Exploring ctDNA: A Non-Invasive Biomarker for Cancer Diagnosis

Circulating Tumor DNA (ctDNA) refers to small fragments of genetic material shed by tumor cells into the bloodstream. ctDNA is one of the most promising cancer tools to monitor the progression of cancer, detect only mild residual disease, and locate genetic variants for targeted treatment. The liquid biopsy market has changed drastically since the discovery of ctDNA in the 1990s with its high-sensitivity detection. Unlike the open surgery of tissue biopsy, ctDNA is detectable in blood plasma, and thus provides an excellent method to follow tumour progression in real time.

What is ctDNA?

ctDNA is DNA fragments that are released into the bloodstream by tumour cells when they die (programmed cell death) or become necrotic. These DNA chunks are generally shortened — around 100 to 200 base pairs in length. ctDNA is packed with tumor-specific mutations, deletions and amplifications, which replicate the tumour's genetic code. Hence, ctDNA acts as a "liquid biopsy" that gives molecular information on the tumour without the need for invasive tissue.

In contrast to normal DNA, which lives in healthy people's blood, ctDNA comes from cancer cells. You can see it in the blood plasma of many types of cancers such as breast, lung, colorectal, and prostate cancers. The ctDNA in the bloodstream can be well-correlated with tumor burden, which makes ctDNA a potential biomarker for cancer detection, monitoring and even therapeutic response.

Three potential origins of ctDNA: apoptotic tumor cells, Living tumor cells, and circulating tumor cells. (Cheng, F.; et al, 2016)

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ctDNA Functions in Oncology

ctDNA plays several key roles in oncology, including:

Early Detection and Diagnosis

One of the most significant advantages of ctDNA is its potential for early cancer detection. Unlike traditional imaging techniques or tissue biopsies, ctDNA can be detected at the earliest stages of cancer, even before tumors become clinically apparent. By detecting tumor-specific mutations or alterations in ctDNA, oncologists can identify cancers at a much earlier stage, leading to improved prognosis and the possibility of less aggressive treatment.

Monitoring Disease Progression and Therapy Response

Exosomes play crucial roles in intercellular communication by transferring bioactive molecules, such as proteins, lipids, and nucleic acids, between cells. The RNA content of exosomes is reflective of the cellular state of the donor cell, providing insights into both normal physiological conditions and pathological states, such as cancer. Exosomal RNA, including both coding and non-coding RNA, is particularly stable, protected from degradation by RNases due to the lipid bilayer membrane encapsulating it. This stability, combined with their presence in easily accessible body fluids, makes exosomal RNAs ideal candidates for biomarker discovery in early cancer detection.

Detection of Minimal Residual Disease (MRD)

Minimal Residual Disease (MRD) refers to the small number of cancer cells that remain in the body after treatment, which may lead to relapse. ctDNA can be used to detect MRD, even when traditional imaging techniques fail to reveal signs of cancer. By detecting residual ctDNA, oncologists can intervene early and potentially prevent relapse.

Identifying Genetic Mutations and Resistance Mechanisms

ctDNA testing can identify specific genetic mutations and alterations in the tumor, allowing for more targeted therapies. For instance, mutations in EGFR, KRAS, or PIK3CA can guide treatment choices, such as the use of specific inhibitors targeting these mutations. Additionally, ctDNA testing can identify mutations associated with resistance to current treatments, such as resistance to tyrosine kinase inhibitors in lung cancer, enabling clinicians to switch to alternative therapies before treatment failure occurs.

ctDNA Testing

Circulating Tumor DNA (ctDNA) testing is revolutionizing the way clinicians approach cancer detection, monitoring, and treatment. Unlike traditional tissue biopsies, ctDNA testing offers a non-invasive alternative to obtain molecular insights about the tumor's genetic composition. This test has significant potential in early cancer detection, identifying genetic mutations, monitoring disease progression, and evaluating therapeutic response. With advances in ctDNA testing technologies, it is now possible to detect even small quantities of ctDNA, which is crucial in cases of minimal residual disease (MRD) or early-stage cancer where tumor burden may be low. The utility of ctDNA testing extends beyond traditional oncology diagnostics. It can also be used for dynamic monitoring of tumors over time, offering an opportunity to tailor treatment strategies based on real-time molecular changes.

ctDNA Extraction

Once plasma is collected, ctDNA is extracted from the sample using specialized extraction kits. These kits are designed to isolate ctDNA with high efficiency and minimal contamination. The extraction process typically follows several steps:

• Cell Lysis: The plasma is subjected to a lysis buffer, breaking open any cells that might be present in the plasma and releasing cellular DNA.
• DNA Binding: The released DNA is then bound to specific capture agents (e.g., silica-based membranes or magnetic beads).
• Elution: The ctDNA is eluted from the capture agents, resulting in a purified sample of ctDNA ready for downstream analysis.

One of the key challenges in ctDNA extraction is the low concentration of ctDNA in plasma, particularly in early-stage cancers or minimal residual disease.

ctDNA Analysis

Once ctDNA is detected, it undergoes extensive analysis to identify specific mutations, genetic alterations, and tumor characteristics. This process often involves bioinformatics tools that analyze large datasets generated by NGS or other high-throughput techniques. The results are interpreted in the context of the clinical data, aiding clinicians in making informed decisions about treatment options. The interpretation of ctDNA analysis involves several steps:

• Mutation Identification: Identifying specific mutations or alterations associated with the tumor.
• Variant Allele Frequency (VAF) Determination: Measuring the frequency of mutated alleles in the ctDNA sample. VAF is critical for assessing tumor burden and monitoring therapy response.
• Bioinformatics Pipeline: Advanced algorithms and software tools are used to filter out false positives, identify rare mutations, and prioritize clinically relevant alterations.
• Clinical Integration: The final analysis integrates the genetic findings with the clinical information, including tumor type, stage, and treatment history, to provide actionable insights for personalized therapy.

