Expert consensus released! After the HPV centralized procurement, methylation has become a hot topic!

Cervical cancer is the fourth most common malignant tumor among women worldwide and an important cause of cancer-related deaths among women in China. The World Health Organization (WHO) has proposed a global strategy of "Accelerating the Elimination of Cervical Cancer", emphasizing the core role of early screening. However, the screening coverage rate in our country is less than 40%, and traditional methods such as cytological examination and high-risk HPV (hrHPV) detection are facing bottlenecks such as low sensitivity, high false positive rate, and strong subjective dependence. Against this backdrop, DNA methylation detection, as a novel molecular marker technology, has become an important direction to break through the existing screening predicament due to its high specificity and objectivity in precancerous lesions and early carcinogenesis.

The principle and advantages of detection technology

DNA methylation is an important mechanism of epigenetic modification, and abnormal methylation is closely related to tumorigenesis. The dual-gene methylation (PAX1m/JAM3m) detection of PAX1 (pairing box gene 1) and JAM3 (ligation attachment molecule 3) enables precise identification of cervical intraepithelial neoplasia (CIN) and carcinogenesis by analyzing the methylation levels of these two genes in cervical exfoliated cells.

Core excellenceTrend

Early warning: Elevated levels of PAX1m/JAM3m can indicate persistent hrHPV infection (with a significant increase in methylation levels in cases lasting for more than 3 years), which occurs earlier than morphological lesions.

2. High sensitivity and shunt value: Multicenter studies have shown that the sensitivity of PAX1m/JAM3m in detecting CIN3+ is 87.6%, and the sensitivity and specificity in diagnosing CIN2+ are 74.1% and 95.9%, respectively. In the triage of HRHPV-positive or cytological ASC-US (atypical squamous cells of unknown significance), colposcopy referrals can be reduced by 57% to 79.5%.

3. Standardization and non-inventiveness: The detection process based on real-time fluorescent quantitative PCR (qPCR) can achieve standardized laboratory operations, convenient sample collection, and avoid invasive examinations.

Technical specifications for the entire testing process

1. Sample collection and processing

Collection requirements: Avoid sampling during menstruation, within 24 hours after medication or sexual activity. Use a dedicated cervical sampler to rotate and brush the shed cells.

Storage and transportation: Samples should be immediately placed in the preservation solution and the test should be completed within one week at room temperature to avoid DNA degradation caused by repeated freezing and thawing.

2. Laboratory operation procedures

Nucleic acid extraction: It is recommended to enrich short DNA fragments by magnetic bead method to ensure the integrity of methylation sites.

Bisulfite conversion: The conversion of unmethylated cytosine to uracil requires verification of conversion efficiency (such as Sanger sequencing comparison).

qPCR detection: Set the ΔCt threshold (PAX1/JAM3 methylation positive determination standard), and each batch must include negative/positive quality control products.

3. Quality Control System

Internal quality control: For each batch of testing, it is necessary to verify the stability of weakly positive quality control products (close to the detection limit), negative controls, and internal reference genes.

External quality control: Participate in external quality assessment to ensure consistency of results among laboratories.

Out-of-control handling: When the results are abnormal, it is necessary to investigate for contamination, reagent failure or equipment error, and trace back to the sample after the last valid quality control.

Clinical application scenarios and management paths

1. Joint screening strategy

hrHPV primary screening and triage

Positive for HPV16/18 and PAX1m/JAM3m: Direct referral for colposcopy.

Non-hpv16/18 type positive and methylation negative: Refer in combination with cytological results (if ASC-US or above is referred, recheck after one year if negative).

Cytological ASC-US triage: Methylation testing can replace repeated cytological or HPV testing, reducing colposcopy referrals by 79.5%.

2. Management of special groups

Postmenopausal women: For the difficulty of cervical canal sampling (TZ type III transformation zone), methylation testing can reduce the risk of missed diagnosis.

Follow-up after treatment: Persistent positive PAX1m/JAM3m after surgery indicates residual lesions or the possibility of recurrence.

3. Key Points of Report Interpretation

Positive result: It indicates the risk of CIN2+ and needs to be evaluated in combination with colposcopy, with particular attention paid to cases where TZ is not visible.

Negative result: A high negative predictive value (>95%) supports extending the screening interval and reducing excessive medical treatment.

Limitations and Future Directions

Current limitations

The detection is a qualitative analysis and cannot quantify the methylation level and the severity of the lesion.

When the colposcopy results are inconsistent with the pathological results (such as positive methylation but negative biopsy), TZ classification and multi-point biopsy should be combined.

Future outlook

Expand prospective cohort validation and explore the application of dynamic methylation monitoring in the assessment of treatment response.

Develop multi-omics joint detection models (such as methylation + mutation + protein markers) to increase the detection rate of adenocarcinoma.

Promote the construction of standardized testing capabilities at the grassroots level and incorporate it into the national public health project for cervical cancer screening.

Summary