The application of WT1 and P53/Ki-67 to predict the recurrence of epithelial ovarian cancer

Purpose: To evaluate the ability of WT1 alone, and in combination with P53/Ki-67, to predict recurrence in patients with epithelial ovarian cancer (EOC). Methods: In this retrospective study, we performed immunohistochemistry on surgical specimens acquired from 137 patients with EOC. We analyzed the sensitivity of WT1 alone, and in combination with P53 and Ki-67, to predict the recurrence of EOC by applying Chi-squared and layered Chi-squared tests. The Kaplan–Meier method was used to predict progression-free survival(PFS). Results: The proportion of WT1-positive patients in the recurrent group (80.4%) was signicantly higher than that in the non-recurrent group (60.4%; P=0.019). In stratied cohort analysis, the recurrence rate of WT1-positive patients was signicantly higher than that of WT1-negative patients in the P53-positive group (P=0.034); similar results were obtained for the group with Ki-67 index >30% (P=0.026). Kaplan–Meier curves showed that WT1-negative patients presented with a signicantly longer PFS than WT1-positive patients in the P53(P=0.006) and Ki-67(P=0.016) positive groups. Conclusion: A high level of WT1 is a potent predictor of recurrence for EOC. Our analyses showed that the predictive ability of WT1 was more sensitive when combined with P53/Ki-67. Monitoring the levels of WT1 could have signicant therapeutic implications for patients with EOC. relapse. The combination of WT1 expression with P53 or Ki-67 expression represents an ecient prognostic indicator for EOC patients. WT1-positive patients presented with a signicantly shorter PFS than WT1-negative patients in the P53-positive and Ki-67-positive (>30%) groups. In future, the targeting of WT1 could have therapeutic implications for the treatment of EOC.


Introduction
Epithelial ovarian cancer (EOC) is a gynecological tumor with the highest degree of malignancy and a poor prognosis. The 5-year survival rate of patients with EOC is only 30% [1]. An important factor affecting the prognosis of EOC is the high recurrence rate; previous research has shown that most patients with EOC will eventually relapse within two years of treatment [2]. The main factors responsible for the recurrence of EOC is the high degree of malignancy, poor differentiation, and rapid proliferation [3]. P53 and Ki-67 are common clinical immunopathological indicators of tumor cell proliferation and differentiation [4,5]. However, these two indicators do not have strong sensitivity [6]. In addition to P53 and Ki-67, Wilms Tumor 1 (WT1) has been used as an immunological marker for the pathological detection of a variety of tumors over recent years. The WT1 gene has been identi ed as a tumor suppressor gene in Wilms tumors; however, there is increasing evidence that WT1 is overexpressed and exerts an oncogenic role in different types of cancers, including leukemia and myelodysplastic syndrome [7], lung cancer [8], and breast cancer [9]. Although WT1 is a known pathological marker for EOC, it is not clear whether the expression levels of WT1 are speci cally related to recurrence.
In this study, we used strati ed cohort analysis to investigate whether WT1 expression alone, or in combination with P53 and Ki-67, could predict recurrence in patients with EOC.

Materials And Methods
Patients and materials: This study included a total of 137 patients undergoing surgery for EOC in the First A liated Hospital of Soochow University from 1st January 2015 with follow-up dates to 31st March 2020. The study was approved by the Institutional Ethics Review Board of the Soochow University School of Medicine. Because this was a retrospective study, and the names of patients were not divulged, the Institutional Ethics Review Board waived the need for informed consent. All surgeries were performed by accredited gynecological oncologists; these physicians also determined the residual tumor size. Pathological analysis revealed that all patients had EOC; 102 patients received 6 or more cycles of chemotherapy. Thirty-ve patients did not complete chemotherapy due to other basic diseases or serious complications. Chemotherapy was mainly performed with Paclitaxel + Platinum (Cisplatin or Carboplatin). The period of chemotherapy was approximately 21 days. Five patients received targeted therapy (Olaparib and Niraparib). Neoadjuvant chemotherapy (NACT) was performed with Paclitaxel + Platinum drugs. Disease stage was assigned in accordance with the International Federation of Gynecology and Obstetrics (FIGO) surgical staging criteria. Histological grades were categorized as either high or low. The clinicopathological features are summarized in Table 1.
Immunohistochemistry: All specimens were xed in formalin, embedded in para n, serially sectioned at a thickness of 5µm, and mounted on electrostatically charged slides. Para n sections were then dewaxed and dehydrated before being incubated with 3% H2O2 at room temperature for 15 min to remove endogenous peroxidase. Citrate buffer solution (0. 1 mol/L, pH 6.0) was then applied for 10 min in a microwave for antigen retrieval. Sections were then blocked with normal goat serum for 15 min at room temperature. Then, sections were incubated with rabbit antihuman WT1 monoclonal antibody (Ab89901, Abcam, 1:100 dilution), rabbit anti-human P53 monoclonal antibody (Ab32389, Abcam,1:100 dilution), or rabbit anti-human Ki-67 monoclonal antibody (Ab92742, Abcam, 1:100 dilution) at 4℃ overnight and rinsed three time with PBS (5 min per wash). The following morning, sections were washed and incubated with a biotinylated goat anti-rat IgG secondary antibody (1∶200 dilution) at 37℃ for 1 h. Sections were then rinsed three times in PBS (5 min per wash) and positive antibody binding was visualized by DAB color development. Finally, sections were stained in hematoxylin, hydrated with a gradient series of alcohols, cleared with xylene, sealed onto glass slides with neutral gum, and observed by microscopy.
Statistical analyses: All statistical analyses were performed with SPSS version 25.0 (IBM Corp., Armonk, NY, USA). The association of WT1 with speci c clinical characteristics was analyzed by the t test and by Chi-squared tests. The association between WT1, P53, and Ki67 expression, and the recurrence of EOC was analyzed by layered Chi-squared tests. Kapla-Meier survival curves were used to determine progression-free survival (PFS). The log-rank test was used to compare survival between different groups. All statistical tests were two-sided and signi cance was set at P < 0.05.

