Circulating Tumor DNA (ctDNA) Sequencing: A Liquid Biopsy Approach for Cancer Detection, Monitoring, and Personalized Treatment

Table of Contents

• Introduction to Circulating Tumor DNA (ctDNA)
• What is Circulating Tumor DNA (ctDNA)?
• Liquid Biopsy
• Principle of Circulating Tumor DNA (ctDNA) Sequencing
• Steps of Circulating Tumor DNA (ctDNA) Sequencing
• Methods for Circulating tumor DNA (ctDNA) Detection
• Advantages of Circulating Tumor DNA (ctDNA) Sequencing
• Limitations of Circulating Tumor DNA (ctDNA) Sequencing
• Applications of Circulating Tumor DNA (ctDNA) Sequencing
• References

Introduction to Circulating Tumor DNA (ctDNA)

• Circulating tumor DNA (ctDNA) sequencing is a technique used to sequence fragments of tumor-derived DNA that are present in the bloodstream.
• The levels of ctDNA are linked to tumor size, stage, type, treatment response, and recurrence, making ctDNA measurement a real-time method for tumor detection and monitoring in cancer patients.
• Advances in sequencing technology have enabled the development and application of ctDNA sequencing in recent years.
• As a form of liquid biopsy, ctDNA sequencing involves minimally invasive sample collection, making it suitable for repeated testing.
• ctDNA can be extracted from various body fluids, offering a more accessible and cost-effective diagnostic alternative to traditional biopsies or screening methods.

What is Circulating Tumor DNA (ctDNA)?

• Circulating tumor DNA (ctDNA) is a part of cell-free DNA (cfDNA) that comes specifically from tumor cells.
• cfDNA consists of fragmented DNA that circulates in the cell-free portion of blood and can also be found in other body fluids.
• In cancer patients, ctDNA originates from four primary sources: primary tumor cells, metastatic tumor cells, circulating tumor cells (CTCs), and healthy cells.
• ctDNA enters the bloodstream through various mechanisms including apoptosis, necrosis, CTC lysis, active release, and shedding from healthy cells.
• Because ctDNA carries tumor-specific mutations, it serves as a biomarker for early cancer detection and monitoring treatment response.

Liquid Biopsy

• Cancer remains a major global health concern, but early detection significantly improves survival rates.
• Tissue biopsy, the most commonly used method for cancer diagnosis, involves extracting a sample from the tumor for testing.
• Tissue biopsy is not practical for early diagnosis or large-scale cancer screening and can be challenging in advanced cancers where obtaining a tumor sample is difficult.
• Existing screening tools like mammograms and low-dose CT scans are limited to certain cancer types and may not always provide accurate results.
• There is a strong need for a more effective approach to enable early and widespread cancer detection.
• Liquid biopsy presents a promising, non-invasive alternative by analyzing ctDNA, RNA, or tumor cells in body fluids.
• Unlike traditional tissue biopsies, liquid biopsies are simpler and can detect genetic material from multiple tumor locations.
• This method is particularly beneficial for tumors that are difficult to access through conventional biopsy techniques, such as brain tumors.

Principle of Circulating Tumor DNA (ctDNA) Sequencing

• Circulating tumor DNA (ctDNA) sequencing is based on analyzing small DNA fragments that are released by tumor cells into the bloodstream.
• These DNA fragments are shed due to processes like apoptosis, necrosis, or active secretion by tumor cells.
• ctDNA contains genetic mutations that are specific to cancer, making it a useful biomarker for cancer detection.
• Because ctDNA exists in very small quantities in the bloodstream, highly sensitive sequencing techniques are necessary for accurate detection.
• Both targeted sequencing and whole-genome sequencing approaches can be applied for ctDNA analysis.

Steps of Circulating Tumor DNA (ctDNA) Sequencing

• Sample Collection: Blood samples are collected from patients using specialized tubes such as EDTA tubes or stabilizing tubes that contain preservatives to prevent cell lysis and contamination.
• Plasma Separation: Plasma, the source of ctDNA, is separated from blood cells through a two-step centrifugation process. The first centrifugation separates plasma from the cells, and the second removes cellular debris and residual fragments. The final plasma is stored at -80°C before DNA isolation.
• DNA Isolation and Quality Control: Plasma samples are processed to extract cell-free DNA (cfDNA), which includes both ctDNA from tumor cells and normal DNA from healthy cells. Various commercial kits and protocols are used for extraction. The quality and quantity of cfDNA are assessed using spectrophotometry or fluorometry. Due to the low concentration of ctDNA, enrichment methods like targeted capture or amplification are employed for effective analysis.
• Library Preparation and PCR: The extracted ctDNA undergoes end repair and ligation of short DNA adapters to the fragment ends, allowing them to bind to the sequencing flow cell. These adapter-ligated fragments are then amplified using PCR and purified to eliminate leftover adapters, primers, and other impurities.
• Sequencing: The purified library is loaded onto a sequencing platform such as Illumina, Ion Torrent, or Oxford Nanopore. The platform generates millions of short DNA reads, which are then analyzed to identify mutations and genetic alterations associated with cancer.
• Data Analysis: The sequencing data is first processed through base calling and quality control steps. Reads are aligned to a reference genome or specific target regions. After alignment, variant calling is performed to detect mutations that are unique to cancer cells.

