GYNECOLOGY

Methylation testing: is this the new frontier in cervical cancer screening?

 Testarea metilării: o nouă frontieră în screeningul cancerului de col uterin?

First published: 30 septembrie 2024

Editorial Group: MEDICHUB MEDIA

DOI: 10.26416/Gine.45.3.2024.10082

Abstract

Cervical cancer represents a critical public health issue, with persistent challenges in screening and early detection that affect mortality rates globally. DNA methylation, an epigenetic modification that alters genes expression, has emerged as a key biomarker in the early detection and stratification of cervical cancer risk. We reviewed the scien­tific basis of DNA methylation in cervical oncogenesis, dis­cus­sing the current methylation tests and exploring the po­ten­tial of these biomarkers in revolutionizing cervical can­cer diagnostics.
 

Keywords
cervical cancer, HPV, DNA methylation, disease progression

Rezumat

Cancerul de col uterin reprezintă o problemă critică de sănătate publică, existând provocări permanente privind screeningul şi detectarea timpurie a leziunilor, afectând ratele de mortalitate la nivel global. Metilarea ADN-ului, o modificare epigenetică ce alterează expresia genelor, a apărut ca un biomarker-cheie în detectarea timpurie şi stratificarea riscului de cancer de col uterin. Acest review abordează mecanismele metilării ADN-ului în oncogeneza cervicală, metodele actuale de testare a metilării şi explorează potenţialul acestor biomarkeri în diagnosticarea cancerului de col uterin.
 

Cervical cancer is the fourth most common type of cancer among women worldwide, with high incidence rates particularly in less developed regions. Traditional screening methods, such as the Pap smear and human papillomavirus (HPV) testing, have contributed significantly to the reduction of mortality rates. However, these methods, although still very useful, have some limitations regarding sensitivity and specificity, which can lead to underdiagnosis or mismanagement of the disease. Cervical cancer management is shifting toward a more molecular-based approach (Figure 1).
 

Figure 1. Screening of cervical lesions, from traditional to modern
Figure 1. Screening of cervical lesions, from traditional to modern

Traditional Pap smears and HPV testing have significantly increased the detection of HPV infections, but these tests often fall short of conclusively determining which patients require treatment and which should be monitored. This challenge undermines the evolving nature of prevention strategies in cervical cancer care.

The World Health Organization (WHO) has set ambitious goals for 2030 to combat cervical cancer more effectively. These targets include ensuring that 90% of girls worldwide are fully vaccinated against HPV by the age of 15 years old. Vaccination is a critical preventive measure, as it can significantly reduce the incidence of the high-risk HPV types that cause the majority of cervical cancers. Additionally, WHO aims for 70% of women to be screened with a high-performance test by the ages of 35 and 45 years old. High-performance screening tests, such as HPV DNA testing, offer greater accuracy and reliability in detecting precancerous conditions and HPV infections likely to lead to cervical cancer if untreated. Finally, WHO’s strategy emphasizes that 90% of women identified with cervical disease receive appropriate and timely treatment. This goal highlights the importance of not only detecting the disease, but also ensuring that interventions are accessible and effective, thereby reducing the global burden of cervical cancer(1). These targets are part of a broader global health initiative to eliminate cervical cancer as a public health problem, demonstrating the shift towards integrating more molecular diagnostics and targeted interventions in the fight against this disease.

The search for effective diagnostic methods for cervical cancer has led to the exploration of methylation biomarkers as a promising frontier. Methylation, a biochemical process that modifies the DNA and affects gene expression, has been closely linked to the development and progression of various cancers, including cervical cancer. Studies have identified specific methylation patterns that are predominantly associated with malignant transformations in cervical cells(2).

