A total of 230 patients with EC who underwent surgical resection in Handan Central Hospital (Handan, China) between January 2015 and December 2019 were retrospectively screened. The inclusion criteria were as follows: i) EC diagnosis by histopathological examination; ii) ≥18 years of age; iii) underwent surgical resection; iv) accessible and available tumor formalin-fixed paraffin-embedded (FFPE) specimens; and v) adequate clinical characteristics data and information for at least one follow-up. Patients who had other malignancies, were pregnant or were lactating were excluded. The present study was approved by the Ethics Committee of Handan Central Hospital (approval no. 2023020615; Handan, China), and informed consent (written or oral) was obtained from every patient or their family member.

Data on demographics, disease, treatment and follow-up were obtained from the patients with EC. Disease-free survival (DFS) and OS were calculated. The last follow-up was in January 2023. Additionally, 230 FFPE specimens of tumor tissue and 50 FFPE specimens of non-tumor tissue were obtained from the hospital for examination of KIF2A expression.

KIF2A expression was assessed using IHC staining. Stored specimens were cut into 4-µm thick sections, deparaffinized with xylene, and rehydrated using a descending ethanol series. Microwave heating (100°C for 7 min) was used for antigen retrieval. Endogenous peroxidase activity was quenched using 3% H₂O₂. Sections were then blocked using 5% goat serum (cat. no. ab7481; Abcam) at room temperature for 10 min. In preliminary experiments, the sections were incubated with rabbit polyclonal anti-KIF2A antibodies (cat. no. ab197988; Abcam) at three different concentrations (1:100, 1:200 and 1:300) overnight at 4°C. In the subsequent experiments, the slides were incubated overnight with rabbit polyclonal anti-KIF2A antibodies (1:200) at 4°C. After incubation, the slides were washed three times with TBST (containing 0.1% Tween-20). The slides were then incubated with goat anti-rabbit IgG H&L (HRP) secondary antibodies (1:1,000; cat. no. ab6721; Abcam) at room temperature for one hour. After incubation, the slides were washed three times with TBST. The slides were then stained using a DAB Peroxidase Substrate Kit [Absin (Shanghai) Biotechnology, Co., Ltd.) and hematoxylin at room temperature for 30 min. Sections were then sealed with Glycerol Jelly Mounting Medium and imaged. A section in which the primary antibody was omitted served as a negative control. IHC scoring was performed by two investigators separately using a light microscopy and Image-Pro Plus 6.0 (Media Cybernetics Inc.), which was used to assess KIF2A density and intensity scores. Briefly, staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong), and staining density was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%) or 4 (76–100%). The mean of the KIF2A density and intensity scores obtained by the two investigators was taken. The final IHC score (ranging from 0–12) was calculated by multiplying staining intensity and staining density. For subsequent analysis, tumor KIF2A expression was divided into high (IHC score >3) and low (IHC score ≤3) (20–22). The present study ensured consistency in personnel, experimental conditions, instrumentation and reagents to control for possible batch effects.

To further verify the correlation between KIF2A expression and OS, information from 541 patients with EC was gathered from The Human Protein Atlas database (https://www.proteinatlas.org), in which the KIF2A expression was classified as low or high with a cut-off value of 5.2.

SPSS v24.0 (IBM Corp.) was used for statistical analysis, and GraphPad Prism v7.01 (Dotmatics) was utilized for graphing and statistical analysis. Comparisons were performed using an unpaired t-test or ANOVA with the Tukey's post hoc test. Correlations were analyzed using Spearman's rank correlation test. Kaplan-Meier curves with the log-rank test were used for DFS and OS assessment. Time-dependent receiver-operating characteristic analyses for relapse and death risk were performed and the area under the curve (AUC) was calculated (AUC >0.7 was considered to indicate good predictive utility). Independent factors related to DFS or OS were screened using forward-step multivariate Cox's proportional hazard regression analyses. P<0.05 was considered to indicate a statistically significant difference.

There were 230 patients with EC with a mean age of 58.1±8.0 years. There were 46 (20.0%) and 184 (80.0%) premenopausal and postmenopausal patients with EC, respectively. Regarding the histological subtype, there were 163 (70.9%) patients with endometrioid carcinoma G1/G2, 17 (7.4%) patients with endometrioid carcinoma G3, 34 (14.8%) patients with serous endometrial carcinoma and 16 (7.0%) patients with clear cell endometrial carcinoma. Additionally, 66 (28.7%) patients demonstrated lymphovascular invasion. There were 137 (59.6%) patients with the International Federation of Gynecology and Obstetrics (FIGO) stage I, 27 (11.7%) patients with FIGO stage II, 45 (19.6%) patients with FIGO stage III and 21 (9.1%) patients with FIGO stage IV (FIGO 2009 revision) (23). Additional information on patients with EC was presented in Table I.

In non-tumor tissue, KIF2A was expressed in glandular cells but not in endometrial stroma cells. Meanwhile, in tumor tissue, KIF2A was expressed in the cytoplasm, membranes and nucleus (Fig. 1A). An unpaired t-test analysis showed that KIF2A expression was significantly higher in tumor tissue compared with non-tumor tissue in patients with EC (P<0.001; Fig. 1B).

KIF2A expression in tumor tissue was significantly related to lymphovascular invasion (P=0.004) and higher FIGO stage (P=0.001) in patients with EC. However, there was no linkage of KIF2A expression in tumor tissue with other clinicopathologic features in patients with EC, including menopausal status, diabetes, hypertension, histological subtype and cervical invasion (all P>0.05; Table II). Furthermore, the results for the comparison by Tukey's post hoc test regarding histological subtype and FIGO stage data are shown in Table SI.

High tumor KIF2A expression was associated with significantly lower DFS in patients with EC [P=0.014; hazard ratio (HR)=2.338; Fig. 2A]. Moreover, the AUC of KIF2A expression in tumor tissues, for predicting relapse risk over 6 years remained stable at ≥0.7 (Fig. 2B). High KIF2A expression in tumor tissues was also correlated with significantly reduced OS in patients with EC (P=0.012; HR=3.577; Fig. 2C). Furthermore, the AUC of KIF2A expression in tumor tissues, for predicting death risk over 6 years also maintained stable at ≥0.7 (Fig. 2D).

KIF2A expression in tumor tissues (high vs. low; HR=2.506; P=0.013), age (≥60 years vs. <60 years; HR=2.485; P=0.015), histological subtype (clear cell endometrial carcinoma vs. endometrioid carcinoma G1/G2; HR=4.211; P=0.005) and cervical invasion (stromal vs. none or epithelial; HR=4.361; P<0.001) independently estimated shorter DFS in patients with EC (Fig. 3A). KIF2A expression in tumor tissues was not independently associated with OS. However, age (≥60 years vs. <60 years; HR=5.670; P=0.001), diabetes (yes vs. no; HR=2.912; P=0.011) and higher FIGO stage (HR=1.969; P<0.001) were independently linked with shorter OS in patients with EC (Fig. 3B).

Data from the Human Protein Atlas database indicated that high tumor KIF2A expression was significantly associated with decreased OS in patients with EC (P=0.027; HR=1.585; Fig. 4).