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

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.

Research advances of MAL family members in tumorigenesis and tumor progression (Review)

“…non-invasive molecular detection techniques for cancer screening are rapidly advancing, with Mal family members serving as important cancer biomarkers in clinical testing (Table V). These members have been extensively utilized in non-invasive tests, such as Mal methylation assays in blood, urine and feces-derived samples from patients with various tumors (82,(196)(197)(198)(199), as well as the analysis of Mal2 transcript levels in blood from patients with gynecological and metastatic breast cancer (200,201). Integration of antibodies targeting MAL family members into existing antibody panels used in standard clinical practice for identifying cancer biomarkers in biopsies and surgical specimens, along with their application in liquid biopsies, may offer valuable avenues for enhanced prognostic and diagnostic insights in cancer cases.…”

“…CADM1/MAL methylation in high-risk human papillomavirus (HPV) positive Pap smears associates with extent and duration of underlying cervical pathology, increasing in invasive cervical cancer (79,80). Combined detection plays an important diagnostic role in identifying precancerous cervical intraepithelial neoplasia (81)(82)(83). MAL also serves as an indicator distinguishing long-term and short-term ovarian cancer prognoses, with silencing conferring treatment resistance (84,85).…”

The MAL Family of Proteins: Normal Function, Expression in Cancer, and Potential Use as Cancer Biomarkers

“…Detection of circulating tumor cells, cancer biomarkers, cell-free DNA, DNA methylation, and specific mRNA or miRNA levels in biofluids (liquid biopsies) are used as non-invasive detection tests for cancer screening [168]. Examples of the potential use of the MAL-family members as cancer biomarkers in these types of test are MAL methylation assays in blood-derived samples from patients with COAD-READ [169], BRCA [170,171], and CESC [172], and in urine and feces from BLCA [89,90] and COAD-READ [173] patients, respectively; and analysis of MAL2 transcript levels in blood from gynecological and metastatic breast cancer patients [174,175].…”

The construction and preliminary validation of methylation haplotype load markers of multiple genes for cervical cancer screenings

“…Notably, hypermethylation of tumor suppressor genes, which regulate cell adhesion, signaling pathways, and the cell cycle, is a common occurrence in cervical lesions [8][9][10], with varying degrees associated with the histological grade of cervical lesions [11][12][13]. This nding underscores the signi cance of DNA methylation in the initiation, development, and progression of cervical cancer [14,15]. While DNA methylation holds potential for cervical cancer screening [16][17][18][19], robustness may be compromised if only a single gene or a simple combination of multiple genes is applied [20][21][22].…”