Barriers to cervical cancer prevention and triage strategies: a study of knowledge, attitudes, and p16/Ki-67 dual-staining utility among high-risk women in Tuoli and Fuyun counties, Xinjiang

Study design and participants

A cross-sectional study was conducted on Chinese women aged 20-65 years with cytological abnormalities or HPV positivity who received treatment or were hospitalized in the Department of Obstetrics and Gynecology at local hospitals in Tuoli and Fuyun counties, Xinjiang Province, during the period from January to December 2023.

Eligible participants were local residents with an intact cervix, sexual exposure history, non-pregnant status, no history of cervical cancer or hysterectomy, and no prior records of cone biopsy, cryotherapy, laser ablation, loop electrosurgical excision procedure (LEEP), or pelvic radiation. All participants demonstrated comprehension of study procedures and provided voluntary written informed consent.

The screening protocol integrated a standardized gynecological examination with concurrent questionnaire administration (Fig. 1). During the examination, cervical specimens were collected using liquid-based cytology (ThinPrep^®) and systematically allocated for three parallel diagnostic approaches: (1) conventional cytological evaluation, (2) p16 immunocytochemical staining, and (3) p16/Ki-67 dual-staining. From each patient sample, three independent slides were prepared under identical fixation conditions to ensure analytical consistency across all testing modalities.

Data collection

Five researchers from Xinjiang Medical University and all research assistants completed a standardized one-week training program covering questionnaire administration, interview techniques, and ethical requirements. All participants provided written informed consent after receiving detailed verbal explanations of the study objectives and procedures.

The questionnaire was developed in Chinese by a panel of researchers and medical practitioners based on a detailed examination of published literature, and has been used with established reliability and accuracy in a number of cervical cancer studies (Zhang et al., 2021). It comprised: (1) demographic information of participants, including marital status, level of education, employment status (classified as: 1 = unemployed; 2 = service sector workers (e.g., retail and hospitality professionals such as mall salespersons, supermarket cashiers, restaurant staff); 3 = public servants (government and state employees, including military personnel, civil servants); 4 = general laborers (agricultural and factory workers such as crop farmers, factory assembly-line workers)), and income; and (2) an assessment of the participants’ awareness and attitudes towards cervical cancer and its screening. The questionnaire took an average of 20–30 min to complete.

Two members of the research team entered the survey responses into Excel. To ensure data quality, all surveys were double-entered to keep the error rate below 0.5%.

p16 staining

The detection of p16 protein in cervical cytology specimens was performed using PathCIN p16 liquid-based cell preservation solution with an automated immunohistochemical staining system (Shenzhen Senying Biotech Co., Ltd., Shenzhen, China) according to the manufacturer’s protocol. Positive staining was identified by the presence of brownish-yellow cytoplasmic and/or nuclear coloration in cervical epithelial cells. Staining interpretation followed the standardized scoring system established by Wentzensen et al. (2005).

p16/Ki-67 dual staining

Dual staining was conducted using the CINtec PLUS kit (CINTec^® Plus Cytology Kit; Ventana Medical Systems, Inc., Oro Valley, AZ, USA) with monoclonal antibodies against p16 (clone E6H4) and Ki67 (clone 274-11 AC3). A positive result was defined as concurrent red cytoplasmic staining (p16-positive) and brownish-yellow nuclear staining (Ki-67-positive) within the same epithelial cell on cytology slides, while a negative result included any of the following patterns: nuclei with only light blue hematoxylin counterstaining, isolated red cytoplasmic staining (p16-positive alone), or isolated brownish-yellow nuclear staining (Ki-67-positive alone). Interpretation criteria adhered to the standardized protocol described by Celewicz et al. (2018).

Liquid-Based Cytology (LBC) testing

Cervical cytology specimens were processed using ThinPrep^® LBC (Hologic, Marlborough, MA, USA). Slides were independently interpreted by board-certified cytopathologists following the 2015 Bethesda Classification (Wilbur et al., 2015). All slides underwent blinded dual review by senior cytopathologists at Xinjiang Tumor Hospital, with discordant cases resolved by consensus. Final reports were quality-controlled by expert cytopathologists from the Cancer Hospital of the Chinese Academy of Medical Sciences, ensuring diagnostic consistency with national standards.