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What are the Methods of Detecting ctDNA?

Several techniques are used to detect and analyze ctDNA. These methods vary in terms of sensitivity, specificity, and the type of mutations or alterations they can detect.

RT-PCR (Reverse Transcription Polymerase Chain Reaction)

RT-PCR is a widely used technique to detect specific mutations in ctDNA. It is based on amplifying DNA sequences that contain mutations, followed by quantitative analysis. RT-PCR is highly sensitive for detecting known mutations, such as EGFR mutations in non-small cell lung cancer, but it has limited capacity to detect unknown mutations or low-frequency variants.

Digital PCR

Digital PCR (dPCR) is an advanced form of PCR that allows for absolute quantification of ctDNA. By partitioning the sample into thousands of individual reactions, digital PCR enables the detection of rare mutations with higher precision and sensitivity. This method is particularly useful for measuring low-frequency mutations, such as those found in early-stage cancers or in the presence of minimal residual disease.

Mass Spectrometry

Mass spectrometry can be used to detect ctDNA by measuring the mass-to-charge ratio of DNA fragments. It is particularly useful for detecting methylation changes and other modifications in ctDNA. Mass spectrometry is highly sensitive and can provide valuable insights into the molecular landscape of cancer, although it is less commonly used for ctDNA analysis compared to PCR-based techniques.

Next-Generation Sequencing (NGS)

Next-generation sequencing (NGS) is a powerful and high-throughput technique used to sequence ctDNA at a deep level, allowing for the detection of a wide range of mutations, including single nucleotide variants (SNVs), insertions/deletions (indels), copy number variations (CNVs), and fusion genes. NGS is highly versatile and can analyze both targeted gene panels and whole-genome ctDNA, providing a comprehensive overview of tumor genetics. NGS has revolutionized ctDNA analysis, offering both high sensitivity and the ability to detect novel mutations.

Methylation Analysis

Methylation analysis detects epigenetic changes in ctDNA, such as DNA methylation patterns associated with tumorigenesis. These changes can be detected using techniques like bisulfite sequencing, which can identify tumor-specific methylation markers in ctDNA. Methylation markers can provide diagnostic and prognostic information, as well as insights into the molecular mechanisms underlying cancer development.

Hybrid Sequencing

Hybrid sequencing, such as NanoString technology, uses a unique approach to detect genetic alterations in ctDNA. This method combines targeted capture with high-throughput sequencing to provide high sensitivity and specificity for detecting mutations and gene expression profiles. NanoString is particularly useful for detecting gene fusions and other complex alterations that may not be easily detected by traditional NGS.

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ctDNA Testing FDA Approved

Several FDA-approved ctDNA assays have demonstrated clinical utility in detecting cancer-associated mutations. These assays are used for various purposes, including early detection, monitoring therapeutic response, and detecting genetic mutations that may guide treatment decisions. Some of the FDA-approved assays include:

FoundationOne Liquid CDx

FoundationOne Liquid CDx is an FDA-approved ctDNA test that analyzes 324 cancer-related genes for genetic alterations in ctDNA. It detects mutations, insertions/deletions (indels), and copy number variations (CNVs), providing a comprehensive molecular profile for treatment decisions. Key Features:

• Broad Cancer Indications: Covers a wide range of cancers, including lung, breast, and colorectal.
• Comprehensive Mutation Detection: Identifies mutations, CNVs, and indels, aiding in targeted therapy selection.
• Non-invasive: Requires only a blood sample.

Guardant360 CDx

Guardant360 CDx is a ctDNA assay that profiles 73 genes associated with cancer using next-generation sequencing (NGS). It detects mutations, gene fusions, and microsatellite instability (MSI), providing essential insights for treatment decisions. Key Features:

• Comprehensive Coverage: Detects alterations in 73 genes, including KRAS, EGFR, and TP53.
• MSI Testing: Identifies tumors with high microsatellite instability, which may respond to immune checkpoint inhibitors.
• Non-invasive: A blood test that provides rapid results, typically within a week.

Signatera by Natera

Signatera is a personalized ctDNA assay designed to monitor minimal residual disease (MRD). It utilizes a custom-designed panel tailored to each tumor, enabling the detection of ctDNA at very low levels. Key Features:

• Personalized Panel: Targets tumor-specific mutations for highly sensitive MRD detection.
• Early Relapse Detection: Can identify recurrence long before symptoms or imaging changes appear.
• Low Detection Limits: Highly sensitive to low ctDNA levels, useful for MRD tracking in various cancers.

Circulating tumor DNA represents a groundbreaking advancement in the field of oncology, offering a non-invasive method to monitor cancer progression, detect genetic mutations, and guide therapeutic decisions. With advancements in detection methods, such as NGS and digital PCR, and the availability of FDA-approved assays, ctDNA is poised to become an essential tool in cancer diagnosis and management, helping to pave the way for a new era of oncology.

Reference

1. Cheng, F.; et al. Circulating tumor DNA: A promising biomarker in the liquid biopsy of cancer. Oncotarget. 2016, 7(30): 48832–48841.

Exploring ctDNA: A Non-Invasive Biomarker for Cancer Diagnosis

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