INTRODUCTION

Cervical cancer remains the fourth most common type of cancer in women. In 2020, there were an estimated 604,000 new cervical cancer cases worldwide and about 342,000 deaths from the disease.[1] Human papillomavirus (HPV) infection is widely recognized as one of the main causes of cervical cancer, which is generally manifested in four stages: latent infection, subclinical infection, clinical symptoms, and HPV-related tumors. However, differences in the specific type of HPV infection may have a different impact on the progression of the above staging.[2-5] DNA methylation is one of the important molecular mechanisms of epigenetics, which, as studies have established, accumulates with the development of tumors. For example, the degree of methylation of paired boxed 1 (PAX1) and zinc finger protein 582 (ZNF582) is consistent with the progression of cervical intraepithelial neoplasia (CIN).[6-8] PAX1/ZNF582 thus can be used as a molecular biomarker for the early diagnosis of cervical cancer. Hence, this study attempts to establish a new triage, diagnosis, and treatment model for accurate screening and hierarchical management of cervical cancer based on HPV typing and quantification as a primary screening method as well as PAX1/ZNF582 methylation detection.

MATERIAL AND METHODS Information and grouping of patients

Totally 115 patients treated at the Department of Cervical Surgery of Xuzhou Maternity and Child Health Care Hospital from October 2018 to October 2020 were selected according to the following inclusion and exclusion criteria. Inclusion criteria: A 20–65-year-old married women were included in the study. Exclusion criteria: History of cervical biopsy, history of reproductive system malignancy or immune system disease, menstruation, or pregnancy were excluded from the study. The study was approved by the Ethics Committee of the hospital, and all subjects signed informed consent. The diagnosis was based on pathological evaluation of colposcopy biopsy as the gold standard, and the patients were classified into four groups: Chronic cervicitis (inflammation, n = 21), low-grade cervical squamous intraepithelial lesion (LSIL, n = 20), high-grade cervical squamous intraepithelial lesion (HSIL, n = 41), and cervical cancer (n = 33). All the enrolled patients underwent thinprep cytologic test (TCT) of cervical exfoliated cells, HPV typing and quantification, and PAX1/ ZNF582 methylation detection, and those who were positive for any of these tests were required to undergo pathological diagnosis of colposcopy biopsy.

Methods Specimen collection

Subjects had no sexual activities, vaginal douching, or vaginal medication within 48 h before specimen collection. Biopsy specimens were collected during non-menstrual periods for examination and before the biopsy started. The cervix was exposed and the secretion as well as blood on the cervical orifice was cleaned before the cervical cytology brush for TCT, HPV and methylation tests was inserted into the cervical squamocolumnar junction and swirled clockwise or anticlockwise for 4–6 times to collect the cervical exfoliated cells. After that, the brush head was placed in a test tube containing the preservation solution marked with the subject code. Three specimens were used for TCT, HPV, and methylation tests. The specimens for typing and quantification of 21 HPV subtypes were collected and transported using the special specimen transport medium provided by Jiangsu Bioperfectus Technologies Co., Ltd.

TCT

Specimens were processed using an automated liquid-based cytology slide preparation system to make thin-layer smears. The cytology results were evaluated by The Bethesda System, and abnormal results had to be interpreted by a second pathologist, which included: Negative for intraepithelial lesion or malignancy, atypical squamous cells of undetermined significance (ASCUS), atypical squamous cells-cannot exclude HSIL, LSIL, HSIL, and squamous cell carcinoma. A positive result of ASCUS and above was considered to be cytologically positive for follow-up analysis.

Typing and quantification of 21 HPV subtypes

21 HPV subtypes (HPV16+, HPV18+, HPV16/18+, 11 high-risk HPV+, and 14 high-risk mixed HPV) could be quickly and accurately distinguished in the tested specimens within 2.5 h, and their viral loads could also be standardized and quantified using the standardized patented technology of cervical exfoliated cell sampling and multiplex quantitative fluorescent polymerase chain reaction (PCR) at the same time. In other words, the typing and quantification of 21 HPV subtypes (viral load value/10000 cells) could be achieved simultaneously.

Detection of gene methylation in cervical cancer

The real-time quantitative methylation-specific PCR was utilized to detect the methylation level of PAX1/ZNF582, which included two steps: (1) DNA extraction and bisulfite conversion: Cervical exfoliated cells were placed in a liquid-based cell preservation solution and stored in liquid nitrogen before DNA was extracted and treated with bisulfite. All procedures followed the instructions of the Cervi-M Detection Kits. (2) The methylation levels were detected by real-time quantitative PCR.

Statistical analysis

SPSS 20.0 was used for statistical analysis. The categorical data were tested by χ2, and the quantitative data were expressed as ( ± s). The pairwise comparison was performed by the t-test, and P < 0.05 is statistically significant.

RESULTS Histocytology

In TCT, the positive rates of chronic cervicitis, LSIL, HSIL, and cervical cancer groups were 42.9%, 45%, 70.7%, and 54.5%, respectively (sensitivity: 59.3%, specificity: 57.89%, diagnostic coincidence rate: 60.8 %), as shown in [Figure 1].