Statistical analyses

The population was categorized in the statistical analysis based on participant characteristics, including screening behavior and awareness of cervical cancer. Univariate analysis of demographic information was performed using the chi-squared test and Fisher’s exact test. Logistic regression models included variables associated with P-values <0.05 (Hosmer Jr, Lemeshow & Sturdivant, 2013), with results presented as 95% confidence intervals (95% CI) and odds ratios (OR). We assessed potential multicollinearity among covariates using variance inflation factors (VIF), with all VIF values <2.0, well below the conventional threshold of 5, indicating no substantial multicollinearity in our model. To account for multiple comparisons, we applied the Bonferroni correction. We evaluated model fit using Hosmer-Lemeshow tests (grouped by deciles) and McFadden’s pseudo R². The mediation effects of educational attainment and employment status on cervical cancer screening through health awareness pathways were examined using bootstrap analysis with 5,000 resampling iterations. Fisher’s exact test was performed to determine whether LBC testing was associated with p16 staining or p16/Ki-67 dual staining. Data from the study were analyzed, and radar plots were generated using STATA 18.0 software. Bar charts were drawn using Microsoft Excel software. All P-values <0.05 were considered statistically significant.

Ethical consensus

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Affiliated Tumor Hospital of Xinjiang Medical University (approval code: K-201802). Written informed consent was obtained from all participants prior to screening. Participants were assigned unique project identifiers, with all data and medical records maintained under strict confidentiality. Individualized written screening reports were provided, and cases with abnormal findings received prompt clinical follow-up.

Sociodemographic correlates of cervical cancer awareness and screening

This study of 260 high-risk women (HPV+/LBC abnormalities) in Xinjiang revealed 67.31% (175/260, 95% CI [61.50–72.90]) screening awareness and 56.15% (146/260, 95% CI [50.23–62.17]) participation rates, with significant sociodemographic variations (Table 1). Unmarried women (single/divorced/widowed) demonstrated substantially higher awareness than married counterparts (94.44%, 95% CI [84.55–100.00] vs 65.29%, 95% CI [59.26–71.34], P = 0.009). A pronounced education gradient was observed, with college-educated women showing both greater awareness (87.37%, 95% CI [80.63–94.17] vs 38.46% [23.23–53.77] in primary-educated, P < 0.001) and screening participation (67.37%, 95% CI [58.55–76.25] vs 50.00% , 95% CI [33.76–66.24], P = 0.029). Employment status differences were particularly striking, with public servants exhibiting the highest rates of both awareness (89.19%, 95% CI [80.55–97.85]) and screening (70.27%, 95% CI [58.16–82.44]), compared to 25.00% (95% CI [15.29–34.71]) awareness and 26.79% (95% CI [17.23–36.37]) screening among unemployed women (all P < 0.001). Although higher income (≥100,000 RMB) initially correlated with awareness (81.82%, 95% CI [72.15–91.49] vs 58.95–72.73% in lower income groups; P = 0.045), this association became non-significant after multiplicity adjustment (α = 0.0125).

Factors associated with cervical cancer awareness and screening: multivariable analyses

Multivariable logistic regression analysis (Tables 2–3) revealed that college-educated women had significantly higher odds of cervical cancer screening awareness compared to those with primary education (aOR = 7.575, 95% CI [2.318–24.751]). Employment status disparities persisted after adjustment, with public servants demonstrating the strongest association (aOR = 11.230, 95% CI [2.637–47.826]), followed by service workers (aOR = 9.160, 95% CI [3.537–23.723]) and general laborers (aOR = 8.503, 95% CI [3.704–19.517]). For screening behavior, service workers (aOR = 5.671, 95% CI [2.520–12.763]), public servants (aOR = 5.118, 95% CI [1.869–14.015]), and general laborers (aOR = 3.751, 95% CI [1.817−7.745]) all showed significantly higher participation rates than unemployed women.

Both regression models demonstrated excellent calibration (awareness model: χ² = 7.010, df = 8, P = 0.535; screening model: χ² = 3.330, df = 6, P = 0.767). The awareness model explained a moderate proportion of variance (McFadden’s pseudo R² = 0.256), considerably higher than the screening model (pseudo R² = 0.081; typical range: 0–1). This difference suggests the potential influence of additional non-sociodemographic factors on screening behavior.