Despite the vast diversity in cellular structure and function, all cells in an organism share the same DNA sequence. The differences arise because certain genes are expressed or utilized differently among cell types. Chemical agents can target DNA to alter gene expression, with DNA methylation being one of the most extensively studied epigenetic modifications. Methylation of promoter regions typically correlates with gene silencing and is integral in maintaining differentiation among cell types. In pathological conditions such as cancer, normal patterns of DNA methylation are often disrupted, leading to altered gene functions that contribute to disease progression. Both the DNA in our cells and the histone proteins that make up chromatin undergo various patterns of chemical modifications, which differ across cell types. These modifications play a crucial role in the epigenetic regulation of gene expression. Histone proteins, particularly at the N-terminal residues, may undergo modifications such as methylation of lysines and arginines and acetylation of lysines. In contrast, DNA modifications are primarily limited to the methylation of specific cytosines(3).

DNA methylation involves the addition of a methyl group to the 5-carbon position of the cytosine ring within CpG dinucleotides, resulting in alterations to chromatin structure and gene expression. This methylation, together with histone modification and nucleosome positioning, represents a primary epigenetic mechanism that influences genomic function. While RNA nucleotides can undergo a range of chemical modifications, DNA primarily experiences methylation, specifically the conversion of cytosine into 5-methylcytosine (5-meC)(4). Though less common, N6-methyladenine also occurs in DNA, averaging 6-7 adenines per million across the genome. This 5-methylcytosine forms base pairs with guanine just as its unmethylated counterpart does, leaving the encoded genetic information intact. However, the added methyl group, positioned in the major groove of the DNA double helix, attracts methyl-binding proteins which can modify gene expression either directly or through interaction with other proteins(4).

The methylation pattern of each cell is different, and this reflects the gene expression pattern of the cell. Methylation typically occurs at cytosine residues adjacent to guanine, known as CpG sites. CpG sites are found all over our DNA. In a normal adult cell, most CpG are methylated. However, CpG sites within promoter regions, particularly those in CpG islands, usually remain unmethylated to allow for the active transcription of genes. These promoter regions are crucial as they contain regulatory elements that control the transcriptional activity of genes.

Promoter DNA methylation is a critical epigenetic mechanism associated with the regulation of gene expression, playing an important role in maintaining cellular differentiation and identity. This process is essential for ensuring that specific genes are active in some cell types while remaining silent in others.

In healthy cells, DNA methylation patterns are tightly regulated to preserve normal function and development. The enzymes responsible for adding methyl groups to DNA are known as DNA methyltransferases (DNMTs). The three major types of DNMTs – DNMT1, DNMT3a, and DNMT3b – have distinct roles. DNMT1 is primarily involved in the maintenance of methylation patterns following DNA replication during cell division, ensuring that the daughter cells have the same methylation pattern as the parent cell. DNMT3a and DNMT3b, on the other hand, are responsible for the de novo methylation, establishing new methylation patterns during cell development and differentiation(5).

An additional layer of complexity in the regulation of DNA methylation is provided by the TET (Ten-Eleven Translocation) enzymes, which promote DNA demethylation. They play an essential role in the dynamic regulation of methylation status, enabling cells to adapt to changes and ensuring proper gene function(6).

In cancer, these finely tuned mechanisms of DNA methylation and demethylation are often disrupted. Typically, cancer cells exhibit abnormal hypermethylation of CpG islands in promoter regions. This hypermethylation leads to the silencing of crucial tumor suppressor genes, contributing to uncontrolled cell growth and the evasion of apoptosis. The inactivation of tumor suppressor genes via hypermethylation is a common hallmark of many cancer types and highlights the critical role of altered epigenetic landscapes in cancer development and progression.

Methylation testing detects abnormal methylation patterns in cervical cell DNA, which can serve as early indicators of malignancy. The selection of appropriate biomarkers is critical for the test’s efficacy. The patterns of DNA methylation are typically analyzed by comparing DNA samples that have been treated with sodium bisulfite against untreated samples. This method is based on the principle that bisulfite treatment transforms unmethylated cytosines into uracil, leaving methylated cytosines unchanged(7). While whole-genome bisulfite sequencing (WGBS) is a comprehensive but costly approach, reduced representation bisulfite sequencing (RRBS) provides a more efficient, high-throughput alternative. RRBS focuses on gene-rich regions by combining restriction enzymes with bisulfite sequencing, allowing for the detailed analysis of genome-wide methylation patterns at single-nucleotide resolution. On average, RRBS can quantify methylation profiles at about 1.2 million CpG sites across 82 different cell lines and tissue types(8). On average, RRBS can quantify methylation profiles at about 1.2 million CpG sites across 82 different cell lines and tissue types(9).

