Tissue biopsies, which are currently the gold standard molecular diagnostic technique for oncologists, are not only painful to cancer patients but expose an already immune-comprised population to increased rates of nosocomial infections. Furthermore, it is not uncommon for the typical clinical pathology workflow to yield samples that are not suitable for routine analysis strategies, e.g., next-generation sequencing (NGS), due to severe degradation. Therefore, researchers at Abramson Cancer Center at the University of Pennsylvania (ACC) are among the many investigators pursuing reliable noninvasive tests to replace traditional tissue biopsies for the detection of reliable biomarker signatures and clinically relevant mutations. Recently, results from a study evaluating cell-free circulating tumor DNA (ctDNA) with blood samples derived from 102 advanced non-small-cell lung cancer patients (NSCLC) were published in Clinical Cancer Research in an article entitled, “Detection of Therapeutically Targetable Driver and Resistance Mutations in Lung Cancer Patients by Next Generation Sequencing of Cell-Free Circulating Tumor DNA” (Thompson et al., 2016). Of the patients in the study, 96% had stage IV disease, 81% had adenocarcinoma tumors, and 68% were women.

The study compared results from sequencing of focused gene panels that were independently developed for the analysis of tissue DNA and ctDNA. The tissue DNA samples were analyzed at the University of Pennsylvania (UP) using either the Illumina TruSeq Amplicon Cancer Panel (covers hotspots or exons of 47 genes) or a smaller UP Precision panel covering the 20 most commonly mutated genes, depending on the amount of DNA available. The ctDNA was analyzed using 68 or 70 gene versions of the Guardant360 panel; the sample processing and sequencing were performed by Guardant Health. The Illumina MiSeq instrument was used for the tissue DNA amplicon sequencing using an unstated sequencing depth, whereas the ctDNA amplicons were sequenced to a depth of 10,000X depth using an Illumina HiSeq 2500. Given the 4.2X higher yield of nucleotides from a single lane of the HiSeq 2500 versus an entire MiSeq run, the sequencing depth was likely lower for the tumor DNA samples. Sequencing was successfully completed using ctDNA template for all 102 patients. In contrast, sequencing of tissue DNA was successful in only 49% of patients due to poor quality of specimens, insufficient DNA yield, or because tissue biopsies were unobtainable.

Learn more about Frontage’s Cell-free DNA Methylation Sequencing Services.

Assuming faithful representation of the tumor genomes in the circulating DNA, because of the lower number of genes sequenced as well as the likely reduced coverage depth for the analysis of tissue DNA versus ctDNA, one would expect that the number of known mutations identified in the ctDNA would be higher than in the tissue DNA when comparing the same samples. This expectation was borne out by the focused comparison of 50 patients for which both tissue DNA and ctDNA were analyzed. Of these 50 common patients, 78% had at least one variant call from tissue DNA versus 84% for ctDNA. In keeping with the larger gene panel size used for the characterization of the ctDNA, an average of 2.8 variants per patient were detected in the ctDNA versus 1.5 in the tissue DNA. The overall concordance between the two sample types for variant detection was 60% when considering only polymorphisms that were covered by both the ctDNA and tissue DNA panels. When narrowing the scope to the therapeutically targetable EGFR mutations, the overall concordance in polymorphism detection between the matched tumor and circulating DNA samples improved to 79%.

The lower-than-desirable concordance rate (60%) should not be taken to indicate limitations in the reliability of the sequencing-based tests. The authors provide several plausible reasons for the reduced concordance between the ctDNA and tissue DNA assays. First, the time between the collection of the tissue sample and the blood sample for the study was up to 2 years, and major changes in the genetic makeup of the tumor could occur during this time. Another reason for reduced concordance could be the differing contribution of specific tumor lineages to tumor versus circulating DNA; in general tumor DNA may be diluted by non-tumor circulating DNA in the blood leading to a reduced frequency of specific mutations. Conversely, cell types with specific mutations may more aggressively shed DNA in to circulation and therefore be over-represented in the ctDNA relative to the tissue DNA. Overall, these results contribute to the promise that as sequencing technology continues to advance with the ability to profile lower amounts of DNA, with coverage of greater numbers of genes at greater depth, patients will be able to benefit greatly from non-invasive monitoring of the genetic make-up of ctDNA. Further improvements in databases of actionable mutations will allow for better clinical decision-making based on the review of ctDNA sequencing results, ultimately improving patient survival and quality of life.

Editor’s Note: The original article first appeared on the Ocean Ridge Biosciences website, authored by Sheena Knight, which, after the acquisition of Ocean Ridge Biosciences, was modified for use on the Frontage website.

Article, Genomics

Through liquid biopsy, studying cfDNA informs us on various considerations for drug treatment.

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 

Low and variable yield of cfDNA: 10 nanograms of DNA, equivalent to 1520 diploid genomes, is the absolute minimum mass for reliably successful detection of somatic polymorphisms. 
Low percentage of circulating tumor DNA relative to total cfDNA: The percentage of tumor-derived DNA in circulation may be as low as 1%. One must sequence deep enough and use strong methods to prevent DNA loss during library preparation (point #4). 
Poor sample quality due to blood sample handling: This can cause contamination with DNA derived from the lysis of hematopoietic cells and inefficient removal of platelets. 
Loss of DNA during the preparation of libraries for sequencing: DNA loss during processing reduces the number of unique sequencing molecules represented in the sequencing library.

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. 

If you’re enjoying this article, check out our Cell-Free DNA Methylation Sequencing services.

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.

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. 

Biomarkers, cfDNA, NGS

Article, Genomics