Since the term was first coined more than a decade ago by Pantel and Alix-Panabières, liquid biopsies have become increasingly important to cancer research. This article highlights the impact of liquid biopsies, challenges in the space, and how current technologies are helping to push the boundaries of precision medicine.
What is ctDNA and why is it important?
Circulating tumor DNA (ctDNA) is a type of cell-free DNA (cfDNA) that is shed into the bloodstream by tumor cells, making it a valuable resource for cancer detection and monitoring. Due to short half-life of cfDNA (less than 2.5 hours), it reflects the current state of the tumor cells, making ctDNA analysis particularly useful for tracking changes in tumors over time.
The application of liquid biopsies in precision medicine
Liquid biopsies are transforming several aspects of cancer management:
1. Targeted Therapy: With the rise of targeted cancer therapies, identifying specific genetic mutations is crucial for selecting the most effective personalized treatment. Liquid biopsies can often detect these targetable mutations more reliably than traditional biopsies, which may miss mutations due to tumor heterogeneity. This knowledge can help direct personalized treatment plans tailored to specific mutations, leading to more effective outcomes.
2. Early Diagnosis: Liquid biopsies could lead to earlier cancer detection and, in some cases, identify cancer before symptoms appear or when the tumor is operable, leading to higher long-term survival rates.
3. Monitoring Response and Minimal Residual Disease (MRD): After surgery, ctDNA levels typically drop significantly. If ctDNA remains elevated or rises again, it could indicate residual disease or recurrence. Depending on the tumor type and stage, detecting MRD using ctDNA from liquid biopsies can precede imaging-based relapse detection by several months. Furthermore, liquid biopsy can detect resistance mutations, allowing for timely adjustments to treatment strategies.
ctDNA detection technologies
Several techniques are employed to detect ctDNA in liquid biopsies, primarily:
1. Next-Generation Sequencing (NGS): NGS is a powerful tool for sequencing specific cancer genes or even entire genomes. Its high sensitivity allows for the detection of low-frequency mutations (below 0.1 percent). Recent advancements in AI applied to whole-genome sequencing (WGS) data from ctDNA have further enhanced the sensitivity to detect tumor fractions as low as 5x10-5.
2. Digital PCR (dPCR): dPCR is an ultra-sensitive method for quantifying specific DNA sequences. It is useful for detecting known mutations, but is less suitable for analyzing genes with diverse mutation sites.
3. Other Enrichment Technologies: Techniques like ICE-COLD PCR and Boreal Genomics' OnTarget can further enhance detection sensitivity by enriching mutant alleles.
Challenges and considerations
Although liquid biopsies have helped to improve cancer diagnosis and management, there are still challenges to overcome to realize their full potential. The most important considerations are pre-analytical factors, such as the collection and processing of blood. Careful handling is essential to prevent DNA release from leukocytes, which can confound the results. Most studies recommend rapid centrifugation of blood to separate plasma within six hours of collection when using standard lavender-top tubes containing EDTA as an anticoagulant. The use of tubes containing leukocyte-stabilizing factors can extend the processing time by up to 48 hours.
Replication is a simple and effective method to increase the robustness of liquid biopsy results. By comparing duplicate samples from the same individual and using mutations detected in both, false positives can be significantly reduced.
Using unique molecular identifiers (UMIs), such as NEXTFLEX UDI-UMI barcodes, can improve the accuracy of mutant allele detection by reducing sequencing errors. By comparing reads with the same UMI, a consensus call can be made. Comparing across UMIs can identify errors in PCR amplification.
The use of ctDNA reference materials for assay development, verification, and routine analysis can contribute to reliable and consistent workflows. Controls, such as the cell line-derived Mimix OncoSpan cfDNA Reference Standard, can help researchers to monitor if the assay is performing as expected and to determine the limit of detection in cfDNA assays.
Combining these methods for limiting errors can allow liquid biopsies to detect mutations at below 0.1 percent variant allele frequency, which could lead to a greater chance of detection and diagnosis using ctDNA.
Summary
The field of liquid biopsy is rapidly evolving, with research focused on improving ctDNA detection at lower allelic frequencies and expanding its applications across various cancers. Standardized protocols and quality control measures, including the use of cell line-based reference materials, can provide a consistent control for assay development and verification, with the goal of integrating liquid biopsy into routine cancer care and advancing precision medicine.
NEXTFLEX and Mimix are registered trademarks of Revvity, Inc.
Mimix reference standards and NEXTFLEX barcodes are for research use only. Not for use in diagnostic procedures.