Enabling the detection and quantification of cancer mutations with diagnostic and prognostic value across different cancer types is one the hottest areas in oncology. One new technology is a blood-based genomic profiling assay, known as liquid biopsy. Liquid biopsies have the power to detect predictive biomarkers for cancer therapies without the need for a tissue sample. With this non-invasive assay, cancer diagnosis and personalized care could be tailored with a simple blood draw. But, challenges remain.
Droplet Digital™ PCR is a useful technology and has shown promise for both invasive and non-invasive (liquid biopsy) genetic profiling and real-time monitoring of solid tumors. It has been widely applied in translational research for the early detection of mutations that confer resistance to standard therapeutic intervention. A significant limitation of droplet digital™ PCR, however, is that tracking circulating tumor DNA often requires personalized assays. These assays, which require both mutation and wild-type probes to detect single nucleotide variants (SNVs), are costly, especially when multiple mutations need to be tracked.
To find out about cutting edge research taking place to overcome these limitations, we spoke to Miguel Alcaide, Research Associate, Simon Fraser University and recent winner of the Bio-Rad Droplet Innovators Award.
To start us off, could you tell me about your work on tracking cancer associated mutations in liquid biopsy?
The Morin lab is very focused on the development of ultrasensitive techniques for the detection and quantification of somatic mutations with diagnostic and prognostic value across different cancer types. For example, early detection of subclonal mutations conferring resistance to standard therapeutic treatments from liquid biopsy specimens has great implications regarding the management of cancer patients. Noninvasive genotyping of solid tumors may also guide patient stratification and the selection of certain drugs to counteract the effects of activating mutations at key genes driving tumor growth and progression. Tracking of circulating tumor DNA levels by means of personalized assays also enables us to monitor tumor burden and early response to therapeutic interventions, including detection of minimal residual disease. Circulating tumor-derived genetic aberrations are often highly diluted in a background of wild-type molecules and, as a result, the development of ultrasensitive and highly specific assays to support the growing field of personalized medicine is a must.
What’s the significance of this research?
Our recent research on droplet digital™ PCR (ddPCR), widely considered as one of the “gold standards” for rare allele detection within complex mixtures by the scientific community, brings into focus a novel set of assays that further leverage the enormous benefits of sample partitioning. In essence, we have demonstrated that ddPCR™ enables the discrimination, at single base pair resolution, and simultaneous quantification of both mutant and wild-type alleles with one single probe. Standard ddPCR™ assays typically rely on two sets of hydrolysis probes conjugated with dyes having different emission spectra to simultaneous quantify mutant and wild-type alleles. By overcoming this limitation, it is now possible to screen for mutations at two independent genomic positions in one single assay, which decreases both overall costs and the amount of clinically precious samples needed to find support for the presence (or absence) and relative abundance of certain mutations.
Furthermore, our provocative “inverted” ddPCR™ assays allow the detection and quantification of multiple mutations with the aid of one single hydrolysis probe that mimics the sequence of the wild-type allele of the region of interest. This finding not only has important implications regarding the number of probes needed to run a given assay, but may also enable the detection of novel mutations that would be otherwise missed by using probes targeting the most commonly observed mutant alleles.
What challenges need to be overcome to enable widespread adoption of ddPCR™ in liquid biopsy?
Droplet digital™ PCR technology has a bright future for its widespread adoption in the clinical setting. However, only a few pre-designed assays capable of screening for multiple mutations in one single assay are currently available (including commercially) to the clinical and scientific community. Assay design and optimization is often expensive and time-consuming. The multiplexing capabilities of ddPCR™ (i.e. our capabilities to detect multiple clinically informative mutations in one single assay) are also quite limited in comparison with other techniques such as targeted hybridization capture coupled with NGS. It must be noted that the amount of DNA obtained from the plasma is usually quite low, and one may need to use up that entire DNA sample to guarantee maximum sensitivity. Moreover, many patients will not display mutations at any of the most widely investigated cancer-associated hotspots. For these cases, we need to design personalized sets of probes to track ctDNA levels. Hydrolysis probes to run these assays are expensive, and one may want to use multiple probes per patient to control for the confounding effects of tumor heterogeneity and clonal evolution. We hope our new set of assays help minimizing these limitations in the near future by decreasing overall costs and boosting the multiplexing capabilities of ddPCR™.
Based on your findings, what do you think the future holds for droplet partitioning technology?
In my opinion, instruments relying on the droplet partitioning technology will become an essential part of any clinical laboratory around the world as they enable a cost-effective and straightforward screening of common and rare genomic aberrations with diagnostic and prognostic value in cancer patients. Turnaround times, one of the most important factors within the clinical setting, definitively favor ddPCR™ in comparison with other ultrasensitive techniques for rare allele detection. Droplet digital™ PCR assays targeting different types of cancer and disease-associated mutations such as single nucleotide polymorphisms, indels, structural variants and copy number alterations will be routinely performed, together with the advent of novel applications such as those supporting single-cell omics.
Miguel Alcaide was talking to Jack Rudd, Senior Editor at Technology Networks