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Liquid Biopsy

“Liquid biopsy” is a term coined to describe diagnostic procedures done on nucleic acids in the blood or in other bodily fluids (e.g. urine or cerebrospinal fluid (CSF)) of patients.

Dying cells release DNA from their fragmented genomes into the bloodstream. This “cell-free” DNA can be broadly divided into two groups:

The smaller size fragments (140 – 160 and 2-3x multiples of this) that originate from apoptotic breakdown of genomic DNA inside a cell.
Larger size fragments that originate mainly from necrotic cell death (necrosis), but also exosome shedding, and other less understood processes.

DNA fragments of apoptotic origin can be detected in healthy people, whereas the larger size fragments do not normally occur in healthy people, and thus appear to be the more diagnostically relevant species, specially in the oncology field. Indeed, the proportion of larger DNA fragments increases with progressing tumor stage.

Recent data indicate that in most cases analysis of circulating tumor DNA (ctDNA) is faithfully reflecting mutations found in all known metastases of a cancer, or is even superior to such an approach (e.g. detecting mutations if standard biopsies fail, or showing more mutations than the standard tissue biopsies), suggesting that sequencing circulating tumor DNA can give a much more complete molecular picture of the systemic cancer disease than standard biopsies.

Therefore, the analysis of the long DNA fragments from the ctDNA fraction offers huge opportunities in oncology to improve diagnosis and treatment in cancer patients. However two main handicaps should be overcome:

Limitations due to the low amount of ctDNA.

Several studies of liquid biopsy approaches in cancer patients have revealed that the success rate of this approach is related to the tumor mass burden and tumor stage of a patient at the time of liquid biopsy, and the approach is not very successful in instances when tumor mass is low, because there are not so many tumor cells dying and releasing DNA into the blood. Therefore, a key problem is the difficulty to obtain results from plasma samples with very low ctDNA content. Moreover, the approach works well in some tumor types (e.g. colon carcinoma), but not in others (e.g. glioblastoma) presumably also due to sensitivity issues.

“Contamination” of ctDNA with DNA coming from unrelated processes.

Current techniques for cell-free DNA analysis are composed of two principal types of methodologies:

A) Next-generation sequencing (NGS): targeted sequencing, exome sequencing or whole genome sequencing.
B) Digital PCR: BEAMing (beads, emulsions, amplification and magnetics), digital PCR ligation assay and emulsion-based ddPCR with technology from RainDance or Bio-Rad.

However, these techniques do not provide the possibility to distinguish between these two fragment populations, or may even favour the smaller fragment size population (e.g. by not size fractionating cell-free DNA and directly adding library adapters for sequencing). Therefore, diluting even more the amount of ctDNA in the background of healthy DNA.

Therefore, sensitivity and specificity limitations of current liquid biopsy approaches are an area in need of technical improvement. The TruePrime™ Liquid Biopsy kit addresses this liquid biopsy sensitivity and specificity issues by an adapted amplification of cancer-disease specific large fragment cell-free DNA based on the technology TruePrime™ and the combination of the enzyme TthPrimPol and Phi29 DNA polymerase. TruePrime™-mediated ctDNA amplification will specifically amplify and therefore select the larger, more cancer-specific, DNA fragment size population from human plasma, providing a extremely useful tool for assessing cancer genome information from all patients, regardless of tumor burden, with superior sensitivity and specificity.