Figure 1:: Histocytological examination results of the four groups (%).

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Helicobocton pyloni (HP)-HPV

In the HP-HPV test, the HPV genotypes with infection rates from high to low were HPV16, 52, 58, 33, 18, and 31, of which HPV16+ accounted for 41.3%. The positive rates of chronic cervicitis, LSIL, HSIL, and cervical cancer groups were 14.2%, 30%, 53.6%, and 90.9%, respectively (sensitivity: 89.71%, specificity: 18.92%, diagnostic coincidence rate: 65.2%), as shown in [Figure 2].

Figure 2:: HP-human papillomavirus detection results of the four groups (%).

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Correlation between viral loads and cervical lesions

According to the Bio perfectus multiplex real time (BMRTHPV) test, there were statistically significant differences in HPV 16, 33, and 52 between different grades of cervical lesions (P < 0.05), and their viral loads increased with the grade of cervical lesions. The viral loads of HPV 16, 33, and 52 in high-grade lesions were significantly higher than those in low-grade lesions (P < 0.05). This indicated that the viral loads of HPV 16, 33, and 52 were positively correlated with the grade of cervical lesions, as shown in [Figure 3].

Figure 3:: Correlation between the viral loads of HPV 16, 33, and 52 and the grade of cervical lesions. HPV: Human papillomavirus.

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PAX1

The positive rates of chronic cervicitis, LSIL, HSIL, and cervical cancer groups were 0%, 5%, 73.1%, and 84.8%, respectively (sensitivity: 79.41%, specificity: 97.3%, diagnostic coincidence rate: 85.71%), as shown in [Figure 4].

Figure 4:: PAX1 detection results of the four groups (%).

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PAX1/ZNF582

The positive rates of chronic cervicitis, LSIL, HSIL, and cervical cancer groups were 4.7%, 15%, 80.4%, and 97%, respectively (sensitivity: 88.24%, specificity: 91.89%, diagnostic coincidence rate: 89.52 %), as shown in [Figure 5].

Figure 5:: PAX1/ZNF582 detection results of the four groups (%).

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DISCUSSION

The principle of cervical cancer screening should be to maximize screening benefits and minimize potential harms. Simply put, it is to identify precancerous lesions that may progress to infiltrating cancer, and avoid excessive examination and unnecessary treatment for transient HPV infections and their corresponding benign lesions. More than 90% of HPV infections will be cleared by the body within 2 years,[9] while infection with high-risk HPV for more than 2 years, if not treated, may progress to CIN. High-grade cervical lesions may further progress into cervical cancer.

The most common HPV genotypes in cervical precancerous lesions among the Chinese population are HPV16, 52, 58, 33, 18, and 31.[10] The higher the amount of HPV infection, the longer it takes the body to clear it.[11] Since the persistent infection of the virus is a necessary condition for the occurrence of cervical cancer, it can be considered that the higher the amount of HPV infection, the greater the risk of cervical cancer. The best screening concept at present is risk stratification and quantitative management. Although HPV is highly sensitive, the large number of transient infections in the results may increase the patients’ unnecessary psychological stress and follow-up treatment. Therefore, given the high sensitivity and low specificity of primary HPV screening, certain measures should be taken to triage those who tested positive in primary screening.

HPV infection itself does not necessarily lead to the occurrence of cervical cancer, but some studies have shown that after HPV infection, the main risk factor contributing to the occurrence of malignant tumors is the abnormal expression of oncogenes, such as abnormal cell proliferation and division. The underlying mechanism is that the abnormal expression of methylation of tumor suppressor genes reduces their overall content, resulting in abnormal downstream genes and finally cervical cancer.[12,13] Studies have demonstrated that PAX1 can be used as a potential biomarker for the early diagnosis and screening of cervical cancer.[14] The methylation level of ZNF582 is also correlated with the occurrence and development of cervical cancer,[15,16] and the overall sensitivity and specificity of ZNF582 methylation detection are 70% and 82%, respectively.[17,18]

CONCLUSION

The present study reveals that the sensitivity and specificity of TCT are 59.30% and 57.89%, respectively; those of HPV16 and 18 are 72.06% and 78.38%, respectively; those of PAX1/ZNF582 are 88.24% and 91.89%, respectively, and the combination with PAX1/ZNF582 methylation detection can make up for the low specificity of HPV. PAX1/ZNF582 methylation detection enjoys several advantages. Firstly, DNA methylation is relatively stable and less affected by subjective factors. Secondly, the cervical exfoliated cells can be used for methylation detection, which is easy to obtain, non-traumatic, and easily accepted by the patients. Thirdly, the combined methylation detection of the two genes improves the sensitivity and specificity. Therefore, typing and quantification of HPV in primary screening, together with PAX1/ZNF582 methylation detection and shunting can make up for the deficiencies of methylation detection, HPV, or cytology alone, and can be considered as a promising means of accurate cervical cancer screening.