The medical data of patients with stage I-III EC who received preoperative biopsy and initial surgical treatment in the First Affiliated Hospital of Chongqing Medical University from January 2015 to May 2020, and in the Women and Children’s Hospital of Chongqing Medical University from January 2016 to May 2020 were collected, including age, body mass index (BMI), preoperative biopsy method, surgical procedures, pathological examination results (including tumor size, tumor location, tumor invasion range, histological type and grade of tumor, etc.), postoperative adjuvant treatment, and P53 immunohistochemical expression.

The inclusion criteria for patients were as follows: (1) Patients underwent preoperative biopsy and was initially diagnosed with EC; (2) Patients underwent initial surgical treatment and was ultimately diagnosed with EC by final pathology, with FIGO staging ranging from stage 1 to stage III (Most stage IV patients did not receive surgical treatment, and the assessment of recurrence in stage IV patients is difficult to determine because they have already experienced distant metastasis, so stage IV patients were not included in this study). The exclusion criteria were as follows: (1) Without standard surgery; (2) Receiving adjuvant therapy before surgery; (3) With incomplete medical records; (4) With other malignancies; (5) Lost follow-up. The study was approved by the Institutional Review Board (IRB) of the First Affiliated Hospital of Chongqing Medical University and the Women and Children’s Hospital of Chongqing Medical University (IRB number: 2021-676 and 2023-002).

All patients included in this study underwent preoperative biopsy, which can be divided into two main methods: (1) Blind biopsy, including blind Division and Curettage (D&C) and Pipelle (suction biopsy); (2) Hydroscopic guided endometrial biopsy, including hydroscopic guided diagnostic curettage and hydroscopic guided “grab” endometrial biopsy ( Figure 1 ). Hysteroscopic biopsy is performed by professional hysteroscopic physicians at their respective medical centers. The patient subsequently underwent a comprehensive staged surgery that included at least abdominal total hysterectomy + bilateral salpingo-oophorectomy + pelvic lymph node dissection ± para-aortic lymph node dissection (3). After surgery, it was recommended that patients receive corresponding adjuvant treatment according to international guidelines and multidisciplinary discussions (3, 9). The specific adjuvant treatment plans refer to similar previous studies (10, 11).

Follow-up was performed every 3 months for the first 2 years after surgery, every 6 months for the next 3 years, and annually thereafter (3). The follow-up plan included regular physical examinations and necessary auxiliary examinations. The follow-up deadline for this study was May 2023, and each patient has been guaranteed a follow-up period of more than 3 years. Two or more gynecological oncologists confirmed recurrence through physical examination, biochemical indicators, imaging examination, and pathological biopsy (10). Recurrence included vaginal stump recurrence, central pelvic region recurrence, upper para-aortic lymph node metastases, peritoneal metastases, and metastases to other organs (11). Recurrence-free survival (RFS) was defined as the time from the surgical date to the confirmed recurrence date, and overall survival (OS) was defined as the time from the surgical date to the death (10).

Preoperative biopsy tissues and postoperative surgical specimens of patients were immediately fixed with standard 10% neutral formalin tissue fixative after being removed from the body (the entire process usually does not exceed 10 minutes). The specimens were completely immersed in the fixative (the volume of the fixative is usually more than 5 times that of the specimens), the fixing time was between 24 hours and 48 hours. Finally, the specimens were sent to the Pathology Experimental Center of Chongqing Medical University for subsequent processing (dehydration, paraffin embedding, sectioning, H&E staining, and immunohistochemical analysis) within 24 hours. The evaluation of pathological results (including tumor size, histological type and grade, depth of myometrial invasion, cervical stromal invasion, LVSI, lymph node involvement, etc.) were performed by professional pathologists. The pathological type I of EC was defined as G1 and G2 endometrioid adenocarcinoma, while type II was defined as G3 endometrioid adenocarcinoma and non-endometrioid adenocarcinoma, including serous carcinoma, clear cell carcinoma, and other special histological types (12).

According to a unified and optimized immunohistochemical protocol, immunohistochemical analysis of P53 protein was performed on an immunohistochemical automatic staining machine (Leica Bond Max, Milton Keynes, UK). P53 antibody (MAB-0674, Maixin Biotech, China) was used as a primary antibody for immunohistochemical analysis of P53 (specific immunohistochemical steps can be found in references) (10, 13).

The immunohistochemical results of P53 were evaluated based on the staining intensity (weak staining, medium staining, and strong staining) and the proportion of positive cells. According to the criteria of P53 immunohistochemical interpretation, overexpression (generally >75% strongly positive staining) or complete loss of expression (no significantly positive tumor cells) of P53 was defined as P53 abnormality (P53abn), while positive expression between these two extremes was defined as P53 wild-type (P53wt) expression ( Figure 2 ) (14, 15).

