Combined Liquid Biopsy Methylation Analysis of CADM1 and MAL in Cervical Cancer Patients

Simple Summary In cervical neoplasia, tissue methylation of CADM1 and MAL has been identified as a promising triage tool for HPV-positive women. Here, we performed an exploratory study on the potential of the methylated genes as combined markers for the analysis of cervical smears and peripheral blood samples in cervical cancer patients. The combination of both markers in liquid biopsy resulted in a sensitivity of 83.3% and specificity of 95.5% for discrimination of cervical cancer from healthy controls. The robustness of the assay was proven by a respectably high sensitivity in stage I-II patients. Cervical cancer is the fourth most common cancer in women, which is associated in >95% with a high-risk human papillomavirus (HPV) infection. Methylation of specific genes has been closely associated with the progress of cervical high-grade dysplastic lesions to invasive carcinomas. Therefore, DNA methylation has been proposed as a triage for women infected with high-risk HPV. Methylation analyses of cervical cancer tissue have shown that cell adhesion molecule 1 (CADM1) and myelin and lymphocyte protein (MAL) methylation are present in over 90% of all cervical high-grade neoplasias and invasive cervical cancers. Here, we established a liquid biopsy-based assay to detect MAL and CADM1 methylation in cell free (cf)DNA of cervical cancer. Methylation of the target gene was validated on bisulfite converted smear-DNA from cervical dysplasia patients and afterward applied to cfDNA using quantitative real-time PCR. In 52 smears, a combined analysis of CADM1 and/or MAL (CADM1/MAL) showed methylation in 86.5% of the cases. In cfDNA samples of 24 cervical cancer patients, CADM1/MAL methylation was detected in 83.3% of the cases. CADM1/MAL methylation was detected already in 81.8% of stage I-II patients showing the high sensitivity of this liquid biopsy assay. In combination with a specificity of 95.5% towards healthy donors (HD) and an area under the curve (AUC) of 0.872 in the receiver operating characteristic (ROC) analysis, CADM1/MAL cfDNA methylation detection might represent a novel and promising liquid biopsy marker in cervical cancer.