Methods for Circulating tumor DNA (ctDNA) Detection

a. PCR-based methods

• PCR-based methods are cost-effective and highly sensitive but are limited to detecting known mutations or specific targets.
• Quantitative PCR (qPCR) or real-time PCR amplifies and quantifies specific DNA targets in real time using fluorescence. A common qPCR method for ctDNA detection is Allele-Specific Oligonucleotide PCR (ASO-PCR).
• ASO-PCR/ARMS uses mutation-specific primers to amplify only mutant alleles. It exploits the lack of 3′ to 5′ exonuclease proofreading activity in Taq polymerase, which reduces amplification when there is a mismatch at the 3′ end.
• Digital PCR (dPCR) increases sensitivity by partitioning the sample into thousands of reactions to reduce background noise.
• Digital Droplet PCR (ddPCR), a type of dPCR, uses water-oil emulsion droplets where each droplet contains one nucleic acid molecule that is individually amplified.
• BEAMing (Beads, Emulsion, Amplification, and Magnetics) is another dPCR method that uses magnetic beads, biotinylated oligonucleotides, and flow cytometry for high-sensitivity mutation detection. It is complex and costly.

b. NGS-based methods

NGS-based methods can detect both known and unknown mutations, offering broad analysis but are less sensitive and more expensive than PCR-based methods.

i. Targeted NGS methods

Targeted NGS methods focus on specific genes or mutations.

• Tagged-Amplicon Deep Sequencing (Tam-seq) uses primers with molecular tags for pre-amplification and selectively amplifies mutated amplicons.
• Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) identifies recurrent mutations in a cancer type and uses biotinylated oligonucleotide selectors to detect these mutations in ctDNA.
• Immunoglobulin High-Throughput Sequencing (IgHTS) is used for minimal residual disease (MRD) monitoring in blood cancers by tracking immune receptor gene sequences.
• Safe-Sequencing System (Safe-SeqS) uses DNA barcodes called Unique Identifiers (UIDs) to correct errors before amplification and detect rare mutations.
• Targeted Error Correction Sequencing (TEC-Seq) uses molecular barcodes and start/end mapping positions for high-accuracy mutation detection, particularly in early-stage cancers.

ii. Untargeted NGS methods

Untargeted NGS methods are genome-wide and not limited to predefined regions.

• Whole-Genome Sequencing (WGS) sequences the entire genome to detect large-scale genomic alterations but is costly and less sensitive.
• Whole-Exome Sequencing (WES) targets only protein-coding exonic regions, making it cheaper than WGS, but it cannot detect mutations in non-coding regions and requires high DNA input.

c. Oxford Nanopore Technology (ONT)

• Oxford Nanopore Technology (ONT) is a third-generation sequencing method that provides rapid, low-cost, and flexible sequencing.
• ONT has a higher error rate and struggles with short ctDNA fragments.
• A new method, CyclomicsSeq, improves ONT accuracy by circularizing and concatemerizing short DNA molecules before sequencing.

Advantages of Circulating Tumor DNA (ctDNA) Sequencing

• Circulating tumor DNA (ctDNA) sequencing is a minimally invasive method that only requires a simple blood draw, avoiding the discomfort and risks of surgical tissue biopsies and making sample collection easier.
• It is highly sensitive and capable of detecting very low levels of ctDNA in the bloodstream.
• PCR-based methods like digital droplet PCR (ddPCR) offer an affordable and fast approach to ctDNA detection.
• While NGS methods can be initially expensive, they are scalable and efficient, especially when processing multiple samples simultaneously.
• ctDNA sequencing is particularly valuable for tumors that are difficult or inaccessible for conventional biopsy methods.
• It enables real-time tumor monitoring, allowing clinicians to track tumor progression, detect relapse, and assess treatment response without repeated tissue biopsies.

Limitations of Circulating Tumor DNA (ctDNA) Sequencing

• Detecting ctDNA in blood is challenging because both cancerous and noncancerous cells release cell-free DNA (cfDNA) into the bloodstream, making it hard to distinguish ctDNA.
• ctDNA fragments are small, highly fragmented, and prone to degradation or loss, which makes their isolation and analysis difficult.
• The concentration of ctDNA in blood is very low, requiring highly sensitive and cost-effective detection technologies for accurate results.
• Pre-analytical steps such as sample collection, handling, transport, and storage can significantly impact the accuracy and reliability of ctDNA sequencing results.

Applications of Circulating Tumor DNA (ctDNA) Sequencing

• Circulating tumor DNA (ctDNA) sequencing can aid in early cancer detection by identifying cancer-related mutations even before symptoms develop or tumors are visible on imaging scans.
• It can be applied at various stages of cancer care, including screening, diagnosis, treatment planning, and monitoring.
• ctDNA sequencing supports personalized treatment by revealing tumor-specific genetic mutations that help guide targeted therapies.
• Due to the short half-life of ctDNA, it is effective for real-time monitoring of treatment response and for tracking the development of drug resistance, allowing timely adjustments to therapy.
• ctDNA sequencing can detect minimal residual disease after treatment and help predict the risk of cancer relapse, improving long-term patient management.

References

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