For the convenience of subsequent research, based on the different expression states of P53 in preoperative biopsy and final pathology, 4-tier P53 expression was defined: P53 immunohistochemistry showed P53wt expression in both preoperative biopsy and final pathology was defined as P53wt/P53wt; Similarly, P53 immunohistochemistry showed P53abn expression in both preoperative biopsy and final pathology was defined as P53abn/P53abn; P53 immunohistochemistry showed P53wt expression in preoperative biopsy while showed P53abn expression in final pathology was defined as P53wt/P53abn; P53 immunohistochemistry showed P53abn expression in preoperative biopsy while showed P53wt expression in final pathology was defined as P53abn/P53wt. In order to reduce subjective interpretation errors, a secondary review was conducted by a professional pathologist for cases with differences in P53 between preoperative biopsy and final pathology in this study.

Firstly, the Cohen’s kappa coefficient and Sankey diagram were used to compare the consistency of P53 immunohistochemistry results between preoperative biopsy and final pathology (16). The kappa coefficient (κ) could be explained as follows: κ <0.01 indicated no consistency, 0.01-0.20 indicated slight consistency, 0.21-0.40 indicated general consistency, 0.41-0.60 indicated moderate consistency, 0.61-0.80 indicated substantial consistency, and 0.81-1.00 indicated almost complete consistency (17). The prognostic value of 4-tier P53 expression, P53 expression in preoperative biopsy and P53 expression in final pathology was compared by using receiver operating characteristic (ROC) curve and area under the curve (AUC). Univariate and multivariate Cox regression analysis was used to determine the correlation between 4-tier P53 expression and the prognosis of patients. On this basis, the nomogram models for predicting patient prognosis were established by combining 4-tier P53 expression and classic clinicopathological parameters, and the models were validated using calibration curves. ROC curve and the maximum value of Youden index (Youden index = sensitivity +specificity -1) were used to determine the optimal risk threshold of the models and then risk stratification was performed on patients (18). Kaplan Meier analysis and log-rank test were used to compare the prognosis between patients in high-risk group and in non-high-risk group patients, and stratified analysis based on different adjuvant treatment methods was further conducted. Categorical variable was expressed in frequency (%), continuous variable of normal distribution was expressed in mean (± SD), and continuous variable of non-normal distribution was expressed in median (P25, P75). SPSS software (version 25.0, IBM Statistics, Chicago, IL, USA) and R software (version 4.0.3, http://www.r-project.org) were used for data analysis.

As shown in Figure 3 , a total of 1186 patients were included in this study through inclusion and exclusion criteria. The baseline data of the patients was shown in Table 1 . The average age of the patients was 53.67 (± 9.28) years old, of which 845 (71.2%) patients were with FIGO stage I and 185 (15.6%) patients had lymph node metastasis (LNM). A total of 737 (62.1%) patients received adjuvant therapy after surgery, of which 389 (32.8%) patients received radiotherapy, 309 (26.1%) patients received chemoradiotherapy, and 39 (3.3%) patients only received chemotherapy due to personal reasons. The median follow-up time of the patients was 44.00 (34.00, 61.00) months, and a total of 183 (15.4%) patients experienced recurrence during the follow-up period. The main form of recurrence was distant metastasis (32.2%), followed by recurrence in the central pelvic region (28.4%); A total of 139 (11.7%) patients died, of which 132 (11.1%) died due to recurrence.

534 (45.0%) patients obtained tissue samples through hysteroscopic guided biopsy before surgery, while the remaining patients obtained tissue samples through D&C or Pipelle biopsy before surgery. Overall, 830 (70.0%) EC patients were assessed as pathological type I through preoperative biopsy, while 849 (71.6%) EC patients were confirmed as pathological type I in the final pathology. Similarly, 359 (30.3%) and 385 (32.5%) patients were diagnosed as P53abn expression in preoperative biopsy and final pathology, respectively.

As shown in Table 2 and Figure 4 , there was inconsistency in the expression of P53 immunohistochemistry between preoperative biopsy and final pathology. Overall, 9.2% of patients diagnosed as P53wt in preoperative biopsy were diagnosed as P53abn in final pathology, while 7.0% of patients diagnosed as P53abn in preoperative biopsy were diagnosed as P53wt in final pathology. The consistency of P53 expression between preoperative biopsy and final pathology was 83.8%, with a kappa coefficient of 0.624. Layered analysis of preoperative biopsy methods revealed that the consistency of P53 expression between preoperative specimens obtained through hysteroscopic biopsy and final pathology was 89.1%, with a kappa coefficient of 0.735. While the consistency of P53 expression between the preoperative specimens obtained through blind biopsy (D&C or Pipelle biopsy) and the final pathological results was relatively low (79.4%), with a kappa coefficient of 0.539.

