hospital of Zunyi Medical University,according to the diagnostic criteria of ovarian serous tumors in the 2014 World Health Organization(WHO)classification of female reproductive organ tumors:including 14 cases of OSBT and 100 cases of HGSOC,and 23 cases of UNF were selected as normal controls.2.Immunohistochemistry(IHC)Envision method was used to detect the protein expression of P53,VEGF,PARP-1 in UNF,OSBT and HGSOC respectively,and the protein expression intensity were analyzed using the image analysis software Image-ProPlus.3.Next-generation sequencing(NGS)was used to detect the mutations of the P53gene,VEGF gene and parp-1 gene in 10 cases of UNF,14 cases of OSBT,and 38cases of HGSOC.4.The analysis of the relationship between the protein expressions and gene mutations of P53,VEGF,PARP-1,and the clinical-pathological and prognosis characteristics of patients in HGSOC.5.SPSS 17.0 software was used for statistical analysis of data.Count data were tested by theχ~2 test and Fisher’s exact probability method.ANOVA was used for comparison of multiple sets of quantitative data.The T-test was used for quantitative data of two independent samples.Kaplan-Meier method was used for single factor survival analysis.The test level was indicated byα=0.05,P<0.05 indicated that the difference was statistically significant.Results:1.Expressions and intensities of P53,VEGF and PARP-1 in HGSOC,OSBT and UNF:The positive expression rates of P53 in UNF,OSBT,and HGSOC were 0%,28.6%,and 94.0%,respectively.With the increase of pathological grade,the positive expression of P53 increased gradually(P<0.05).The positive expression rates of VEGF in UNF,OSBT,and HGSOC were 17.4%,71.4%,and 92.0%,respectively.With the increase of pathological grade,the positive expression of VEGF increased gradually(P<0.05).The positive expression rates of PARP-1 in UNF,OSBT,and HGSOC were 17.4%,64.3%,and 91.0%,respectively.With the increase of pathological grade,the positive expression of PARP-1 increased gradually(P<0.05).The MOD values of P53 in UNF,OSBT,and HGSOC were 0.01,0.04,and 0.14,respectively.With the increase of pathological grade,the expression intensity of P53protein gradually increased(P<0.05)The MOD values of VEGF in UNF,OSBT,and HGSOC were 0.02,0.07,and 0.17,respectively.With the increase of pathological grade,the expression intensity of VEGF protein gradually increased(P<0.05).The MOD values of PARP-1 in UNF,OSBT,and HGSOC were 0.02,0.09,and 0.15,respectively.With the increase of pathological grade,the intensity of PARP-1 protein expression gradually increased(P<0.05).2.Mutations of p53,vegf,parp-1 genes in UNF,OSBT,HGSOC:The mutation rates of P53,VEGF,parp-1 genes in UNF and OSBT were all 0.The mutation rates of P53,VEGF,and parp-1 in HGSOC were 97.4%,2.6%,and10.5%,respectively.The difference between P53 and VEGF mutation rates was statistically significant(P<0.05),and the difference between P53 and parp-1 mutation rates was statistically significant(P<0.05),and the difference between VEGF and parp-1 mutation rates was not statistically significant(P>0.05).The mutation type of p53 gene in HGSOC was a systematic mutation,and the mutation sites were exon 4,exon 5,exon 6,exon 7,exon 8 and intron 4 and intron6,of which the mutation rate was the highest in the fifth exon was 29.7%(11/37),the mutation model was missense or frameshift mutation.The mutation type of the parp-1gene was a systematic mutation,and its mutation sites were exon 7,exon 9 and exon15,among which the highest mutation rate was 50.0%in exon 7(2/4),the mutation model was missense mutation.Vegf was a copy number variation(CNV),and there was no clear mutation site.Therefore,the p53 gene mutation rate of HGSOC was the highest in this study,followed by the parp-1 gene,and Vegf was a copy number variation.3.Correlation analysis and Kappa consistency test of P53,VEGF and PARP-1 protein expressions and gene mutations in HGSOC:Spearman correlation analysis showed that the correlation coefficient(r=0.702)between P53 protein and VEGF protein expressions was positively correlated,indicating that the higher the