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Fluid biopsies (and NGS)

Posted on April 24, 2020 By Matthew No Comments on Fluid biopsies (and NGS)

The existence of cell-free DNA was established as early as the late 1940s, but liquid biopsy samples and their analysis are still a very acute research topic, especially due to their diagnostic utility.

What are fluid biopsies and what are they studied for?

Liquid biopsy samples most often refer to human venous blood samples, i.e., serum or plasma. Other body fluids can also be used as sample material, e.g., urine, cerebrospinal fluid (CSF), saliva, etc. These contain a wide variety of molecules that can be used as biomarkers.
Circulating biomarkers include, for example, cell-free DNA (cfDNA) and molecules contained in extracellular vesicles, or exosomes. The cancer also secretes circulating tumor DNA and / or RNA (circulating tumor DNA ctDNA or ctRNA) and circulating tumor cells (CTCs) into the bloodstream and other body fluids. Cell-free DNA is also secreted from other tissues in the body, or from a placenta and fetus when a woman is pregnant.

However, very low concentrations of these markers pose their own challenges for analysis: in 10 ml of whole blood, the proportion of genomic DNA is about 50 µg, of which less than 0.1% (50 ng) is cfDNA. Of this cell-free DNA, in turn, the proportion of ctDNA is 1-10%, i.e. only about 0.5-5 ng. Thus, for example, DNA from cancer cells represents only a fraction of the total amount of cell-free DNA. In addition, the short half-life of extracellular DNA, which is only minutes or hours, is also noteworthy.

Why fluid biopsies?

Fluid biopsies are non-invasive specimen material. A patient sample can be easily collected during blood sampling, and no solid tissue / specimen or cell sample is required, e.g., in the case of lung cancer or brain tumor.

Each patient’s tumor is different, and each tumor may have different cell types, i.e., the cancers are very heterogeneous. Instead of tumor samples / tissue biopsies, fluid biopsies represent the full profile of the disease and thus the overall situation can be analyzed. For example, the amount of ctDNA varies at different stages of the disease, and pathogenic mutations in ctDNA can be analyzed and utilized in cancer diagnosis, monitoring, and response assessment.

What should be considered in sample handling?

To ensure the best result, it is essential to collect a blood sample in tubes suitable for this purpose, which ensure optimal preservation of cell-free DNA and RNA. Nucleic acids must also be successfully isolated for further analysis, e.g., genetic testing and mutation testing.

Fluid biopsies and NGS

To date, fluid biopsies have mostly been analyzed using PCR-based methods such as digital droplet PCR (ddPCR). For the first time, ddPCR was able to identify genomic, cancer-related brain tumors in children with a rare brain tumor type from liquid biopsies. Thus, in this study, no invasive tissue sample was required at all, and the finding and association with cancer was significant in this particular sample type.

In addition to PCR-based methods, liquid biopsy samples can also be analyzed by next-generation sequencing, or NGS. This method and its reliable utilization are under active development work. In the analysis of genomic data and the study of genetic alterations, an important criterion is the detection sensitivity of the method used. A detectable marker (e.g., CTC) from fluid biopsy samples is challenging to identify due to its scarcity. Therefore, identifying changes requires greater sequencing depth than tissue samples and also requires optimization of bioinformatics. A good detection sensitivity found in NGS is the 1-2% allele frequency of the mutation, but the method makes it possible to access even lower frequencies.
In particular, by utilizing MID / UMI (unique Molecular identifiers) with NGS, it is possible to identify genomic changes with an allele frequency of only 0.1%. An example of this is the UMI Lung cancer panel from Paragon Genomics or the Swift Biosciences HS High Sensitivity Panel. Paragon Genomics UMI panels will soon also be available customized, enabling, for example, extensive tumor mutation burden (TMB) analyzes of different types of cancer or from a patient at different stages of cancer.

For example, Bolivar et al. successfully utilized a customized Swift Biosciences NGS panel in patients with endometrial cancer and were able to identify cancer-associated mutations in the cfDNA of plasma samples from these patients.

Targeted and specifically optimized for liquid biopsy samples, amplicon-based NGS panels are thus ideally suited for the analysis of genomic changes in cfDNA or ctDNA and allow the identification of even rarer variants in a specific and cost-effective manner.

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