Although there are many variations in liquid biopsy assay technology, the following steps are typically involved in a liquid biopsy:
Sample collection, preparation, and stabilization
When blood is collected in EDTA tubes and stored, the white blood cells begin to lyse and release genomic wild-type DNA in quantities typically many-fold higher than the ctDNA present in the sample. This makes the detection of mutations or other ctDNA biomarkers more difficult. Therefore, sample collection for liquid biopsies usually involves the use of commercially available cell stabilization tubes which can prevent or delay the lysis of white cells thereby reducing the dilution effect of ctDNA. Plasma is better than serum for ctDNA recovery. The use of cell stabilization collection tubes can be advantageous in situations where the blood cannot be processed immediately into plasma.
Isolation and extraction of CTCs, ctDNA, and exosomes
CTCs, ctDNA, or exosomes are extracted from the plasma using several commercially available kits. The extracted sample is then ready for amplification and sequencing.
Analysis of CTCs, ctDNA, and exosomes via amplification and sequencing
The amplification and sequencing of CTCs, ctDNA, or exosomes are usually performed using advanced next-generation sequencing (NGS) and/or polymerase chain reaction (PCR) technologies. This can be done in one of two approaches based on the genes targeted for analysis:
- Untargeted methods: The goal of this method is to interrogate all genes to have an untargeted approach. This involves whole-genome or whole-exome sequencing to discover new mutations in tumor DNA while monitoring disease burden or tracking drug resistance. Untargeted approaches may be useful in research to observe tumor heterogeneity or to discover new drug targets. However, untargeted approaches are more expensive and have lower resolution. This approach typically uses high-throughput DNA sequencing technologies.
- Targeted methods: In a targeted approach, sequencing is often directed towards a genetic panel constructed based on mutational hotspots for the cancer of interest. This is especially important for informing treatment in situations where mutations are identified in drug-based targets. Targeted approaches allow for more personalizing targeted analysis for each patient by combining liquid biopsies with standard primary tissue biopsies. Targeted approaches typically amplify ctDNA through PCR or digital PCR. This approach allows for higher sensitivity for ctDNA detection and is especially important because there are relatively low levels of DNA circulating in the blood.
Data analysis and interpretation
Most liquid biopsy systems have dedicated bioinformatics instrumentation that analyzes the data for the presence of CTCs, ctDNAs, or exosomes and interprets the results to assess the patient’s status and prognosis.
The following sections will describe three commercial liquid biopsy assays that use the following biomarkers:
- CTCs (CELLSEARCH® CTC Test)
- ctDNA (cobas® EGFR Mutation Test v2)
- Exosomes (ExoDx™ Lung (ALK) test)
