Circulating Tumor DNA (ctDNA) Sequencing: Principle, Steps, Uses microbiologystudy

Circulating tumor DNA (ctDNA) sequencing is a method that sequences the tumor-derived ctDNA fragments circulating in the bloodstream.

ctDNA levels are associated with tumor size, stage, type, treatment response, and recurrence so measuring ctDNA in a cancer patient’s sample provides a real-time method for detecting and monitoring tumors.

Circulating tumor DNA (ctDNA) sequencing has been possible due to the advancement of sequencing in recent years. Since it is a type of liquid biopsy, sample collection for ctDNA sequencing is minimally invasive and is also suitable for repeated testing. Also, ctDNA can be taken from different body fluids so using it as a diagnostic marker is more accessible and affordable option compared to traditional biopsies or screening methods. 

What is Circulating Tumor DNA (ctDNA)?

ctDNA is a component of cell-free DNA (cfDNA) that originates from tumor cells. cfDNA consist of fragmented DNA circulating in the cell-free portion of blood. It can also be found in other body fluids. In cancer patients, ctDNA originates from four main sources: primary tumor cells, metastatic tumor cells, circulating tumor cells (CTCs), and healthy cells. ctDNA is released into the bloodstream through different pathways including apoptosis, necrosis, CTC lysis, active release, and shedding from healthy cells. Since ctDNA carries tumor-specific mutations, it is used as a biomarker for early cancer detection and treatment monitoring.

Liquid Biopsy

Cancer is a major health issue worldwide but early detection can increase survival rates. The most widely used method for cancer diagnosis is tissue biopsy which involves taking a sample of the tumor for testing. However, this method is not practical for early diagnosis and large-scale cancer screening. It is also difficult to obtain tumor samples in many cases like in advanced cancers. Current screening methods like mammograms and low-dose CT scans are also limited to specific cancer types and may not always be accurate. So, a more effective method is needed for large-scale early detection.

Liquid biopsy is a promising alternative. It is a non-invasive test that studies ctDNA, RNA, or tumor cells in body fluids. Unlike traditional tissue biopsy, liquid biopsy is simple and can capture genetic material from multiple tumor sites. It is especially helpful for tumors that are hard to access with traditional biopsy methods. 

Principle of Circulating Tumor DNA (ctDNA) Sequencing

Circulating tumor DNA (ctDNA) sequencing works on the principle of studying small DNA fragments released by tumor cells in the bloodstream. Tumor cells continuously release fragmented DNA into the blood due to apoptosis, necrosis, or active secretion so ctDNA carries genetic mutations specific to cancer. This allows ctDNA to be used as a biomarker to detect cancer. Highly sensitive sequencing methods are required to detect the small amount of ctDNA present in the sample. Targeted or whole-genome sequencing methods can be used for ctDNA sequencing.

Steps of Circulating Tumor DNA (ctDNA) Sequencing

1. Sample Collection: At first, blood samples are collected from patients in a specialized collection tube like ETDA tubes or stabilizing tubes with preservatives. These blood collection tubes are designed to prevent cell lysis and contamination.
2. Plasma Separation: Plasma is used as the source of ctDNA so it is carefully separated from blood cells using centrifugation. The sample undergoes two rounds of centrifugation. The first centrifugation separates the plasma from blood cells and the second one removes cellular debris and fragments from the plasma. The processed plasma is then stored at -80°C before DNA isolation.
3. DNA Isolation and Quality Control: Then, the plasma sample is processed to extract cfDNA which includes both ctDNA from tumor cells and normal DNA from healthy cells. Different commercial kits and protocols can be used for extraction. The quantity and quality of the extracted cfDNA are checked using spectrophotometry or fluorometry. Since ctDNA is present in very small amounts, highly sensitive methods such as targeted capture or amplification are used to enrich ctDNA for analysis.
4. Library Preparation and PCR: The extracted ctDNA is processed by repairing its ends and ligating short DNA adapters to the ends of the fragments. Adapters help the fragments to bind to the sequencing flow cell. The resulting fragments are amplified by PCR and purified to remove any residual adapters, primers, and other contaminants.
5. Sequencing: The prepared library is loaded into a sequencing platform like Illumina, Ion Torrent, or Oxford Nanopore. The sequencing reads generate millions of short DNA fragments that are processed to detect mutations and genetic changes associated with cancer.
6. Data Analysis: The raw sequencing data are processed through base calling and quality control. Then, reads are mapped to a reference genome or specific target regions. Once the data is aligned, the next step is variant calling which identifies potential mutations that are specific to cancer cells.

Methods for Circulating tumor DNA (ctDNA) Detection

a. PCR-based methods

These methods are cost-effective and highly sensitive but they are limited to detecting known mutations or specific targets. Some of the commonly used PCR-based methods for ctDNA detection are:

• Quantitative PCR (qPCR), also known as real-time PCR, amplifies specific DNA targets and measures them in real time using fluorescence detection. One commonly used qPCR method for ctDNA detection is ASO-PCR.
• Allele-specific oligonucleotide PCR (ASO-PCR) or amplification refractory mutation system (ARMS) is a qPCR method that detects known mutations by using mutation-specific primers to selectively amplify mutant alleles. This method utilizes the lack of 3′ to 5′ exonuclease proofreading activity of Taq polymerase, which reduces amplification if there is a mismatch at the 3′ end of the primer.
• Digital PCR (dPCR) is a method that splits the sample into thousands of reactions to reduce background noise. It is more sensitive than qPCR. Digital droplet PCR (ddPCR) is a widely used method for ctDNA detection. It is a type of dPCR that uses water-oil emulsion droplets to partition the sample. Each droplet contains a single nucleic acid molecule that undergoes PCR amplification.
• Beads, Emulsion, Amplification, and Magnetics (BEAMing) is another type of dPCR that uses magnetic beads and flow cytometry to identify specific mutations. It uses streptavidin-coated beads and biotinylated oligonucleotides. It can detect genetic variations with high sensitivity but is it complex and costly. 

