Abstract
Objective
Human papillomavirus (HPV) gene transcription activity and methylation could be promising tools for cervical cancer (CC) screening. This study compared HPV E6/E7 messenger RNA (mRNA) + ThinPrep cytology test (TCT) vs. HPV genotyping + TCT in screening CC and explored the relationship between gene methylation and cervical lesions.
Methods
Women who visited gynecology outpatient clinics between January 2023 and November 2024 were enrolled and assigned to 2 screening strategies based on their wishes: HPV DNA + TCT or HPV E6/E7 mRNA + TCT. Methylation testing of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 genes was performed on the biopsy specimens from 100 patients.
Results
Ultimately, 1,399 and 991 women underwent HPV DNA + TCT and HPV E6/E7 mRNA + TCT screening, respectively. The sensitivity, specificity, and accuracy in diagnosing high-grade squamous intraepithelial lesion (HSIL)+ were 72.09%, 83.05%, and 81.7% for HPV DNA + TCT, and 77.97%, 86.37%, and 85.37% for HPV E6/E7 mRNA + TCT, respectively. The areas under the receiver operating characteristic curves were 0.777 for HPV DNA + TCT and 0.822 for HPV E6/E7 mRNA + TCT. No HSIL+ lesions were observed in 65 patients with HPV 16/18 (+) but HPV E6/E7 mRNA (−). In cases with HPV 16/18 (+) and HPV E6/E7 mRNA (+), the immediate risks for cervical intraepithelial neoplasia (CIN) 2+ and CIN3+ were 64.06% and 50%, respectively. Methylation levels of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 genes increased with lesion severity.
Conclusion
The sensitivity and specificity of HPV E6/E7 mRNA + TCT appeared higher than HPV DNA + TCT. Simultaneous positivity for HPV 16/18 and HPV E6/E7 mRNA suggests a high immediate risk of CIN2+ and CIN3+. These results may improve CC screening.
Keywords: Human Papillomavirus Infection; RNA, Messenger; Cytodiagnosis; Cervical Cancer; Risk
Synopsis
Human papillomavirus (HPV) E6/E7 messenger RNA (mRNA) testing with cytology outperformed HPV DNA genotyping. Dual positivity for HPV 16/18 and mRNA predicted high cervical lesion risk. Six gene methylation levels increased with advancing cervical lesions.
INTRODUCTION
Cervical cancer (CC) is the fourth most common cancer among women worldwide and ranks second among female malignancies. It remains a leading cause of cancer-related mortality in women, significantly affecting women’s lives and quality of life [1,2]. CC is one of the few cancers that can be effectively prevented through early intervention through human papillomavirus (HPV) vaccination and screening for precancerous lesions, both of which are strongly recommended by the World Health Organization (WHO) and have been shown to significantly reduce incidence and mortality of CC [3,4,5]. High-risk HPV infection is the main cause of precancerous cervical lesions and CC [6].
The WHO guidelines on CC prevention recommend HPV nucleic acid testing for identifying women at risk of developing CC or its precursors [7,8]. Currently, clinical HPV nucleic acid detection methods include testing for HPV DNA and HPV E6/E7 messenger RNA (mRNA). While HPV DNA testing detects viral genetic material, E6/E7 mRNA testing identifies transcripts of viral oncogenes E6 and E7, which are closely associated with the virus’s oncogenic activity and can more effectively assess the risk of CC development [8,9]. Still, HPV nucleic acid testing for CC screening is limited primarily by lower specificity, risk of overdiagnosis, and reduced sensitivity for certain lesion types, especially in younger populations and for adenocarcinoma precursors [10,11]. In addition, cytology testing for CC screening is limited by lower sensitivity, the risk of false-negative and false-positive results, potential for overdiagnosis and overtreatment, technical and interpretive variability, and psychosocial harms [10,12].
According to the China Cervical Cancer Screening Guidelines (Part I), co-testing (HPV nucleic acid testing combined with cytological testing) is recommended as the primary method for opportunistic screening. These 2 methods complement each other. Emerging methods such as methylation testing also show promise for application [13,14,15]. Methylation testing in CC screening refers to the analysis of DNA methylation patterns in specific host or viral genes to detect cervical precancerous lesions and CC, especially in women who test positive for high-risk HPV. This molecular method identifies alterations in gene methylation associated with disease progression, serving as a triage or primary screening tool to refine risk stratification and reduce unnecessary procedures [14,16].
Still, the CC screening strategies remain to be optimized, and data on CC screening through gene methylation remain scarce. There is a need for data to formulate relevant guidelines [14]. Therefore, this study aimed to compare HPV E6/E7 mRNA + ThinPrep cytology test (TCT) vs. HPV genotyping + TCT for screening and diagnosis of CC, and to explore the relationship between gene methylation and the occurrence and progression of cervical lesions.
MATERIALS AND METHODS
1. Study population
This prospective cohort study enrolled women who presented at the gynecology and physical examination outpatient clinics of the author’s Hospital between January 2023 and November 2024 and accepted and underwent colposcopy-guided biopsy. The study was approved by the ethics committee of Luoyang Maternal and Child Health Hospital (approval #KY2022122218.0, approved on December 26, 2022). The patients provided written informed consent to participate in the study. All methods were performed in accordance with the relevant guidelines. All procedures were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.
