A Preliminary Study On The Pathogenesis Of Endometriosis-associated Ovarian Cancer

Endometriosis(EMs)is one of the most common gynecological disease.Although EMs is a benign disease in histology,but it has some malignant behaviors such as invasion,metastasis and recurrence.In 1925,Sampson firstly reported that endometriosis may be a precancerous lesion of epithelial ovarian cancer(EOC),and the concept of endometriosis-associated ovarian cancer(EAOC)was also proposed.With the "binary disease model" proposed and the second generation high-throughput sequencing technology developed,a growing number of studies have confirmed that endometriosis is a risk factor of ovarian cancer,EAOC patients may have distinct characteristics and ovarian clear cell carcinoma(OCCC),ovarian endometrial adenocarcinoma(OEC)are the most common types of EAOC.However,the clinicopathological characteristics of EAOC patients are still unclear and the effect of EMs on the prognosis of patients with EOC are controversial,the pathogenesis of EAOC is still unclear by far.In the first part of this study,we retrospectively analyzed the clinicopathological features and prognosis of 256 patients with OCCC or OEC,aiming to find some distinct clinicopathological characteristics of EAOC patients and the influence of endometriosis on the prognosis of OCCC and OEC patients.On this basis,whole-exome sequencing(WES)was performed on paraffin-embedded cancer tissues of 7 patients with endometriosis-associated OCCC and 10 patients with non-endometriosis-associated OCCC.The normal paraffin-embedded ovarian tissue of the same patient was used as control.It was designed to detect the significant mutated genes of the EAOC patients,and to preliminously explore the role in EAOC patients.Part ? Clinicopathological characteristics of patients withendometriosis-associated ovarian cancer Objective:To investigate the clinicopathological features of patients with endometriosis-associated ovarian cancer(EAOC)and the effect of endometriosis on prognosis of EAOC patients.Methods:In Women's Hospital,School of Medicine,Zhejiang University and Zhejiang Cancer Hospital,256 patients diagnosed with OCCC,OEC and mixed epithelial ovarian cancer composed with OCCC or OEC were collected.Patients were divided into two groups: EAOC and NEAOC group.After that,the clinicopathological characteristics and prognosis between the two groups were compared.Results:1.Endometriosis was found in 27.3% OCCC and OEC patients.2.Compared with the NEAOC group,patients in the EAOC group were more likely with OCCC,FIGO I and II stage,no residual lesions after primary surgery,and the preoperative serum CA125 level was usually normal or slightly elevated.3.The 5-year progression-free survival(PFS)rates in EAOC and NEAOC groups were 83.3% and 54.7%,respectively(p<0.001).The 5-year overall survival(OS)rates in EAOC and NEAOC groups were 89.7% and 63.9%,respectively(p=0.001).Conclusion:1.Compared with patients with NEAOC,patients with EAOC were mostly OCCC,the FIGO stage was mostly in the early stage,the preoperative serum CA125 level was usually normal or slightly elevated and more likely with no residual lesions after primary surgery.2.Endometriosis may be one of the factors affecting the prognosis of ovarian cancer patients.Part ? A preliminary study on the pathogenesis of endometriosis-associated ovarian cancerObjective: To investigate the possible pathogenesis of EAOC and to verify it preliminarily.Methods: Firstly,whole-exome sequencing(WES)analysis was performed on paraffin-embedded cancer tissues of 7 patients with endometriosis-associated OCCC and 10 patients with non-endometriosis-associated OCCC.The normal paraffin-embedded ovarian tissue of the same patient was used as control and the significantly mutated genes(SMG)in endometriosis-associated OCCC patients were screened and their functions were verified.Secondly,according to the result of WES,we chose the two most common mutated genes in EAOC patients: ARID1 A and PIK3 CA for further study.Paraffin-embedded tissues of 76 OCCC and 9 OEC patients were chose to perform immunohistochemical staining(IHC).IHC of ARID1 A,PIK3CA p110? and the downstream proteins of PI3 K were performed to verify and analyze the relationship between the expression of BAF250 a,PI3K pathway related proteins and the clinicopathological characteristics,prognosis of EAOC patients.Thirdly,the expression of BAF250 a was verified in four human ovarian cancer cell lines(ES-2,HO-8910,A2780 and TOV-21G),and it was found that BAF250 a was absent in A2780 and TOV-21 G cell lines,while it was present in ES-2 and HO-8910 cell lines.Therefore,lentivirus-wrapped sh RNA was used to down-regulate the expression of ARID1 A gene in ES-2 and HO-8910 cell lines in vitro.The down-regulation effect was verified by RT-q PCR and Western Blot.CCK-8 experiment was used to measure cell proliferation.Results: 1.A total of 13 significantly mutated genes were found to be associated with OCCC,among which 58.8%(10/17)patients had somatic mutation of ARID1 A,and 50% were transcoding mutations.7 patients had somatic mutation of PIK3 CA,with a mutation rate of 41.2%,and all of them were missense mutations.2.Among the 7 endometriosis-associated OCCC patients,4 of them carried somatic mutation of ARID1 A gene,with a mutation rate of 57.1%,of which 2 patients were transcoding mutations and 2 were nonsense mutations.There were 4 patients with somatic mutation of PIK3 CA gene,with mutation rate of 57.1%,all of which were missense mutations.3.Immunohistochemical experiments confirmed that among 76 patients with OCCC,45(59.2%)patients had BAF250 a deletion,and there was no significant difference in BAF250 a deletion rate between patients in EAOC and NEAOC groups(69.6% vs.44.8%,p=0.062).4.Among the 9 patients with OEC,6(66.7%)patients had BAF250 a deletion,and there was no significant difference in BAF250 a deletion rate between patients in EAOC and NEAOC groups(66.7% vs.57.1%,p=0.778).5.In OCCC patients,no significant difference was found in PFS(82.7% vs.79.5%,p=0.577)and OS(84.6% vs.89.4%,p=0.869)between patients with or without BAF250 a deletion.6.In OCCC patients,no significant difference was found in PI3 K pathway related protein expression between patients in EAOC and NEAOC groups.7.In OEC patients,no significant difference was found in PI3 K pathway related protein expression between patients in EAOC and NEAOC groups.8.The expression of ARID1 A was down-regulated in human ovarian cancer cell lines ES-2 and HO-8910,with no significant changes in cell proliferation.Conclusion: 1.Mutations of ARID1 A and PIK3 CA genes may be highly associated with EAOC.2.High frequency of BAF250 a deletion was found in the tissues of OCCC and OEC patients.3.The deletion of BAF250 a caused by the mutation of ARID1 A may be an early molecular event in the development of ovarian cancer,but the mutation of ARID1 A alone may not lead to carcinogenesis,which requires the combination of other gene variations.