b. NGS-based methods

Unlike PCR-based methods, next-generation sequencing (NGS) can analyze large amounts of DNA and can detect unknown mutations but they are less sensitive and more expensive. There are targeted and untargeted methods of NGS.

i. Targeted NGS methods

NGS-based methods can be used with targeted panels to detect specific ctDNA mutations. Some of these methods are:

• Tagged-amplicon deep sequencing (Tam-seq) uses specific primers with unique molecular tags to pre-amplify target regions. This preamplification step reduces sampling errors. This is followed by another amplification step that amplifies only the amplicons with mutations and eliminates non-specific products.
• Cancer Personalized Profiling by deep sequencing (CAPP-Seq) detects mutations in cancer by targeting common mutations in a specific cancer type. First, a population analysis is done to find recurrent mutations and based on these mutations, a selector made of biotinylated oligonucleotides is designed. These custom oligonucleotides are first used to identify mutations in tumors and later used to test ctDNA.
• Immunoglobulin High-Throughput Sequencing (IgHTS) is a method that is used for minimal residual disease (MRD) monitoring in blood cancers. It can predict recurrence of disease by tracking specific immune receptor gene sequences.
• Safe-sequencing system (Safe-SeqS) is an amplicon-based method that uses DNA barcodes called Unique Identifiers (UIDs) to track and correct errors before amplification. This method can detect very rare mutations.
• Targeted Error Correction Sequencing (TEC-Seq) combines targeted sequencing with error correction to detect mutations in early-stage cancers. It uses molecular barcodes to tag DNA fragments before amplification like Safe-SeqS but is also uses start and end mapping positions of sequenced fragments as barcodes to improve accuracy.

ii. Untargeted NGS methods

NGS can also be used for genome-wide mutation analysis. Some of the genome-wide sequencing methods are:

• Whole-genome sequencing (WGS) is used to analyze the complete genetic profile of tumor DNA. It sequences the entire genome and is useful for identifying large-scale genomic changes and mutations. However, it has limitations like high sequencing costs and low sensitivity.
• Whole-exome sequencing (WES) targets only exonic regions. It focuses on sequencing only the protein-coding regions of the genome. It is less expensive than WGS but requires high sample input like WGS and cannot detect mutations in non-coding regions.

c. Oxford Nanopore Technology (ONT)

NGS methods give detailed genetic information but they are more expensive and take longer to process. Third generation sequencing methods like ONT is a more recent method that provides speed, low cost, and flexibility. However, it has a higher error rate and cannot easily handle the shorter ctDNA fragments. To overcome these challenges, a new method called CyclomicsSeq has been developed which improves sequencing accuracy by using circularization and concatemerization of short DNA molecules.

Advantages of Circulating Tumor DNA (ctDNA) Sequencing

• Circulating tumor DNA (ctDNA) sequencing is a minimally invasive method that requires only a simple blood draw instead of a surgical tissue biopsy. This reduces patient discomfort and risks associated with the procedure. It also makes sample collection easier.
• Circulating tumor DNA (ctDNA) sequencing is highly sensitive in detecting low ctDNA levels.
• PCR-based methods like ddPCR are affordable and fast. NGS methods can be expensive at first but they are also scalable and efficient when multiple samples are analyzed at the same time.
• Circulating tumor DNA (ctDNA) sequencing is useful in cases where certain tumors are difficult or inaccessible to biopsy. 
• Circulating tumor DNA (ctDNA) sequencing allows real-time monitoring of tumor which helps track tumor progression, detect relapse, and check the efficacy of treatment without the need for tissue biopsies.

Limitations of Circulating Tumor DNA (ctDNA) Sequencing

• Detecting ctDNA in blood samples is challenging because both cancerous and noncancerous cells release cfDNA into the bloodstream. 
• It is difficult to isolate ctDNA fragments as they are small, highly fragmented, and prone to degradation or loss.
• The concentration of ctDNA in blood is very low so it requires highly sensitive and cost-effective detection methods. 
• Sample processing steps like sample collection, handling, transport, and storage can affect the accuracy of Circulating tumor DNA (ctDNA) sequencing.

Applications of Circulating Tumor DNA (ctDNA) Sequencing

• Circulating tumor DNA (ctDNA) sequencing can help detect cancer at an early stage. It can identify cancer-related mutations even before symptoms appear or before traditional imaging techniques can detect the tumor. 
• Circulating tumor DNA (ctDNA) sequencing can be used in different stages of cancer screening and treatment.
• Circulating tumor DNA (ctDNA) sequencing also helps guide personalized treatment by identifying tumor-specific genetic mutations. 
• The short half-life of ctDNA is useful in monitoring treatment response and tracking resistance of drugs to modify treatment.
• Circulating tumor DNA (ctDNA) sequencing can also detect residual disease and can predict the risk of relapse.

References

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