The inclusion criteria were: 1) non-pregnant and non-lactating women, 2) no signs of acute genital tract infection (e.g., abnormal vaginal discharge, itching, pain, foul odor, fever, or lower abdominal pain), 3) no severe systemic diseases such as significant hepatic or renal dysfunction, or cardiopulmonary failure that could interfere with the screening or skew the results, 4) no immunodeficiency diseases (e.g., acquired immunodeficiency syndrome or congenital immunodeficiencies) and not under long-term immunosuppressive therapy (e.g., post-organ transplant or autoimmune disease treatment), and 5) no history of malignant tumors. The exclusion criteria were: 1) history of uterine or cervical surgery or 2) incomplete clinical data, including indeterminate/invalid test results.
2. Procedures
The patients selected 1 of 2 co-testing screening strategies after a comprehensive discussion with the study personnel. Strategy 1: HPV genotyping combined with TCT, hereafter referred to as HPV DNA + TCT. Strategy 2: HPV E6/E7 mRNA testing combined with TCT, hereafter referred to as HPV E6/E7 mRNA + TCT. All patients subsequently received colposcopy-guided biopsy and histopathological examination.
The patients were instructed to abstain from sexual intercourse, vaginal douching, and intravaginal medications within 24 hours before sampling to avoid disruption of the vaginal environment and ensure sample accuracy. All patients were placed in the lithotomy position, and the cervix was exposed. Using a sterile cotton ball soaked in normal saline, the cervical surface was gently cleaned to remove secretions. Cervical exfoliated cells were collected using a disposable cervical brush, which was then placed in a vial containing a cell-preservation solution for further analysis.
3. TCT
The ThinPrep 2000 automated processor was used for sample processing, including separation, filtration, adhesion, fixation, and staining. Two pathologists independently reviewed the cytological slides and made diagnoses according to the 2014 Bethesda System. A cytological result of atypical squamous cells of undetermined significance (ASC-US) or more severe was considered positive.
4. HPV E6/E7 mRNA testing
The residual TCT sample was used for HPV E6/E7 mRNA detection via branched DNA signal amplification, using the HPV E6/E7 mRNA Detection Kit (Zhengzhou Kodia Biotechnology Co., Ltd., Zhengzhou, China). Results were interpreted according to the kit’s instructions, with values ≥1.0 considered positive.
5. HPV DNA genotyping
HPV DNA genotyping was performed using a gene chip technology combining polymerase chain reaction (PCR) and reverse dot blot hybridization (Yaaneng Biotech, Shenzhen, China) to detect 15 HPV genotypes: HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68. The sampling method was identical to that for TCT. The test was performed following the manufacturer’s protocol.
6. Methylation detection of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 genes
Tissue samples were preprocessed, and nucleic acids were extracted. The extracted nucleic acids were treated with a bisulfite conversion kit. The promoter methylation status of the FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 genes was determined by PCR. Methylation results were interpreted based on the cycle threshold values of target and internal control genes.
7. Histopathological examination
Two experienced pathologists independently reviewed all pathological specimens at the study institution. In cases of disagreement, a third pathologist was consulted, and a consensus was reached. According to the 2014 WHO histological classification, the pathological diagnoses were categorized as chronic cervicitis, low-grade squamous intraepithelial lesion (LSIL) (cervical intraepithelial neoplasia [CIN]1), high-grade squamous intraepithelial lesion (HSIL) (CIN2 and CIN3), and carcinoma. For analysis purposes, lesions classified as chronic cervicitis and LSIL were grouped as HSIL−, while HSIL (CIN2 and CIN3) and carcinoma were grouped as HSIL+. Histopathological diagnosis served as the gold standard, and HSIL+ was defined as the primary study endpoint. Serial co-testing (i.e., HPV DNA + TCT) was defined as positive only when both HPV DNA and TCT were positive. Parallel co-testing (i.e., HPV DNA or TCT) was defined as positive if either test was positive.
8. Statistical analysis
This study adopted a single-blind design with evaluators: all laboratory testing personnel (responsible for HPV DNA testing, HPV E6/E7 mRNA testing, pathological radiographs, etc.) were blinded to the clinical information (such as age, past medical history) and other test results (such as TCT report). The samples were coded using a random number. Investigators who were not involved in the result assessments collected data that matched the test results of the tested samples. Data analysis was performed by statisticians after all tests were completed to ensure that the test results were not affected by subjective bias.
