Why Liquid Biopsies?

Investors considering new developments in healthcare and science like ideas that have the potential to change existing treatment protocols or dramatically impact on an entire industry segment.

Clayton Christensen of Harvard Business School coined the term “disruptive innovation” to refer to those innovations that “create new markets by discovering new categories of customers.”

The liquid biopsy concept is one of the examples of relatively new ideas that have the potential to change the life science industry, therefore it belongs to this category of innovations that show the main characteristics of a “disruptive innovation”, changing the paradigm and being considered by investors as appealing target.

What is 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.

Cells dying by apoptosis or necrosis in a variety of diseases (cancer, myocardial infarction, transplant rejection) release DNA from their fragmented genomes into the bloodstream. Also, DNA from a fetus can be detected in the blood of the mother. A specific case are exosomes, 30-100 nm microvesicles, that are not “naked” DNA or RNA but also contain proteins and lipids identical to the originating cell.

These nucleic acids in the blood can be detected and analyzed using PCR techniques, next generation sequencing (NGS), or array technologies. Data that have been generated from analyzing liquid biopsies (also called cell-free DNA or cfDNA) have shown the enormous potential in this approach that could have a revolutionary impact on medical diagnosis, maybe similar only to the impact of the introduction of magnetic resonance imaging (MRI).

Clinical applications that look particularly promising for the liquid biopsy approach are:

Diagnosis of chromosomal abnormalities in the fetus (in particular trisomy) by analyzing the blood from the mother (called also non-invasive prenatal screening (NIPT) bassed on cfDNA)
Diagnosis and monitoring of graft rejection in transplantation patients (DNA from donor tissue attacked by immune cells of the host can be detected in the patient’s blood).
Diagnosis and monitoring of cancer disease. Areas with limited data so far are other diseases with tissue necrosis or apoptosis, such as myocardial infarction.

Why Liquid Biopsies?

The use of “liquid biopsy” is most advanced in the detection of fetal chromosomal abnormalities (in particular trisomies) where genomic diagnosis is challenging the traditional combination of nuchal thickness measurement by ultrasound and the triple test (AFP, hCG, and Estriol) (Bianchi et al., 2014). However, the field with the promise of highest medical impact is clearly oncology, where data generated during the last few years have shown that key cancer mutations can principally be detected by liquid biopsy that mirror those present in traditional tumor biopsies

Traditional cancer diagnostic approaches are far from satisfactorily fulfilling clinical needs. Early diagnostic cancer screening approaches are not accurate or sensitive enough for many cancer types (e.g. colon, prostate, breast cancer), or simply do not exist for the majority of tumor types (e.g. glioblastoma, sarcomas, etc.). In many cases, blood markers, imaging and other procedures only detect cancers after significant growth of tumor tissue.

Biopsies can be very invasive, in approximately 20% – 50% of the cases the material is not enough for a conclusive analysis (Anderson, C. 2015) and some tumors are not accessible for biopsy. This makes traditional techniques not suited for proper treatment monitoring.

However, liquid biopsies may be even superior to standard biopsies, as:

  1. All parts of a tumor and all metastases are potentially sampled. Recent data indicate that in most cases analysis of circulating tumor DNA 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)(Lebofsky et al., 2015), suggesting that sequencing circulating tumor DNA can give a much more complete molecular picture of the systemic cancer disease than standard biopsies.
  2. Access to a patient’s blood is unproblematic. Serial liquid biopsies can be easily taken to monitor cancer therapy effects or to screen for reoccurrence of cancer as long as the volume of blood needed for the respective analysis is small (e.g. a few ml’s).
  3. Sensitivity of the method is superior for detecting cancer at a very early stage, e.g. in cases of reoccurrence of cancer disease after curative surgery, or in a population-based screening program. If liquid biopsy can improve early detection of tumors in preventive screening programs will lead to a higher rate of cured cancer disease, especially for tumor types where means of early detection and preventive screenings are limited or non-existent.

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 (Bettegowda et al., 2014; Breitbach et al., 2014). 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 (Bettegowda et al., 2014).

Therefore, sensitivity limitation of current liquid biopsy approaches is an area in need of technical improvement. SYGNIS is actually running a development program to address this liquid biopsy sensitivity issue by amplification of cell free DNA by its core technology, TruePrime®, and hopes to contribute to make the great promise of liquid biopsy come true.

What is TruePrime®?

TruePrime® is the name of a novel technology dedicated to the amplification of genomic DNA. While the current gold standard MDA relies on short oligonucleotides to start off the amplification, TruePrime® is based on a combination of the highly processive Phi29 DNA polymerase with the recently discovered primase/polymerase TthPrimPol. In this setup, TthPrimPol synthesizes the DNA primers needed for Phi29 DNA polymerase which allows for the exponential amplification of genomic DNA. TthPrimPol displays a potent primase activity, preferring dNTPs as substrates unlike conventional primases.

Key parameters that determine the quality of amplification of genomic DNA are:

  • Absence of contaminations and artefacts in the reaction products.
  • Coverage breadth and uniformity.
  • Ability to recover variants (SNVs, CNVs).

Using a series of experimental approaches we have shown that this technology has:

  • An extreme sensitivity down to the femto-/attogram range,
  • Absence of artefacts derived from synthetic oligonucleotides present in random-primed methods.
  • Very low amplification bias (i.e. a more even coverage than other methods), unsurpassed breadth of genome coverage, and high recovery rates of variants (single nucleotide variants (SNVs) and copy number variants (CNVs).

Due to these features TruePrime® is already available as a superior amplification technology for whole genomes from single or few cells and DNA from low amount samples or circular DNA molecules (TruePrime® SC WGA kit, TruePrime® WGA kit and TruePrime® RCA kit).

SYGNIS now aims at leveraging these key advantages also to the amplification of cell free DNA.

Why TruePrime®?

SYGNIS believes that the TruePrime® technology has unique features that are needed for the challenging amplification of cell free DNA: extreme sensitivity at least to the femtogram range, and excellent SNV preservation due to a very low nucleotide error rate, and a low allelic dropout rate (at ~5%). SYGNIS is therefore convinced that TruePrime® amplification of cell free DNA has the prerequisites to preserve most of the original features of the sample, in particular point mutations at cancer hotspots.

The figure above shows a Venn diagram depicting the high concordance of SNVs detected in single Hek293 cells amplified with TruePrime® (brown) and non-amplified DNA from the same cells (blue).

Initial results from pilot approaches for cell free DNA amplification by TruePrime® look very promising. SYGNIS will keep you updated in this blog on future developments in this program.