It was worth noting that we also found a strong correlation between the consistency of P53 and the consistency of pathological type. As shown in Supplementary Table 1 , in the group of patients with consistent P53 expression (n=994), 900 patients (90.5%) had consistent pathological type between preoperative biopsy and final pathology. In the group of patients with inconsistent P53 expression (n=192), 133 patients (69.3%) also showed differences in pathological type (correlation P value<0.001).

The ROC curve suggested that, for the prediction of EC recurrence and death, the AUC (AUC for recurrence and death was 0.702 and 0.695, respectively) of 4-tier P53 expression, which was defined by the combination of P53 expression in preoperative biopsy and P53 expression in final pathology, was superior to the AUC of P53 expression in simple preoperative biopsy (AUC for recurrence and death was 0.635 and 0.621) or the AUC of P53 expression in simple final pathology (AUC for recurrence and death was 0.667 and 0.655) ( Figures 5A, B ). The Kaplan Meier analysis ( Figures 5C, D ) suggested significant survival differences among the four groups of 4-tier P53 expression. The P53wt/P53wt group had the best prognosis (RFS and OS), the P53abn/P53abn group had the worst prognosis, while the P53wt/P53abn group and P53abn/P53wt group had similar prognosis, the prognosis of these two groups was between the P53wt/P53wt group and the P53bn/P53bnn group. The specific prognosis of each group was shown in Supplementary Table 2 .

Univariate and multivariate Cox regression analysis related to RFS and OS was conducted, respectively ( Tables 3 , 4 ). Univariate Cox regression analysis found that multiple clinicopathological factors, including age, FIGO stage, pathological type in final pathology, myometrial invasion, cervical stromal invasion, LVSI, lymph node metastasis, and 4-tier P53 expression, were significantly correlated with RFS and OS. Factors with a P value < 0.05 in univariate analysis were further included in multivariate analysis. The results of multivariate analysis showed that only 6 factors were both significantly correlated with the RFS and OS, namely FIGO stage, pathological type in final pathology, myometrial invasion, LVSI, lymph node metastasis, and 4-tier P53 expression.

The prognostic value of using 4-tier P53 expression alone to predict patient prognosis was still very limited. Based on this, we compared the AUC of 4-tier P53 expression, classic clinicopathological parameters, and their combination (4-tier P53 expression+ classic clinicopathological parameters) for predicting the recurrence and death of EC. The ROC curve showed that the AUC of the combination for predicting the EC recurrence and death were 0.856 (95% CI, 0.828-0.885) and 0.838 (95% CI, 0.804-0.871), respectively, which was higher than the AUC of simple clinicopathological parameters or the AUC of simple 4-tier P53 expression ( Supplementary Table 3 and Figure 6 ).Therefore, based on the results of multivariate analysis, we combined the 4-tier P53 expression with classic clinicopathological factors to construct the nomogram models for predicting EC recurrence and death, respectively. Based on these two models, personalized predictions can be made for the 1-year, 3-year, 5-year RFS and OS rates of patients ( Figure 7 ). The calibration curves related to RFS and OS showed good fitness of these two models ( Figure 8 ).

Due to the fact that most recurrent patients of EC experience recurrence within 3 years after surgery, we calculated the 3-year RFS rate, as well as the corresponding sensitivity and specificity, for each patient using the above model. ROC curve and the maximum value (0.577) of Youden index (Youden index= sensitivity +specificity -1) showed that the optimal risk threshold of the 3-year RFS rate of the model was 0.86 (corresponding sensitivity was 0.776, specificity was 0.801) ( Figure 9A ). Similarly, the optimal risk threshold for the 3-year OS rate of another model was 0.90 (corresponding sensitivity was 0.803, specificity was 0.761) ( Figure 9B ). Then patients with a 3-year RFS rate < 0.86 or a 3-year OS rate < 0.90 were defined as high-risk group, while the remaining patients were defined as non-high-risk group. The Kaplan Meier analysis suggested that the RFS and OS rates of high-risk group were significantly lower than those of non-high-risk group ( Figure 10 and Supplementary Table 4 ).

Based on stratified analysis of adjuvant therapy, it was found that, in the non-high-risk group, there was no significant difference in prognostic outcomes between patients who received adjuvant therapy and those who did not receive adjuvant therapy ( Figures 11A, B ). In high-risk group, the overall prognosis of patients who received adjuvant treatment was to varying degrees better than those who did not receive adjuvant treatment, and the prognosis of patients receiving chemoradiotherapy was better than that of patients receiving chemotherapy or radiotherapy alone ( Figures 11C, D ). The specific prognosis of patients who received different adjuvant treatment methods in high-risk group was shown in Supplementary Table 5 .