Data were analyzed using SPSS v26.0 (IBM Corp., Armonk, NY, USA). Categorical variables were expressed as number (%) and analyzed using the χ2 test. Spearman correlation analysis was used to assess associations. The diagnostic performance of HPV E6/E7 mRNA, HPV DNA, and their respective co-testing strategies for HSIL+ was evaluated using receiver operating characteristic (ROC) curve analysis. The areas under the curve (AUC) values of ROC curves were compared using the DeLong test. Sensitivity, specificity, accuracy, and AUC were calculated. The consistency of HPV DNA, HPV E6/E7 mRNA, TCT (alone and in combination), and the methylation status of the FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 genes with cervical lesion classification was evaluated using the κ test. Spearman correlation was used to analyze the relationship between the positivity rates of TCT, HPV E6/E7 mRNA, and gene methylation (FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3) and the severity of cervical lesions. A positive r-value with p<0.05 indicated a statistically significant positive correlation, with a larger r-value indicating a stronger association. The diagnostic performance of HPV E6/E7 mRNA was compared with other methods (TCT, gene methylation, HPV DNA) in detecting HSIL+ using the χ2 test. Two-sided p-values <0.05 were considered statistically significant.
RESULTS
1. Patients and histopathological results
A total of 1,399 women underwent HPV DNA + TCT testing, and 991 underwent HPV E6/E7 mRNA + TCT testing. The age range of the patients was 17 to 81 years, with a mean age of 42.85±11.40 years.
Among the 1,399 patients, the cytology results indicated 987 cases of negative for intraepithelial lesion or malignancy (NILM), 237 of ASC-US, 108 of LSIL, and 67 of atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H+). Among the 1,399 participants who underwent HPV DNA + TCT testing, the histopathological results revealed 774 cases of chronic cervicitis (55.33%), 453 of LSIL (32.38%), and 172 of HSIL+ (12.29%).
Among the 991 patients, the cytology results indicated 681 cases of NILM, 175 of ASC-US, 89 of LSIL, and 46 of ASC-H+. Among the 991 participants who underwent HPV E6/E7 mRNA + TCT testing, there were 496 cases of chronic cervicitis (50.05%), 377 of LSIL (38.04%), and 118 of HSIL+ (11.91%) (Fig. S1). There were no significant differences between HPV DNA + TCT and HPV E6/E7 mRNA + TCT in age distribution (p=0.999) and TCT screening results (p=0.660) (Table S1). Table S2 shows the age subgroup analyses of cytological classifications and molecular diagnoses.
2. Positivity rates of different detection methods across pathological grades
The positivity rates of both HPV DNA and HPV DNA + TCT increased with lesion severity, and the differences were statistically significant (χ2=276.102, p<0.001; χ2=404.214, p<0.001). The positivity rates of both HPV E6/E7 mRNA and HPV E6/E7 mRNA + TCT also increased with lesion severity, and the differences were statistically significant (χ2=291.344, p<0.001; χ2=327.227, p<0.001) (Table 1).
Table 1. Positivity rates of HPV E6/E7 mRNA, HPV DNA, HPV DNA + TCT, and HPV E6/E7 mRNA + TCT across different histopathological grades.
| Pathological grade | HPV E6/E7 mRNA | HPV DNA | HPV DNA + TCT | HPV E6/E7 mRNA + TCT |
|---|---|---|---|---|
| Chronic cervicitis | 13.7% | 24.5% | 5.6% | 3.6% |
| LSIL | 40.1% | 61.1% | 36.4% | 26.8% |
| HSIL+ | 94% | 83% | 72% | 78% |
| χ2 | 291.344 | 276.102 | 404.214 | 327.227 |
| p-value | <0.001 | <0.001 | <0.001 | <0.001 |
HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; mRNA, messenger RNA; TCT, ThinPrep cytology test.
3. Risk assessment of HSIL+ detection by HPV DNA + TCT and HPV E6/E7 mRNA + TCT
The sensitivity, specificity, and accuracy for detecting HSIL+ were 72.09%, 83.05%, and 81.7% for HPV DNA + TCT, and 77.97%, 86.37%, and 85.37% for HPV E6/E7 mRNA + TCT. The odds ratio (OR) for developing HSIL+ in HPV E6/E7 mRNA + TCT positive patients was 22.42, which was 1.8 times higher than that of HPV DNA + TCT positive patients (OR=12.66) (Table 2; the 2×2 tables are shown in Table S3). The AUC was 0.777 (95% CI=0.728–0.826) for HPV DNA + TCT and 0.822 (95% CI=0.776–0.867) for HPV E6/E7 mRNA + TCT (DeLong test, p<0.001). An AUC between 0.8 and 0.9 indicates good diagnostic performance. Hence, HPV E6/E7 mRNA + TCT has a higher diagnostic accuracy and outperforms the conventional HPV DNA + TCT method (Fig. 1).