DNA methylation involves the addition of a methyl group to the 5-carbon of the cytosine ring within CpG dinucleotides, leading to changes in chromatin structure and gene expression. In the context of cervical cancer, the hypermethylation of promoter regions typically results in the silencing of tumor suppressor genes. These epigenetic alterations are influenced by genetic, environmental and viral factors, particularly oncogenic HPV types, which are known to induce methylation changes as a mechanism of promoting carcinogenesis.

Conservative measures based on DNA methylation offer a promising avenue for refining the management of cervical squamous intraepithelial neoplasia (SIL), especially given the challenges associated with the heterogeneity of treatment responses. Current management strategies for SIL rely primarily on histopathological grading. Lesions classified as HSIL (high-grade squamous intraepithelial neoplasia) are generally treated by excision. However, the reproducibility of this classification is not perfect, and pathologists face difficulties in distinguishing lesions that are likely to regress from those likely to progress, often leading to the overtreatment of lesions that might otherwise regress spontaneously. Studies have shown that between 30% and 60% of HSIL lesions can regress spontaneously, particularly in younger females(10,11). Moreover, the surgical treatment of SIL is associated with risks such as preterm birth due to cervical insufficiency(12). Therefore, there is a critical need for reliable biomarkers that can differentiate lesions with low progression risk from those with a high risk.

DNA methylation markers hold significant potential in this context. Promoter methylation of specific genes has been functionally linked to cervical carcinogenesis. Genes such as CADM1, MAL, miR124-2, and FAM19A4 have been identified as key players(13). The methylation levels of these genes tend to increase with disease progression, indicating their potential utility in risk stratification (Table 1).
 

Table 1. Risk stratification of methylation levels
Table 1. Risk stratification of methylation levels

The use of these methylation markers could dramatically improve patients’ outcomes by preventing unnecessary treatments for those with low-risk lesions and appropriately escalating care for those with high-risk profiles. Furthermore, integrating methylation testing into the current screening process could provide a more nuanced, personalized approach to patient care, ultimately aligning treatment strategies more closely with individual risk profiles. This approach not only aims to enhance the precision of SIL management, but also reduces the potential for adverse effects associated with overtreatment, such as increased rates of preterm birth.

Over the years, several methylation assays have been discovered, and essays have been validated. One of the most critical aspects of using methylation biomarkers is their potential to facilitate early detection of cervical cancer (Figure 2).
 

Figure 2. Commercially available tests for methylation
Figure 2. Commercially available tests for methylation

Methylation biomarkers, such as the hypermethylation of the tumor suppressor gene promoters, have been shown to appear early in the carcinogenesis process, which makes them valuable targets for early diagnostic tests. These biomarkers can be detected using techniques such as Methylation-Specific PCR (MSP), quantitative methylation-specific PCR (qMSP), and next-generation sequencing, which provide the sensitivity and specificity required for clinical diagnostics.

Another advantage of methylation biomarkers is their noninvasive nature. Tests developed to detect these biomarkers can use samples obtained from noninvasive or minimally invasive procedures, such as cervical swabs or liquid biopsies. This aspect is particularly beneficial in increasing patients’ compliance and screening reach, especially in low-resource settings. These methylation assays play a critical role in the triage process by identifying HPV-positive women who are at a higher risk of progression to cervical cancer. Each assay has its unique set of targeted genes and technological approaches, ranging from qMSP to pyrosequencing. The choice of assay may depend on several factors, including the specific clinical setting, availability of technology, cost considerations, and the particular risk profile of the screened population. While promising, the adoption of methylation tests in routine cervical cancer screening faces several challenges, mainly related to cost and accessibility. High-throughput methylation assays may be cost-prohibitive in low-resource settings. Also, there is a need for standardized protocols and cutoff values to interpret methylation levels accurately across laboratories (Table 2).
 

