Methylation markers for risk-stratification in cervical cancer screening and management of CIN
Authors:   , Dick Stefanie 
Published: December 2023

Cervical cancer is one of the most preventable and treatable forms of cancer. The development of cervical cancer is preceded by cervical intraepithelial neoplasia (CIN). This offers possibilities for screening and treatment before CIN lesions progress to cervical cancer. Novel biomarkers offer the opportunity to improve screening and tailor management. The ultimate goal is to mainly refer women for colposcopy and to consequently only treat women with high-grade CIN (CIN2/3) with a high chance of progression to cervical cancer.

Chapter 1 provided a general introduction on cervical carcinogenesis, cervical screening and management of CIN2/3.

Part 1.Screening
Host-cell DNA methylation markers have shown a good performance in the detection of CIN3 and cervical cancer (CIN3+) in HPV-based cervical screening as a triage test. In Chapter 2, six novel methylation markers were evaluated and compared for the detection of CIN3 and cervical cancer. We showed that all six methylation markers had a high performance. ASCL1 and LHX8 showed complementarity in the detection of CIN3 with an AUC of 0.890, resulting in a sensitivity of 84.0% and specificity of 83.2%. The bi-marker panel detected all 50 cervical cancers.

The implementation of HPV-based screening with cytology triage led to an increased colposcopy referral rate. This increase in mainly caused by the direct referral of women with borderline or mild cytological abnormalities. In Chapter 3, we evaluated whether the FAM19A4/miR124-2 methylation test can be applied as an additional triage test in HPV-positive women with borderline or mild dyskaryosis cytology to reduce overreferral. The CIN3+ risk after a positive or negative methylation result was 33.1% and 9.8%, respectively. The direct colposcopy referral rate could be reduced with 66% after additional risk-stratification with FAM19A4/miR124-2 methylation while retaining high CIN3+ sensitivity.

In Chapter 4, we determined the long-term performance of the FAM19A4/miR124-2 methylation test in HPV-positive women. The 14-year cumulative CIN3+ incidence after a positive or negative methylation test was 39.8% and 16.3%, respectively. The 14-year cumulative CIN3+ incidence after a positive or negative cytology test was 46.5% and 15.6%, respectively. These results indicate that a negative methylation test provides a similar long-term safety against CIN3+ compared with a negative cytology.

Part 2. Management
Women with an abnormal screening result are referred for colposcopy. The histological result of colposcopy-guided biopsies largely determines the management of women with CIN. Accurate and consistent histological grading of CIN lesions is important to decide between conservative management and excisional treatment.

In Chapter 5, we described the immunohistochemical expression patterns of p16INK4a, Ki-67 and HPV E4 in relation to the FAM19A4/miR124-2 DNA methylation. A great heterogeneity was found in immunohistochemical expression and DNA methylation status. In Chapter 6, we evaluated the prognostic value of the FAM19A4/miR124-2 DNA methylation test in a longitudinal cohort study of women with untreated CIN2/3 in the prediction of clinical regression. Clinical regression was significantly associated with a negative methylation test. Women with a negative methylation result showed regression in 75% and women with a positive methylation result in 51%. Host-cell DNA methylation can be used to support a wait-and-see policy in women with CIN2/3 to prevent overtreatment. Women treated for CIN2/3 with LLETZ remain at increased risk for the development of recurrent CIN and cervical cancer. Therefore, post-treatment monitoring is necessary. In Chapter 7, methylation of FAM19A4/miR124-2 and ASCL1/LHX8 was in the detection of recurrent CIN2/3. Both methylation marker panels showed a >92% detection rate of recurrent CIN3, while <9% of ≤CIN1 tested methylation positive.

Chapter 8 provided a general discussion of the results of this thesis and presented an overview of the clinical indications of host-cell DNA methylation in cervical screening and management of women with CIN.