Table 2. Comparison of diagnostic performance of different screening strategies for HSIL+ detection.
| Screening strategy | Sensitivity (95% CI), % | Specificity (95% CI), % | Accuracy (95% CI), % | χ2 value | p-value | κ | OR | 95% CI | |
|---|---|---|---|---|---|---|---|---|---|
| Lower | Upper | ||||||||
| HPV DNA + TCT | 72.09 (64.96–78.26) | 83.05 (80.85–85.04) | 81.70 (79.59–83.64) | 253.419 | <0.001 | 0.39 | 12.66 | 8.785 | 18.232 |
| HPV DNA in parallel with TCT | 89.53 (84.07–93.28) | 55.42 (52.63–58.18) | 59.61 (57.02–62.16) | 121.945 | <0.001 | 0.194 | 10.636 | 6.444 | 17.554 |
| HPV E6/E7 mRNA + TCT | 77.97 (69.67–84.5) | 86.37 (83.93–88.49) | 85.37 (83.03–87.43) | 256.736 | <0.001 | 0.48 | 22.42 | 13.924 | 36.1 |
| HPV E6/E7 mRNA in parallel with TCT | 96.61 (91.61–98.67) | 63.92 (60.68–67.04) | 67.81 (64.84–70.65) | 327.227 | <0.001 | 0.208 | 50.486 | 18.451 | 138.136 |
CI, confidence interval; HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; mRNA, messenger RNA; OR, odds ratio; TCT, ThinPrep cytology test.
Fig. 1. ROC curves for HPV DNA + TCT and HPV E6/E7 mRNA + TCT in diagnosing HSIL+.
HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; mRNA, messenger RNA; ROC, receiver operating characteristic; TCT, ThinPrep cytology test.
4. Concordance analysis between HPV DNA and HPV E6/E7 mRNA
Among the 991 participants, 671 had completely concordant results: 227 were HPV E6/E7 mRNA (+) and HPV DNA (+), 444 cases were HPV E6/E7 mRNA (-) and HPV DNA (-). There were 320 discordant cases: 103 were HPV E6/E7 mRNA (+) and HPV DNA (−), and 217 were HPV E6/E7 mRNA (−) and HPV DNA (+). Among these, 129 cases were HPV 16/18 (+) and 862 cases were HPV 16/18 (−). Within the HPV 16/18 (+) group, 64 cases were HPV E6/E7 mRNA (+) and 65 cases were HPV E6/E7 mRNA (−). Within the HPV 16/18 (−) group, 266 cases were HPV E6/E7 mRNA (+), 596 cases were HPV E6/E7 mRNA (−) (Fig. 2).
Fig. 2. HSIL+ detection across HPV 16/18 and HPV E6/E7 mRNA test results. HSIL+ includes CIN2, CIN3/AIS, and cervical cancer.
AIS, adenocarcinoma in situ; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; mRNA, messenger RNA.
No HSIL+ lesions were observed in 65 patients with HPV 16/18 (+) but HPV E6/E7 mRNA (−). In cases with HPV 16/18 (+) and HPV E6/E7 mRNA (+), the immediate risks for CIN2+ and CIN3+ were 64.06% and 50%, respectively. In cases with HPV 16/18 (−) and HPV E6/E7 mRNA (+), the immediate risks for CIN2+ and CIN3+ were 26.32% and 6.39%, respectively. In cases with HPV 16/18 (−) and HPV E6/E7 mRNA (−), the immediate risks for CIN2+ and CIN3+ were 1.17% and 0.50%, respectively. The immediate risks for CIN2+ and CIN3+ were highest in the HPV 16/18 (+) and HPV E6/E7 mRNA (+) group. It suggests that although HPV 16/18 is currently recognized as a key factor in cancer development, the absence of E6/E7 mRNA expression indicates a relatively lower carcinogenic risk compared with HPV with active E6/E7 mRNA expression. Moreover, in cases with HPV 16/18 (−) but HPV E6/E7 mRNA (+), a risk of CIN2+ lesions remains (Table 3).
Table 3. Distribution of HPV 16/18 and HPV E6/E7 mRNA test results in the study population.
| Test | Total number | Chronic cervicitis | LSIL | CIN2 | CIN3/AIS | Cervical cancer |
|---|---|---|---|---|---|---|
| HPV 16/18 (+) | 129 | 31 | 57 | 9 | 30 | 2 |
| HPV E6/E7 mRNA (+) | 64 | 5 | 18 | 9 | 30 | 2 |
| HPV E6/E7 mRNA (−) | 65 | 26 | 39 | 0 | 0 | 0 |
| HPV 16/18 (−) | 862 | 465 | 320 | 57 | 15 | 5 |
| HPV E6/E7 mRNA (+) | 266 | 63 | 133 | 53 | 14 | 3 |
| HPV E6/E7 mRNA (−) | 596 | 402 | 187 | 4 | 1 | 2 |
AIS, adenocarcinoma in situ; CIN, cervical intraepithelial neoplasia; HPV, human papillomavirus; LSIL, low-grade squamous intraepithelial lesion; mRNA, messenger RNA.
