Objectives: Currently, traditional cervical cancer screening methods, such as high-risk human papillomavirus testing and liquid based cytology (LBC), still possess limitations. This study aims to identify new diagnostic biomarkers to achieve the goal of "precision screening" via exploring the clinical value of DNA methylation [ΔCtP: paired box gene 1 (PAX1)and ΔCtJ: junctional adhesion molecule 3 (JAM3)] detection in cervical exfoliated cells for the diagnosis of high-grade cervical lesions.
Methods: A total of 136 patients who underwent gynecological examinations in the vaginal room of the Department of Gynecology at the Third Xiangya Hospital of Central South University from June 2021 to June 2022 were retrospectively studied. Among them, 122 patients had non-high-grade cervical lesions, and 14 patients had high-grade cervical lesions. The variables included general information (age, body mass index, and menopause status), LBC, high-risk human papillomavirus, cervical tissue pathology, vaginal examination results, and the ΔCt values of JAM3 and PAX1 gene methylation. Logistic regression analysis was used to identify the factors affecting the diagnosis of high-grade cervical lesions, followed by correlation analysis and construction of a conditional inference tree model.
Results: Logistic regression analysis showed that the methylation ΔCt values of PAX1 and JAM3 genes and LBC detection results were statistically significant between the high-grade cervical lesions group and the non-high-grade cervical lesions group (all P<0.05). Correlation analysis revealed a negative correlation between cervical pathological changes and ΔCtP (r=-0.36, P<0.001), ΔCtJ (r=-0.448, P<0.001), LBC (r=-0.305, P<0.001), or bacterial diversity (r=-0.183, P=0.037). The conditional inference tree showed that when ΔCtJ>10.13, all of patients had non-high-grade cervical lesions, while ΔCtP>6.22, the number of non-high-grade lesions accounted for 97.5% (117/120), and high-grade lesions accounted for only 2.5% (3/120). When ΔCtJ>8.61 and LBC were atypical squamous cell of undetermined significance or negative for intraepithelial lesions or malignancy (NILM), 105 (99.1%) patients were non-high-grade cervical lesions, only 1 (0.9%) patient was high-grade lesion. When the results of LBC were high-grade lesions, only 9 patients' histopathological examination was the high-grade lesions and 3 non-high-grade lesions. When LBC indicated low-grade lesions, atypical squamous cell of undetermined significance, no intraepithelial lesions, and ΔCtP>6.22, 117 (97.5%) of patients' histopathological examination was the non-high-grade lesions.
Conclusions: The JAM3/PAX1 gene methylation test can be used independently for the stratified diagnosis of high-grade/non-high-grade cervical lesions in women with high-risk human papillomavirus infection, independent of the cytological results of cervical excision. The JAM3/PAX1 gene methylation test can also be used in combination with LBC to make up for the shortcomings of low sensitivity of LBC. In addition, the application of methylation kit in large-scale cervical cancer screening in the future will be good to the detection of more patients with high-grade cervical lesions, and achieve early screening and early treatment for cervical lesions/cancer.
目的: 传统的宫颈癌筛查手段——高危型人乳头瘤病毒(high-risk human papillomavirus，hrHPV)和宫颈脱落细胞学检测存在局限性。本研究通过检测宫颈脱落细胞中连接黏附分子3(junctional adhesion molecule 3，JAM3)/配对盒基因1(paired box gene 1，PAX1)高甲基化水平，并将其与液基薄层细胞学(liquid based cytology，LBC)进行比较，评价JAM3/PAX1甲基化对于宫颈高级别病变的诊断能力，以探索新的宫颈高级别病变的诊断模式，实现宫颈高级别病变“精准筛查”的目的。方法: 回顾性收集2021年6月至2022年6月在中南大学湘雅三医院妇科阴道镜门诊接受检查的患者共136例，包括宫颈非高级别病变122例，高级别病变14例。研究的变量包括：基本临床信息(年龄、体重指数、是否绝经)、LBC、hrHPV、宫颈组织病理、阴道微生态结果、阴道镜结果(宫颈转化区类型)、JAM3(ΔCtJ)和PAX1(ΔCtP)基因甲基化的ΔCt值。首先通过logistic回归分析筛选影响宫颈高级别病变的影响因素，并进行相关性分析，然后用差异有统计学意义的变量构建条件推断树模型。结果: Logistic回归分析提示高级别宫颈病变组与非高级别宫颈病变组的PAX1与JAM3基因甲基化ΔCt值以及LBC检测结果差异均有统计学意义(均P<0.05)。相关性分析发现，宫颈病理结果与ΔCtP(r=-0.360，P<0.001)、ΔCtJ(r=-0.448，P<0.001)、LBC(r=-0.305，P<0.001)、菌群多样性(r=-0.183，P=0.037)呈负相关。条件推断树显示：当ΔCtJ>10.13时，全部为宫颈非高级别病变的患者；当ΔCtP>6.22时，非高级别病变的患者有117例，占97.5%，高级别病变者仅3例，占2.5%。ΔCtJ>8.61且LBC为不明确的非鳞状上皮细胞(atypical squamous cell of undetermined significance，ASC-US)或未见上皮内病变细胞时，105例(99.1%)为宫颈非高级别病变的患者，仅1例(0.9%)检出为高级别病变；当LBC结果为高级别病变时，仅9例患者检出高级别病变，3例检出非高级别病变；而当LBC提示低级别病变、ASC-US、未见上皮内病变细胞，且ΔCtP>6.22时，有117例(97.5%)的患者检出非高级别病变。结论: 在人乳头瘤病毒感染妇女中，JAM3/PAX1双基因甲基化检测可独立应用于宫颈高级别病变/非高级别病变的分层诊断，且不依赖于宫颈脱落细胞学检测结果；亦可与LBC联合使用以弥补LBC敏感度低的缺点。此外，未来将甲基化试剂盒应用于大规模宫颈癌筛查，有利于发现更多宫颈高级别病变患者，并达到早筛、早治宫颈病变/癌的目的。.
Keywords: DNA methylation; cervical lesions; conditional inference tree; junctional adhesion molecule 3; paired box gene 1.