Assays for circulating cell-free DNA

Proxeom is developing chemistry and reagents for highly sensitive detection of circulating cell-free nucleic acids in blood and other body fluids such as urine, semen, spinal fluid, pleural fluid, etc. 

The approach involves the attachment of Next Generation Sequencing Adapters directly onto DNA still bound within nucleosomal particles resulting in a simplified and more efficient conversion of cell free DNA particles into library molecules. 

The proprietary method of adapter attachment and downstream amplification result in library molecules that have self-correcting error modalities built in.  The library molecules constructed from the circulating cell-free DNA are then sequenced by a whole genome paired-end sequencing approach that would provide a comprehensive view of the circulating cell free DNA population present in the sample. Instead of querying the whole genome, a fraction of the genome can be sequenced by targeted hybridization capture approaches to sequence only a subset of genes, for example, genes implicated in breast cancer.

An overview of the lab protocol for a targeted capture approach in development is shown below.

The paired-end sequencing reads are then processed through an algorithm that determines de novo errors through the self-referential library construct that results in a consensus call that is expected to achieve greater than 0.1% accuracy at high sensitivity and specificity thresholds.

The primary advantage of the approach invented by Proxeom is the need for significantly less sequencing depth to achieve similar sequencing accuracy as unique tag based methods. The up-front direct conversion of nucleosomal particles to library molecules result in an increased sensitivity as loss through nucleic acid extraction and purification are substantially reduced. The higher sensitivity could result in smaller sample sizes being amenable to analysis.

Applications for the high-sensitivity approach to sequencing cell-free nucleic acids

Being able to discover low levels of mutations at lower cost expands the application of Next Generation Sequencing into delineating the presence of rare disease occurrences in a body by liquid biopsy approaches. The circulating cell-free DNA in plasma is released from dying and dead cells throughout the body and is collected by the blood stream. 
A milliliter of cell free plasma (from blood) contains genomic DNA from the debris of 1000 to 10,000 cells. That is about < 10 ng to > 100 ng of cell free DNA is found in one ml of blood (based on current research). Most of this cell-free DNA originates from normal cells, with a small fraction representing diseased cells. Based on current research, the circulating tumor DNA has been detected at 0.1% of the total cell free DNA in blood. With the Proxeom method, DNA from a single cell should be detectable in an ml of plasma.

We see five broad areas of applications for high-sensitivity nucleic acid liquid biopsy detection technologies:
  1. Cancer
    1. Early-diagnosis - For monitoring at risk subjects, such as those with genetic predisposition or familial history of cancer. Appearance of mutations in the blood stream (e.g., BRCA1 mutations) would indicate the presence of a tumor somewhere in the body requiring further investigations.
    2. Post-diagnosis Monitoring the effectiveness of surgical intervention and/or chemo- and radio-therapy. DNA with cancer mutations found in the original tissue biopsy would decrease in the cell-free fraction of blood in the case of successful treatment but would remain constant or increase if the response to treatment is poor.
    3. Predicting relapse. Often a suspectible cancer clone is overtaken by a resistant sub-clone that was originally present in a heterogenous tumor or by a sub-clone that was created de novo through new mutations. Sequencing of circulating tumor DNA would show the appearance of new mutations in cancer-related genes. The new mutations could suggest an alternative treatment regimen.
  2. Inflammatory disease. Epigenetic changes are often associated with dramatic changes in phenotypes even when there are no detectable genetic changes. Epigenetic changes can also be used to determine the tissue of origin for the circulating DNA. By detecting changes in DNA base modifications (DNA methylation) over the normal background the source of a diseases tissue may be determinable. Localized inflammation and the associated necrotic and apoptotic cascades result in the release of genomic DNA that carry specific histone and DNA modification marks associated with the tissue of the origin and its response to inflammation.
  3. Tissue transplantation. Alleles from the host tissue can be detected in the recipients blood stream to monitor transplantation success.
  4. Infectious disease. Presence of pathogen nucleic acids in cell-free DNA and changes in the hosts epigenetic status may have utility in detecting pathogens as well as predicting the hosts response to the pathogen.
  5. Fetal health. Analysis of fetal DNA in maternal blood provides a robust non-invasive method to detect potential de novo genetic lesions in the fetus.