5. Correlation between FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 gene methylation and cervical lesions
One hundred tissue samples were randomly selected to investigate the correlation between the methylation of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 genes and cervical lesions. The samples included 20 cases of chronic cervicitis, 20 of LSIL, 58 of HSIL, and 2 of CC. The positive methylation rates of the 6 genes (FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3) in chronic cervicitis, LSIL, HSIL, and CC were 40%, 70%, 74%, and 100%, respectively, with an overall positive rate of 67%. The κ analysis between gene methylation and cervical lesion grade yielded a κ value of 0.206 (p=0.031), indicating a statistically significant but low level of consistency between the 2. The Spearman correlation analysis showed that the positivity rates of TCT, HPV E6/E7 mRNA, and FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 gene methylations were all positively correlated with the grade of cervical lesions. TCT and HPV E6/E7 mRNA positivity were significantly positively correlated with lesion grade (p<0.001). The correlation coefficient r values were in the following order, from highest to lowest: HPV E6/E7 mRNA, TCT, methylation, and HPV DNA. The diagnostic performance of HPV E6/E7 mRNA (sensitivity, specificity, and OR value) was superior to the other methods, with statistically significant differences (χ2=42.438, p<0.001) (Table 4, Fig. S2). (Table 4; the 2×2 tables are shown in Table S4).
Table 4. Comparison of diagnostic performance of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, JAM3 methylation and other screening methods for HSIL+.
| Screening method | Sensitivity (95% CI), % | Specificity (95% CI), % | AUC (95% CI), % | κ | R | χ2 | p-value | OR | 95% CI | |
|---|---|---|---|---|---|---|---|---|---|---|
| Lower | Upper | |||||||||
| TCT | 76.7 (64.56–85.56) | 75.0 (59.81–85.81) | 0.758 (0.463–0.6985) | 0.508 | 0.51 | 26.001 | <0.001 | 9.857 | 3.878 | 25.053 |
| HPV E6/E7 mRNA | 83.3 (71.97–90.69) | 82.5 (68.05–91.25) | 0.829 (0.742–0.917) | 0.65 | 0.651 | 42.438 | <0.001 | 23.571 | 8.157 | 68.118 |
| HPV DNA | 78.3 (66.38–86.88) | 37.5 (24.22–52.97) | 0.579 (0.463–0.695) | 0.167 | 0.173 | 2.984 | 0.067 | 2.169 | 0.893 | 5.267 |
| FAM19A4, ZNF582, EPHA7, SOX17, PAX1, JAM3 methylation | 75.0 (62.77–84.22) | 45.0 (30.71–60.17) | 0.600 (0.485–0.715) | 0.206 | 0.208 | 4.342 | 0.031 | 2.455 | 1.045 | 5.766 |
AUC, area under the curve; CI, confidence interval; HPV, human papillomavirus; HSIL, high-grade squamous intraepithelial lesion; mRNA, messenger RNA; OR, odds ratio; TCT, ThinPrep cytology test.
DISCUSSION
This prospective observational study compared HPV E6/E7 mRNA + TCT versus HPV genotyping + TCT in screening CC and to explore the relationship between gene methylation and cervical lesions. The results suggested that the sensitivity and specificity of HPV E6/E7 mRNA + TCT were superior to those of HPV DNA + TCT. Simultaneous positivity for HPV 16/18 and HPV E6/E7 mRNA suggests a high immediate risk of CIN3+. Methylation levels of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3 increased with lesion severity, but the low κ-value suggests that the clinical significance of their methylation levels should be carefully investigated.
In this study, women undergoing opportunistic screening at a single hospital were tested using various screening strategies. A comparative analysis of the performance of different screening methods for detecting high-grade cervical lesions was conducted. The results showed that combined screening was more effective than single-method screening, indicating that among the combined strategies, HPV E6/E7 mRNA + TCT had the best diagnostic performance for HSIL+. The sensitivity, specificity, and accuracy of HPV E6/E7 mRNA + TCT were all superior to those of HPV DNA + TCT, consistent with the findings of Yuan et al. [17]. The OR of developing HSIL+ among patients positive for HPV E6/E7 mRNA + TCT was 22.42, 1.8 times higher than that among patients positive for HPV DNA + TCT (OR=12.66). It indicates that dual positivity in HPV E6/E7 mRNA + TCT was associated with a significantly higher risk of cervical lesions. Furthermore, the AUC was 0.822 for HPV E6/E7 mRNA + TCT, indicating that the combined screening results more accurately reflect the actual disease status. A study by Al-Shibli et al. [18] showed that even when cytology is negative, a positive HPV mRNA result still indicates a high risk of CIN3+, confirming that HPV E6/E7 mRNA + TCT is more predictive of lesion progression and thus more beneficial as a primary screening strategy.
In addition, the present study revealed discrepancies between HPV E6/E7 mRNA and HPV DNA results. Some patients were HPV DNA-positive but HPV E6/E7 mRNA-negative, indicating viral infection without transcriptional activation of E6/E7, which may represent a transient infection. Conversely, some patients were HPV DNA-negative but HPV E6/E7 mRNA-positive. Possible reasons include cross-mutations among different HPV types that cause the failure of the specific hybridization probes used in the PCR-reverse dot blot method, or the deletion of the L1 gene after HPV DNA integration into the host genome, leading to reduced viral replication and a lower viral load [19].