Mediating role of educational attainment and employment status on the association between awareness and screening

The bootstrap mediation analysis (5,000 resamples, adjusted for marital status and income) demonstrated complete mediation of employment status’ effect on screening through health awareness (indirect effect = 0.061, 95%CI [0.029−0.093], p < 0.001; direct effect = 0.004, p = 0.872), accounting for 93.8% of the total effect (Fig. 2). A secondary reverse pathway showed employment modestly enhanced awareness through prior screening (indirect effect = 0.024, p = 0.026). For educational attainment, health awareness mediated 128.8% of its total effect (indirect effect = 0.103, 95%CI [0.046−0.161], p < 0.001), completely suppressing the direct negative association (direct effect = −0.023, p = 0.655). Educational attainment directly increased awareness (direct effect = 0.173, 95%CI [0.083−0.263], p < 0.001) with negligible mediation through screening (indirect effect = 0.029, p = 0.164).

Cervical cancer screening preferences among high-risk women

According to the questionnaire results from 260 high-risk women, the majority preferred annual cervical cancer screening (128/256, 50%, 95% CI [42.16–57.84]) and expected to receive results within one week (116/258, 44.96%, 95% CI [38.34–51.64]), with written reports (89/249, 35.74%, 95% CI [30.04–41.78]) being the most favored feedback method for screening results. High compliance was demonstrated in retesting willingness, as 234 of 249 respondents (93.98%, 95% CI [90.85–96.27]) agreed to re-test if recommended by doctors. Key barriers to treatment were lack of money (82/246, 33.33%, 95% CI [27.64–39.42]) and the belief that treatment was unnecessary due to no symptoms (75/246, 30.49%, 95% CI [24.97–36.43]). Additionally, 68.99% (178/258, 95% CI [63.03–74.47]) of participants had heard of the HPV vaccine, and 82.95% (214/258, 95% CI [78.33–86.84]) intended to have their children vaccinated against HPV (Fig. 3).

The survey on knowledge sources about cervical cancer and screening showed that the primary source was physician recommendation (112/289, 38.75%, 95% CI [32.76–44.93]), followed by media (84/289, 29.07%, 95% CI [23.99–34.66]) and friend/family recommendation (43/289, 14.88%, 95% CI [10.96–19.63]). Social welfare campaigns accounted for 12.46% (36/289, 95% CI [8.96–16.79]), while slogans/posters and other sources accounted for 2.77% (8/289, 95% CI [1.21–5.65]) and 2.08% (6/289, 95% CI [0.77–4.63]), respectively (Fig. 4).

To convince women reluctant to undergo cervical cancer screening, 37.88% (164/433, 95% CI [33.15–42.76]) of respondents prioritized increased hygiene promotion and education, followed by family/friend persuasion at 25.17% (109/433, 95% CI [21.15–29.51]). Other approaches included mandatory attendance by parent organizations (18.01%, 78/433, 95% CI [14.47–21.94]), reducing inspection frequency alongside transportation support (13.86%, 60/433, 95% CI [10.88–17.40]), and self-collecting cervical samples (3.93%, 17/433, 95% CI [2.33–6.29]), as shown in Fig. 5.

Of those unwilling to be rescreened, 29.91% (35/117, 95% CI [20.96–40.26]) cited excessive testing frequency, while 23.93% (28/117, 95% CI [15.98–33.35]) attributed it to inconvenience or distance. Other reasons included preferring county/city hospital retesting (13.68%, 16/117, 95% CI [7.69–22.47]), the screening process being too arduous (12.93%, 15/117, 95% CI [6.96–21.91]), perceived good health/non-serious results (10.26%, 12/117, 95% CI [5.20–18.17]), and time constraints (8.55%, 10/117, 95% CI [4.01–16.32]), as shown in Fig. 6.

Progressive increase of p16 and p16/Ki-67 positivity correlates with the severity of cytological abnormalities detected in liquid-based cytology

Consistent with cytological findings, both p16 staining and p16/Ki-67 dual-staining exhibited a lesion-grade-dependent increase in positivity rates, escalating from 0% in normal to 100% in HSIL specimens (P < 0.001 for trend). This progressive increase was observed across all diagnostic categories, with statistically significant inter-group differences (Fig. 7).