A thought-leadership review by Dhruvajyoti Roy, Ph.D., on how liquid biopsy-based circulating tumor DNA (ctDNA) methylation is emerging as a promising, non-invasive approach for detecting cancer early.
By Dhruvajyoti Roy, Ph.D. Originally published in Onco’Zine.
The early detection of cancer has been proven to significantly improve patient survival rates and quality of life, as well as reduce the cost and complexity of cancer treatment. Therefore, the discovery and development of novel biomarkers are in critical need for the screening and surveillance of high-risk populations to enable prompt and successful treatment. Although traditional biopsies have been an integral part of cancer care for decades, biopsies are invasive, may induce complications and, depending on the tumor location, are not always feasible to access.
In this context, a liquid biopsy is less burdensome than a tissue biopsy and offers simple, fast and cost-efficient monitoring of disease status or response to treatment. Moreover, tissue biopsies may not correctly reflect the complex molecular profile of a primary tumor because of its intratumoral variations or spatial heterogeneity. In contrast, liquid biopsies can offer more comprehensive cross-sectional information of heterogeneous diseases. Due to the increasing knowledge of the pathways causing cancer and instrumental developments, liquid biopsy has begun to attract interest for implementation in clinical practice and routine molecular diagnostics.
A Multimodal Platform: Source of Genomic, Epigenomic and Proteomic Information
In recent years, several cancer-derived components that circulate in our body fluids — such as circulating tumor cells (CTCs), circulating cell-free nucleic acids (RNA and DNA), extracellular vesicles (EVs)/exosomes, and proteins — have been extensively investigated for cancer research. Isolation and molecular analysis of these tumor-derived components, including genomic, epigenomic and proteomic assessments from liquid biopsy samples, represent a new multimodal diagnostic tool. In particular, circulating cell-free DNA (cfDNA) has emerged as a potential biomarker and is being investigated to distinguish between signals from non-cancer and pre-cancerous populations in translational and clinical research.
A fraction of the total cfDNA in body fluids, called circulating tumor DNA (ctDNA), is derived from tumor cells primarily undergoing apoptosis or necrosis, and it has mostly been used to monitor the response to therapy and to assess therapy guidance. The cobas® EGFR Mutation Test v2 (Roche Diagnostics) is the first FDA-approved test for the detection of the epidermal growth factor receptor (EGFR) gene in DNA derived from plasma samples, as a companion diagnostic for non-small cell lung cancer (NSCLC) therapy. An increasing number of studies demonstrate the potential use of ctDNA as a surrogate marker for multiple indications in various cancer types, including diagnosis, prognosis, and monitoring.
Although the detection of point mutations within ctDNA has great potential to locate known druggable mutations and impact therapy decisions, this mutation-based approach has less sensitivity for early detection of cancer. Furthermore, due to the limited number of recurrent mutations available for discriminating ctDNA from total cfDNA, a large proportion of the genetic changes identified in a potentially relevant gene are not clinically interpretable.
DNA Methylation Classifiers in Circulating Cell-Free DNA
It has been well documented that cancer is not an exclusively genetic disease, but its progression is highly dependent on additional biological processes such as immune activity, tissue microenvironment, and epigenetics. Epigenetic alterations — such as abnormal patterns of DNA methylation, histone post-translational modifications and changes in chromatin composition — have led to new opportunities for cancer care. Researchers have emphasized that epigenetic abnormalities might play an influential role in the earliest steps of cancer initiation and the progression of malignancies.
Epigenetic variants involve a change in the DNA methylation pattern within a gene’s CpG islands that leads to the silencing of tumor suppressor genes and subsequent oncogenesis. Unlike genetic alterations, epigenetic variants have high potential and wide scope to be implemented as early diagnosis biomarkers due to their involvement in the initiation of carcinogenic pathways. Both global hypomethylation and hypermethylation at selected CpG islands have revealed the potential of DNA methylation for non-invasive detection and monitoring of various cancer types. In particular, alterations in methylation status that often occur in the promoter regions of specific transcription factors are among the most frequent molecular changes associated with early molecular events in carcinogenesis.
The Impact of DNA Methylation for Cancer Detection and Clinical Utility
Recent epigenetic biomarker discovery and validation are making well-established epigenetic modifications a promising target as biomarkers for the early detection of cancer and the prediction of clinical outcomes and patient monitoring. Among all studied epigenetic biomarkers, DNA methylation is the most frequently examined in various cancers and has been recognized as most useful for sensitive detection of disease, resulting in improved clinical outcomes. The analysis of ctDNA methylation from bodily fluids, such as blood, saliva or urine, could also be very useful for risk assessment and monitoring of disease progression.
DNA Methylation Techniques and Representative Biomarkers
Numerous methods can be applied for the detection of DNA methylation biomarkers, either at a genome-wide scale or a locus-specific level. Mainly, three principal approaches are exploited for the detection or isolation of DNA methylation: immunoprecipitation, methyl-sensitive restriction enzymes, and sodium bisulfite conversion. Emerging studies have revealed useful diagnostic and prognostic DNA methylation markers across many tumor genes — for example, GSTP1, RASSF1A, and RARB2 for breast cancer; FBN2, TAC1 and SEPT9 for colorectal cancer; and RASSF1A, PRDM1, DAPK1 and 3OST2 for lung cancer.
Two tests have progressed through to FDA PMA approval — the blood-based Epi proColon® (Epigenomics) and the stool-based Cologuard® (Exact Sciences) — and several other liquid biopsy platforms have received breakthrough device designations from the FDA. For example, Laboratory for Advanced Medicine is focused on cancer-specific DNA methylation and also received the FDA’s breakthrough designation for its IvyGene Liver Dx for the early detection of liver cancer. DNA methylation technology preferentially targets the most informative regions of the genome and uses machine-learning algorithms to both detect the presence of cancer and identify the tumor’s tissue of origin when cancer is present.
Artificial Intelligence (AI) and Machine Learning (ML) to De-code Methylation Patterns in Cancer
The development of a successful biomarker requires specific thresholds for test performance and precise clinical utility. Implementation of AI and ML algorithms advances the analysis of large amounts of high-dimensional biological and medical data that could help to de-code weak signals in liquid biopsy markers at an early stage of cancer. Over the years, companies such as Laboratory for Advanced Medicine, Freenome, Grail and Exact Sciences have implemented AI and ML algorithms and used the power of data science to identify and validate cancer-specific DNA methylation markers, significantly speeding up the process of analyzing vast amounts of data to provide faster and better-advised decision-making.
Conclusions and Future Perspectives
As liquid biopsy options grow, the evolving field will bring diagnostic solutions for early detection that go beyond the utilities of tissue biopsy. With high-throughput DNA methylation detection approaches and the continued reduction in the cost of Next-Generation Sequencing (NGS), the use of whole methylomes for biomarker discovery is becoming more common. Given the greater consistency of DNA methylation changes in cancer compared to mutations, methylation is a promising target for the development of biomarkers for early detection. The DNA-methylation-based approach is confirmed to be sensitive and specific enough to detect cancers at an early stage; therefore, ctDNA methylation provides a possible alternative for cancer diagnosis and surveillance for routine clinical applications in the future.
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By Dhruvajyoti Roy, Ph.D. Originally published on Onco’Zine.