Results
The proportions of patients who were positive and negative for WT1, P53, and Ki-67, were determined by calculating the proportions (%) of the total number of tumor cells that showed positive antibody staining in the nuclei ( Figure 1). Statistical analyses showed that WT1-positive staining was present in 92/137 of specimens, P53-positive staining was observed in 92/137 of specimens, and positive Ki-67 staining (>30%) was observed in 106/137 of specimens. The characteristics of the patients in each group are shown in Table 1.
Next, the expression of biomarkers in the recurrent and non-recurrent patients were compared by Chi-squared analysis ( Table 2). The proportion of WT1-positive patients in the recurrent group (80.4%) was signi cantly higher than that in the non-recurrent group (60.4%; P=0.019). There was no signi cant difference in P53-positive expression when compared between the recurrent (63.0%) and non-recurrent group (70.3%; P=0.388). Similarly, the there was no signi cant difference in Ki-67-positive (>30%) expression when compared between the recurrent (80.4%) and nonrecurrent group (75.8%) (P=0.542).

Discussion
Because of its high recurrence rate and poor prognosis, EOC is considered to be the gynecological tumor with the highest degree of malignancy.
At present, the clinical judgment of EOC recurrence depends mainly on an increase in tumor indicators and imaging changes. However, there are no valid methods to predict the recurrence of EOC. Since there is no de nitively effective treatment for recurrent patients, these patients have a very poor prognosis.
The WT1 gene, located on chromosome 11p13, was originally identi ed as a tumor suppressor gene in Wilms tumors in children [10]. Since then, WT1 has been identi ed as an oncogenic factor in a variety of human cancers, including leukemia and myelodysplastic syndrome [11], lung cancer [8], and breast cancer [12]. It has been reported that WT1 enhances the migration and invasive capacity of EOC cells and could be associated with a poor prognosis. However, it is not clear whether WT1 expression is related to recurrence. In the current study, the proportion of WT1-positive patients in the recurrent group was signi cantly higher than that in the non-recurrent group. Many previous studies have reported that epithelial ovarian tumors generally show high levels of WT1 gene expression [13,14]. Han et al. reported that WT1 enhanced the migration and invasion capacity of EOC cells [15]. Furthermore, Taube et al. reported that patients with high expression levels of WT1 were more likely to relapse [16]. There is increasing evidence that WT1 exerts an oncogenic role, rather than a tumor-suppressive role,in the development and progression of cancers. Therefore, the positive expression of WT1 may cause higher recurrence in EOC patients.
It is notable that we detected patients without recurrence in the WT1-positive group. To explore which subgroups were more sensitive to WT1 with regards to predicting recurrence, we performed strati ed cohort analysis. Our data showed that patients with combined WT1-positivity and P53/Ki-67-positivitity (>30%) were more likely to relapse. In a previous study, Bani-Ahmad et al. reported that the enhanced expression levels of WT1 and P53 play a negative role in the pathogenesis and progression of acute promyelocytic leukemia [17]. Similar results were reported for ovarian cancer by Carter et al. who investigated partial or total P53 tumor functional inactivation; under these conditions, WT1 could be overexpressed to promote tumor growth [18]. In another study, Mondal et al. reported that the expression levels of Ki-67 and WT1 may represent indicators of an unfavorable prognosis in patients with advanced serous epithelial ovarian carcinoma [19]. However, these authors did not investigate whether the combination of these factors could indicate a poor prognosis. Further studies on the association between Ki-67 and WT1 expression are now required.
Our analysis showed that WT1-negative patients presented with a signi cantly longer PFS than WT1-positive patients in the P53-positive and Ki-67-positive (>30%) groups. Our data also indicated that WT1 gene expression, in combination with p53 and Ki-67 expression, is signi cantly associated with PFS. Many previous studies have suggested that WT1 expression alone is associated with PFS [20,21]. Qi et al. carried out metaanalysis involving 3620 cases and found that WT1-positive expression was signi cantly associated with a worse overall survival (OS) and PFS [22]. However, previous studies have failed to consider whether the combination of WT1, P53 and Ki-67 expression is related to PFS; further studies are now required to address this possibility.

Conclusion
The results of the present study suggest that WT1 is associated with the recurrence of EOC and that WT1-positive patients are more likely to relapse. The combination of WT1 expression with P53 or Ki-67 expression represents an e cient prognostic indicator for EOC patients. WT1positive patients presented with a signi cantly shorter PFS than WT1-negative patients in the P53-positive and Ki-67-positive (>30%) groups. In future, the targeting of WT1 could have therapeutic implications for the treatment of EOC.

Declarations
Author Contributions YC and FS contributed to the study design, literature search, and data acquisition and interpretation. YC and YZ performed the major data analyses, statistical analysis, and manuscript editing. JW and JZ contributed to data acquisition, statistical analysis, and manuscript revision.  II:Tumor involves one or both ovaries or fallopian tubes with pelvic extension below pelvic brim or primary peritoneal cancer; III:Tumor involves one or both ovaries or fallopian tubes, or primary peritoneal cancer, with microscopically con rmed peritoneal metastasis outside the pelvis and/or metastasis to the retroperitoneal (pelvic and/or para-aortic) lymph nodes; IV:Distant metastasis, including pleural effusion with positive cytology; liver or splenic parenchymal metastasis; metastasis to extra-abdominal organs (including inguinal lymph nodes and lymph nodes outside the abdominal cavity); and transmural involvement of intestine.