Authors: David Willoughby, Jessica Sinha 

Precision medicine has seen many improvements in the current clinical approaches, especially as liquid biopsy methods are being increasingly studied. Liquid biopsy, which is an alternative sampling method for circulating macromolecules, has a particular emphasis on nucleic acids. Of particular interest is cell-free DNA (cfDNA), which is the extracellular DNA circulating in body fluid and can be derived from both normal and diseased cells and has the potential to be used as biomarkers. Patients who are likely to respond and those who do not respond to a drug treatment during a late-stage clinical trial may be distinguished by an assay for specific biomarkers in a process termed patient stratification. With improvements seen in next-generation sequencing (NGS) tools, successful sequencing of cfDNA analysis is possible. It is informative in various areas, from learning about mutations to monitoring targeted drug resistance.  

For more on the current state and future of Precision Medicine and Companion Diagnostics, watch our panel discussion with Frontage expert, Dr. Kai Wang.

Liquid Biopsy: A Tool for Sampling Biomarkers  

Liquid biopsy is a method of biomarker sampling that minimizes invasiveness, allows for routine sampling, and is associated with the analysis of circulating macromolecules with a particular emphasis on nucleic acids. Serum and plasma are the most common sample types, but urine, saliva, and other bodily fluids can also be used. Liquid biopsy is used for the determination of diagnosis or prognosis and to facilitate informed treatment decisions. Types of molecules that can be analyzed in liquid biopsy samples include lipids, carbohydrates, small biomolecules, proteins, circulating nucleic acids, or tumor cells. Particularly the circulating DNA and RNA assayed can be informative about a disease or drug therapies. However, for solid tumors or more organ-specific diseases, cfDNA or RNA is typically much more informative.  

Methods for Cell-Free DNA Analysis  

Frontage offers various methods to analyze cfDNA:  

• Droplet digital PCR helps accurately measure copy number changes, single nucleotide polymorphisms (SNP), and small insertions/ deletions (indels) for one or a few loci. 
• Real-time PCR monitors SNPs, indels, conserved translocations, and methylation changes for one up to a few hundred specific sites (with qPCR panels). This method is highly sensitive with a wide dynamic range and can detect an extremely low % of variants in a population. However, this method is best suited for 1 or a few loci, though there are technologies that offer panels to run for multiple loci. 
• Sequencing Techniques
  • Whole genome sequencing is used mainly for the detection of major chromosomal rearrangements, deletions, and hypomethylation. This is not practiced for most applications for cfDNA given the non-uniform coverage of cfDNA.  
  • Amplicon sequencing is a low-cost and rapid procedure to target up to 200 genomic regions with commonly occurring polymorphisms that inform treatment decisions. Tumor mutational burden and microsatellite instability can also be determined. 
  • Hybridization-based capture sequencing allows high-depth sequencing of up to 100 million nucleotides from the human genome. This technique also enables the detection of potentially pathogenic SNP and indels, as well as some large deletions and gene fusion events. 
  • Methylation sequencing checks the methylation of cytosine residues in the DNA.  

Major Challenges with cfDNA Sequencing  

Frontage teams utilize commercially available amplicon- and hybridization-capture-based multigene sequencing panels from Illumina, Invitae, and other suppliers as well as custom sequencing panels focused on key target genes. A broad range of analysis capabilities such as droplet digital PCR (ddPCR) and qPCR are also available to Sponsors that choose Frontage. 

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Conclusion  

Analysis of cell-free DNA has been evaluated and studied in many fields from precision medicine, oncology, prenatal medicine, and transplant medicine, to cardiovascular diseases. Cell-free DNA analysis is an exciting space in the current drug development and diagnostic space, and cfDNA tests are being developed by many molecular diagnostics companies, the first ones being approved by the FDA in 2020: Guardant 360 CDx and FoundationOne Liquid CDx tests. Analyzing cell-free DNA enables better targeting of the populations that would respond best to the drug and has tremendous value in the early development of companion diagnostics. The rapid development of new molecular techniques propels the various uses and applications of cfDNA. Not only does it open doors to minimally invasive diagnostics, but it also delivers promising data that could improve clinical decision-making and support closer monitoring of drug responses. 

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Contract Research Organizations like Frontage assist sponsors with cfDNA analysis during clinical trials. Some common applications of cfDNA analysis are:   

• Identification of classes of polymorphisms and mutated genes associated with experimental anti-tumor drug efficacy  
• Identification of surrogate biomarkers of anti-tumor drug efficacy  
• Companion diagnostics development  

Frontage provides a wide array of genomic services for protein-, oligonucleotide-, gene-, and cell-based therapeutic discovery and development, and complete solutions for the analysis of RNA expression, DNA polymorphisms, methylation, microbial composition, and protein biomarkers. Our labs are optimized for ultra-low input requirements and challenging sample types, supporting mechanisms of action, lead optimization, biomarker discovery, and the development of companion diagnostics.