It is well-known that high-risk HPV types 16 and 18 cause the majority (about 70%) of CCs [20,21,22], and colposcopy is recommended for HPV 16/18-positive patients. However, HPV screening alone, although highly sensitive, tends to have low specificity. Huang et al. [23] reported that the prevalence of HSIL+ among HPV 16/18-infected women was 29.5%, suggesting that some patients may undergo overtreatment and require individualized triage. The present study found that in patients with both HPV 16/18 and HPV E6/E7 mRNA positivity, the immediate risks of developing CIN2+ and CIN3+ were 64.06% and 50%, respectively. Among those positive for HPV 16/18 but negative for HPV mRNA, none were diagnosed with HSIL+, consistent with data reported by Bruno et al. [24]. It indicates that HPV E6/E7 mRNA positivity has a higher predictive value for CIN2+ lesions. In the study by Sørbye et al. [25], the cumulative incidence of CIN3+ after 6 years was 1.3% among women with negative cytology and only 0.6% among those with both negative cytology and HPV mRNA, confirming the safety of HPV E6/E7 mRNA-negative results. In addition, Bruno et al. [26] suggested that negative HPV E6/E7 mRNA results could serve as a prognostic indicator of CIN2 regression. However, since the present study population consisted of women undergoing initial screening, further follow-up studies on HPV 16/18- and E6/E7-mRNA-positive patients are needed to gain deeper insights into the risk assessment value of these 2 testing methods for high-grade lesions.
Tissue methylation detection and cytology evaluation reflect different dimensions of CC. Methylation is an early indicator of epigenetic changes at the molecular level in cervical cells [27,28], whereas cytological examination evaluates morphological abnormalities at the cellular level. Previous studies have extensively investigated methylation sites in genes such as FAM19A4, ZNF582, and PAX1 [29]. It has been found that the promoter regions of FAM19A4, PAX1, and ZNF582 are significantly methylated in cervical HSIL+, suggesting that this may be an important molecular event in the development of cervical lesions and that these regions have the potential to serve as reliable markers for CC diagnosis [29,30]. According to a study by Li et al. [31], dual-gene methylation testing for JAM3 and PAX1 can be used independently to stratify cervical high-grade lesions from non-high-grade lesions in HPV-infected women, regardless of cervical cytology results. Xiang et al. [32] conducted a study based on the methylation of 6 genes (DLX1, ITGA4, RXFP3, SOX17, ZNF671, and ASTN1) and found that the accuracy of multi-gene methylation testing in distinguishing HSIL+ could reach 97.5%, indicating that multi-gene methylation testing can accurately identify high-grade cervical lesions and cancer. Furthermore, Liu et al. [33] investigated methylation of the PAX1 promoter and HPV E6/E7 mRNA detection, finding that the combined detection of these markers enhances diagnostic performance for HSIL and CC. Due to the influence of clinical interference factors, tissue samples are susceptible to local inflammation and necrotic tissue, which can affect cytological evaluation. Cytological samples are susceptible to menstrual cycles, storage conditions, etc. In addition, different detection timings (screening stage vs. biopsy stage) will also lead to differences in results. Since methylation aberrations precede cytological changes, the methylation results may possibly be used to identify at-risk patients. Therefore, the present study preliminarily investigated the methylation of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3. Spearman correlation analysis showed that the methylation of these genes was associated with cervical lesions, suggesting that the degree of methylation positivity may reflect the severity of cervical lesions and that promoter methylation plays a role in the progression of cervical disease. However, the low κ-value, consistent with biopsy results, may be due to an insufficient sample size, heterogeneity in tissue samples, and potential selection bias in sample inclusion. The diagnostic value of methylation of FAM19A4, ZNF582, EPHA7, SOX17, PAX1, and JAM3, as well as HPV E6/E7 mRNA testing in HSIL+, warrants further large-scale and standardized clinical studies. Still, methylation patterns may help identify subgroups of women at different risk of lesion progression, and their impact could vary across lesions. The methylation subgroup analysis (n=100) of this study was designed as an exploratory study to preliminarily assess the potential association of target markers with clinical outcomes, rather than to validate diagnostic efficacy. Although the sample size is small compared to the total cohort, it has included diverse pathological outcomes, providing an important basis for subsequent large-scale studies. The observed weak agreement (κ=0.206) may suggest that altered methylation is an early event in disease progression. It could allow for a refinement of the triage strategies, but studies are necessary.
A strength of the present study is the large sample size and the comparison of 2 screening strategies. Another strength is the investigation of gene methylation patterns. The HPV mRNA detection kit (branched DNA signal amplification; Zhengzhou Kodia Biotechnology Co., Ltd., Zhengzhou, China) covered HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, which are currently recognized as 14 high-risk types of HPV at home and abroad. These 14 high-risk HPV types are also those defined by WHO, because the expression of the E6/E7 oncogenes is directly related to the progression of cervical lesions [6,7]. HPV DNA genotyping was performed using a gene chip technology combining PCR and reverse dot blot hybridization (Yaaneng Biotech) to detect 15 HPV genotypes. The HPV DNA kit includes HPV 53, which is a type of HPV infection that is often found in Chinese, but its carcinogenic potential is extremely low [34]. The inclusion of HPV 53 in the HPV DNA testing is based on regional epidemiological needs. Hui and Yang [35] showed that HPV 53 is among the five most common HPV subtypes in Henan Province, with a positive rate of 1.32% despite its low carcinogenicity. The broad coverage of HPV DNA testing (including HPV 53) reduces missed diagnoses, while the specificity of HPV mRNA testing avoids overtreatment, forming a complementary strategy.