P53 protein positive expression,the higher the VEGF positive expression and the difference between the groups was statistically significant(P<0.05).The correlation coefficient(r=0.509)between P53 protein and PARP-1protein expression was positively correlated,indicating that the higher the P53 protein positive expression,the higher the PARP-1 positive expression and the difference between the groups was statistically significant(P<0.05).There was no correlation between PARP-1 and VEGF protein expressions,and the difference between the groups was not statistically significant(P>0.05).Kappa consistency test showed that 38 cases of HGSOC underwent NGS sequencing and immunohistochemical detection at the same time.P53 gene:κ=1.000,P<0.05,indicating that the detection results of the two methods were consistent,and the consistency was well.Vegf gene:κ=0.003,P>0.05,suggesting that the detection results of the two methods were inconsistent.Parp-1 gene:κ=0.02,P>0.05,suggesting that the detection results of the two methods were inconsistent.4.The relationship between the protein expressions and gene mutations of P53,VEGF and PARP-1 in HGSOC and the clinicopathological characteristics of patients:The positive expressions of P53,VEGF and PARP-1 proteins were closely related to the patients’International Federation of Gynecology and Obstetrics(FIGO)(P<0.05)and lymph node metastasis status(P<0.05)The higher the positive expression of this molecule,the higher the probability of patients with lymph node metastasis and the higher the FIGO stage,but they were not related to the patient’s age,peritoneal metastasis and chemotherapy resistance and other clinical-pathological features(P>0.05).P53 gene mutations were related to the FIGO stage of patients(P<0.05),the FIGO stage of patients with p53 mutation was higher than that of patients without p53mutation,and p53 mutation was not related to other pathological characteristics of patients(P>0.05).Vegf and parp-1 gene mutations were not related to the clinicopathological characteristics of patients(P>0.05).5.Relationship between P53,VEGF and PARP-1 protein expression and gene mutation and prognosis of HGSOC patients:The 3-year survival rate of HGSOC patients was 36.4%(28/77),and the 5-year survival rate was 12.5%(4/32).Kaplan-Meier method:The positive expression of P53 protein was statistically significant with the total survival time of patients in the HGSOC group,the results showed that the higher the P53 protein,the shorter the patient’s overall survival period.The positive expressions of VEGF and PARP-1 proteins had no statistical significance with the overall survival time of HGSOC patients(P>0.05).The relationship between parp-1 gene mutations and the overall survival time of patients in the HGSOC group was statistically significant(P<0.05).The results showed that the higher the parp-1gene mutation rate,the shorter the patient’s overall survival period.There was no statistical significance between the mutation of p53,vegf gene and the overall survival time of patients(P>0.05).Conclusion:1.P53,VEGF and PARP-1 protein expression may participate in the process of HGOSC development,and it can also be used to assist HGSOC diagnosis and differential diagnosis.2.P53 was positively correlated with VEGF and PARP-1 protein in HGSOC,suggesting that there may be a synergistic effect between P53,VEGF and PARP-1protein in HGSOC.3.The protein expressions and gene mutations of P53 in HGSOC were all related to the FIGO stage of the patient,and the P53 protein expressions were related to the patient’s overall survival time,suggesting that P53 may be used to evaluate the prognosis of HGSOC patients.P53 protein can be used as a surrogate marker for P53mutation by IHC detection.4.The expression of VEGF protein is related to lymph node metastasis and the FIGO stage in patients,suggesting that VEGF may be related to the high invasiveness of HGSOC.5.The higher the mutation rate of the parp-1 gene,the shorter the overall survival of the patient in the HGSOC.The parp-1 gene may be related to the prognosis of the patient. |
