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. 2022 Aug 16;14(16):3954.
doi: 10.3390/cancers14163954.

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

Markus Leffers  1 , Johanna Herbst  1 , Jolanthe Kropidlowski  1 , Katharina Prieske  2 , Anna Lena Bohnen  2 , Sven Peine  3 , Anna Jaeger  2 , Leticia Oliveira-Ferrer  2 , Yvonne Goy  4 , Barbara Schmalfeldt  2 , Klaus Pantel  1 , Linn Wölber  2 , Katharina Effenberger  1 , Harriet Wikman  1
Affiliations

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

Markus Leffers et al. Cancers (Basel). .

Abstract

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.

Keywords: CADM1; MAL; cervical cancer; liquid biopsy; methylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dilution series of the CADM1 (a) and the MAL (b) qMSP for assessing the sensitivity and efficiency of the assay. The x-axis indicates the starting concentration of fully methylated DNA diluted in unmethylated DNA in percent on a logarithmic scale by a total DNA input of 10 ng. Triplicate analyses were performed for each dilution except for 100%. The y-axis shows the Cq at which the threshold value was exceeded (Ct-value). For CADM1, an efficiency of PCR of 97.5%, detection of methylated DNA down to 0.1% is possible. For MAL an efficiency of PCR of 92.1%, detection of methylated DNA down to 0.2% is ensured.
Figure 2
Figure 2
Methylation status of CADM1 and MAL (shown by the y-axis: 0 means negative, 1 means positive) in follow-up samples of three cervical cancer patients. The x-axis shows the time in months. In addition, therapies (lines) and disease progression (dots and diamonds) are shown. (a): Shows the follow-up of a patient in FIGO stage IVB with the progression of osseous metastases. (b): Shows the follow-up of a patient in FIGO stage IIB with a local relapse. (c): Shows the follow-up of a patient in FIGO stage IVB with a relapse in the form of newly appeared distant lymph node metastases.
See this image and copyright information in PMC

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