In this study, no HSIL+ lesions (0/65) were observed in 65 patients with HPV 16/18 (+) and HPV E6/E7 mRNA (−) (95% CI=0.0–4.5). It should be noted that zero events may reflect a truly low risk, or a too-small number of people may have been included due to sample size limitations. Therefore, the ‘zero risk’ conclusion of this study is applicable only to its results, and risk reassessment should be performed in a wider population or over a longer period of observation. In clinical practice, HPV 16/18 and HPV E6/E7 mRNA should be combined with other risk stratification tools to make comprehensive risk assessments.
In 2020, the WHO adopted the Global Strategy to Accelerate the Elimination of Cervical Cancer as a Public Health Problem, recommending a comprehensive prevention and treatment strategy [6,7]. The strategy divides cervical cancer prevention into 3 levels, with secondary prevention covering screening and treatment of precancerous lesions. The WHO encourages countries to conduct HPV testing, including HPV DNA and HPV mRNA testing. In addition, the 2019 American Society for Colposcopy and Cervical Pathology guidelines recommend selecting highly specific products for clinical screening [36]. HPV mRNA testing can reduce the referral rate for colposcopy by 21% compared with HPV DNA testing, indicating greater cost-effectiveness. In addition, patients with positive HPV E6/E7 mRNA tests have a higher risk of progressing to CIN2+ in the early stages of precancerous lesions compared to HC2 methods (24%–29%) [37]. This evidence further highlights the importance of HPV E6/E7 mRNA testing in the early screening stage of cervical cancer. The data from the present study support the critical role of HPV E6/E7 mRNA testing in risk stratification, and HPV E6/E7 mRNA + TCT can be considered for screening in areas with sufficient resources, but more cost-effectiveness data are still needed to fully replace DNA screening. Indeed, primary HPV screening programs increasingly rely on risk-based management to balance cancer prevention against over-referral to colposcopy, and the observed performance of HPV E6/E7 mRNA + TCT in this study is consistent with a strategy that maintains or improves HSIL+ detection while modestly reducing unnecessary procedures compared with HPV DNA + TCT. Within these pathways, the combination of mRNA-based triage and methylation markers provides a framework to more precisely target colposcopy to women with transcriptionally active, transformation-associated infections and epigenetically advanced lesions, which aligns with current efforts to refine triage in HPV-positive populations [36,38,39]. Indeed, HPV E6/E7 mRNA assays identify transcriptionally active infections and have repeatedly shown higher specificity and positive predictive value than HPV DNA when used as triage of HPV-positive women, with fewer colposcopy referrals per CIN2+ detected and preserved sensitivity. In population-based programs, genotype-specific E6/E7 mRNA triage has reduced colposcopy referrals by roughly one-third compared with cytology-based triage, while detecting a similar or higher proportion of CIN2+ lesions, which closely parallels the improved performance metrics observed for HPV E6/E7 mRNA + TCT in this cohort [38,40]. In the present data, women with HPV 16/18 (+)/E6/E7 mRNA (+) had immediate CIN2+ and CIN3+ risks of 64.06% and 50%, respectively, which are far above standard colposcopy thresholds and even approach ranges where expedited treatment without prior biopsy is considered acceptable in current risk-based guidelines. Conversely, the absence of HSIL+ in women who were HPV 16/18 (+)/E6/E7 mRNA (−) suggests that these cases resemble regressing or transient infections and might be managed with repeat HPV-based testing rather than immediate colposcopy, potentially shifting them into a 1-year surveillance category if validated in larger cohorts. The higher specificity and AUC of HPV E6/E7 mRNA + TCT relative to HPV DNA + TCT (86.37% vs. 83.05% for specificity; 0.822 vs. 0.777 for AUC) imply fewer false-positive screens and, therefore, fewer low-yield colposcopies for a given HSIL+ detection rate. It is consistent with larger triage studies, in which HPV mRNA testing reduced colposcopy positivity rates and the number of colposcopies needed per CIN2+ case, without compromising the detection of clinically relevant disease [38,40]. DNA methylation assays targeting genes such as PAX1, JAM3, FAM19A4, ZNF582, EPHA7, and SOX17 have emerged as highly specific markers of advanced cervical intraepithelial neoplasia, with methylation levels rising with lesion severity, as also observed in this cohort. Recent studies show that combined PAX1/JAM3 methylation can achieve sensitivities of 84%–89% and specificities of 95% or higher for CIN2+, substantially reducing colposcopy referrals among HPV-positive or ASC-US women while maintaining or improving the detection of clinically significant disease [39]. Embedding these methylation markers downstream of HPV primary screening and mRNA or cytology triage fits naturally within risk-based frameworks: women with high-risk HPV, positive E6/E7 mRNA, and high methylation levels would occupy the highest risk stratum, warranting immediate colposcopy or even expedited treatment, whereas HPV-positive women with negative mRNA and low methylation could be safely managed with extended surveillance intervals. In partially vaccinated or ageing populations where overall CIN3+ prevalence declines, such multi-step, molecularly anchored triage algorithms may be essential to contain colposcopy volumes while preserving the effectiveness of cervical cancer prevention programs.