Table 2. Pros and cons of methylation in cervical cancer
Table 2. Pros and cons of methylation in cervical cancer

Several large trials and studies have been done with the purpose of understanding the role of methylation analysis in cervical lesions. The studies of De Strooper (2016) and Bonde (2021) compared the clinical performance of DNA methylation analysis against cytology in detecting CIN3+ in hrHPV-positive women. A separate study assessed the 9-year and 14-year CIN3 risks for women using FAM19A4/miR124-2 methylation for triage – the Dutch POBASCAM study. In contrast, the VUSA-Screen study combined both cytology and HPV testing in its management approach(14-16). Data from these trials showed that a negative FAM19A4/miR124-2 methylation assay provides a lower long-term cervical cancer risk in hrHPV-positive women compared to cytology. The long-term risk of developing CIN3+ was similar in hrHPV-positive women with a negative methylation test compared to those with a negative cytology test. Methylation analysis not only identifies the current disease state but also predicts clinical outcomes effectively.

Overall, while DNA methylation offers promising advantages in cervical cancer screening and management, challenges related to sensitivity, standardization and cost need to be addressed for broader clinical application.

Methylation status in cervical cancer can also significantly affect treatment outcomes by serving as a prognostic biomarker. Studies have shown that specific methylation patterns can predict the efficacy of concurrent chemoradiotherapy. Hypermethylation of certain gene promoters is associated with a better tumor response to CCRT, indicating a potential for complete response(24). Methylation-related genes can predict prognosis and response to immunotherapy – although high-risk methylation profiles are linked to shorter survival times, these patients may benefit more from immunosuppressive therapies, such as anti-CTLA-4 treatment(25). Methylation status can also help in stratifying patients for personalized treatment approaches, as patients with specific methylation profiles could turn out to be more likely to experience disease regression, allowing for more conservative management strategies(20,26).

DNA methylation analysis offers a promising tool for the triage of hrHPV-positive women in cervical cancer screening programs. Its high sensitivity for detecting both CIN3+ lesions and cervical carcinoma, coupled with its ability to predict long-term clinical outcomes, supports its integration into current screening protocols. As methylation testing becomes more accessible and cost-effective, it could significantly enhance the early detection and management of cervical cancer, potentially reducing the reliance on more invasive procedures like biopsies and ensuring timely therapeutic interventions.

Ongoing research aims to refine methylation-based assays in cervical lesions and explore their integration with the existing screening modalities. Longitudinal studies are needed to establish the long-term benefits of methyl­ation testing, including its impact on cervical cancer incidence and mortality rates. Furthermore, advancements in bioinformatics and the development of panel-based methylation tests could facilitate personalized screening approaches tailored to individual risk profiles. Methyl­a­tion testing represents a transformative approach in the early detection and management of cervical cancer. By enabling the identification of epigenetic alterations that precede morphological changes, these tests hold the potential to enhance the precision of cervical cancer screening and contribute significantly to the reduction of disease burden. As research progresses, healthcare systems should consider the integration of methylation biomarkers into standard screening protocols to realize the full potential of this technology in improving patient outcomes. Understanding the mechanisms of DNA methyl­ation and its impact on gene expression in cancer not only provides insights into the molecular basis of the disease, but also opens up possibilities for developing targeted therapies. These could involve drugs designed to modify DNA methy­lation patterns or to inhibit the enzymes responsible for inappropriate methylation, thus reactivating silenced tumor suppressor genes and restoring the normal control of cell growth.  

 

 

Autori pentru corespondenţă: Maria Olinca E-mail: comanescu.mv@gmail.com

CONFLICT OF INTEREST: none declared.

FINANCIAL SUPPORT: none declared.

This work is permanently accessible online free of charge and published under the CC-BY.

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