Still, the study also had limitations. The study was conducted at a single center, located in a relatively small geographic area in China. Multicenter studies would allow for a larger sample size. Second, allocation of women to screening arms based on patient choice represents a form of self-selection that can compromise both internal and external validity and should be explicitly acknowledged as a limitation. In diagnostic accuracy research, non-random, non-consecutive participant inclusion is a well-recognized source of selection (spectrum) bias that can lead to over- or underestimation of sensitivity, specificity, and predictive values. Nevertheless, the demographics and lesion distribution across screening methods were comparable. Third, the criteria for biopsy referral could introduce a verification bias. Fourth, the sample size for the methylation analysis was small (n=100), limiting the statistical power and robustness of these specific findings. It could be responsible for the poorer diagnostic value results observed in the subgroup compared with the whole study population. It could also be responsible for the low κ value. Although the study was prospective, strategy assignment was not random, leading to unequal group sizes and possible selection bias. Fifth, no Decision Curve Analysis (DCA) and Net Reclassification Improvement (NRI) were performed. Performing a robust DCA/NRI analysis would require a different trial design focused on predefined management thresholds and larger sample sizes for stable estimates.
In conclusion, as the severity of cervical intraepithelial neoplasia increases, the expression level of E6/E7 mRNA also increases. A positive result indicates that high-risk HPV is in a transcriptionally active phase, making it a more specific risk assessment indicator. Combined screening strategies are superior to single-method screening strategies, particularly the combination of HPV E6/E7 mRNA with TCT, which improves specificity without compromising sensitivity and aligns with China’s CC screening strategy. Cervical DNA methylation markers may have potential value in detecting HSIL+, but the limited sample size and the low κ value indicate the need for further large-scale, multicenter clinical validation. Therefore, for the initial outpatient screening population, it may be recommended to use HPV E6/E7 mRNA testing combined with TCT testing for CC screening, as it enhances sensitivity while maintaining specificity, helps identify high-risk individuals, and improves diagnostic efficiency.
ACKNOWLEDGEMENTS
The authors would like to express their gratitude to Professor Chang Lei from the First Affiliated Hospital of Zhengzhou University for her guidance on this research paper.
We also extend our thanks to Chengdu Norson Medical Laboratory Co., Ltd. for their experimental support in this project.
Special appreciation goes to the gynecology team at Luoyang Maternal and Child Health Hospital for their assistance and support in data collection and literature review.
Footnotes
Funding: This study was funded by the Key Scientific and Technological Project of Luoyang Science and Technology Bureau (grant No. 2302017A) in 2023. The funder had no role in the decision to publish the results.
Conflict of Interest: No potential conflict of interest relevant to this article was reported.
Data Availability: All data generated or analyzed during this study are included in this published article.
- Conceptualization: B.Y.
- Data curation: Z.X., C.L., D.W., L.L., S.Y., F.X., B.Y., W.Y., L.J.
- Formal analysis: Z.X., C.L., D.W., L.L., S.Y., F.X., B.Y., L.J.
- Funding acquisition: S.Y., W.Y.
- Investigation: C.L., L.L.
- Methodology: D.W., F.X., B.Y.
- Project administration: Z.X., D.W., S.Y., W.Y., L.J.
- Resources: C.L.
- Software: Z.X., L.L., L.J.
- Supervision: Z.X., D.W., S.Y., W.Y.
- Validation: Z.X., S.Y., F.X., W.Y.
- Visualization: F.X., B.Y., W.Y.
- Writing - original draft: Z.X., C.L., D.W., L.L., F.X., B.Y., L.J.
- Writing - review & editing: Z.X., D.W., L.L., S.Y.
SUPPLEMENTARY MATERIALS
Age and diagnoses according to the testing strategy
Age subgroup analyses of cytological classifications and molecular diagnoses
Risk assessment of HSIL+ detection by HPV DNA + TCT and HPV E6/E7 mRNA + TCT, complementary to Table 2
Risk assessment of HSIL+ detection by methylation, complementary to Table 4
Patient inclusion flowchart.
Receiver operating characteristic curves for HPV DNA, TCT, HPV E6/E7 mRNA, and methylation in diagnosing HSIL+.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Age and diagnoses according to the testing strategy
Age subgroup analyses of cytological classifications and molecular diagnoses
Risk assessment of HSIL+ detection by HPV DNA + TCT and HPV E6/E7 mRNA + TCT, complementary to Table 2
Risk assessment of HSIL+ detection by methylation, complementary to Table 4
Patient inclusion flowchart.
Receiver operating characteristic curves for HPV DNA, TCT, HPV E6/E7 mRNA, and methylation in diagnosing HSIL+.