June 3, 2019 | In the Soper lab, researchers are developing microfluidic devices out of plastics for the analysis of liquid biopsy markers. They hope that the plastic format will allow for high-scale production at low cost to enable implementation into the clinic.
"We have recognized that enumeration of the liquid biopsy markers is now insufficient in many cases," says Steven Soper, Foundation Distinguished Professor at the University of Kansas, Department of Chemistry and Mechanical Engineering and Cancer Biology. "Clinicians now require molecular information on the cargo the marker possesses to make important decisions on how to manage the patient's disease (precision medicine). Thus, we have now geared our work toward integrating the liquid biopsy enrichment platform to microfluidic and even nanofluidic devices to carry out molecular processing of the liquid biopsy cargo in a fully automated fashion that could also allow for point-of-care testing."
On behalf of Diagnostics World News, Emily Le spoke with Soper about the history of liquid biomarkers, the role they play in precision medicine, and the challenges ahead for companion diagnostic development.
Editor's note: Emily Le, a conference producer at Cambridge Healthtech Institute, is planning a track dedicated to Enabling Technologies for Circulating Biomarkers at the upcoming Next Generation Dx Summit in Washington, D.C., August 20-22. Soper is a featured speaker on the program. Their conversation has been edited for length and clarity.
Diagnostics World News: Hi Steve, can you tell us about your background and what you are working on right now?
Steven Soper: I received my PhD in Bioanalytical chemistry from the University of Kansas followed by a Post-doctoral fellowship at Los Alamos National Laboratory, where I worked on the Human Genome project – developing an innovative single-molecule sequencing strategy. I spent 20 years at Louisiana State University in the Department of Chemistry, 5 years at the University of North Carolina, Chapel Hill (Department of Biomedical Engineering), and am now a Foundation Distinguished Professor at the University of Kansas (Department of Chemistry and Mechanical Engineering and Cancer Biology).
We are currently working on developing microfluidic devices for the analysis of liquid biopsy markers for a variety of diseases. Unique to our program is that we are making the microfluidic devices in plastics to allow for high-scale production at low cost to enable implementation into the clinic (Bench-to-bedside). We have technologies for the analysis of many of the liquid biopsy markers, including circulating tumor cells [CTCs], cell free DNA, and extracellular vesicles [EVs].
For example, we have developed devices for detecting point mutations in the genomic DNA of CTCs, and expression profile the mRNA in both CTCs and EVs for a variety of diseases. We are now working on a new and innovative single-molecule sequencing device that uses nanofluidic technology to process unamplified DNA and RNA to elucidate their primary structure.
What are some big hurdles in developing liquid biopsy technology?
Simple enumeration of the liquid biopsy markers is now insufficient. It is becoming required that the molecular cargo now undergo characterization. The challenge associated with molecular profiling liquid biopsy markers is that following enrichment from clinical samples, the mass of disease-associated molecular markers is low. For example, in a single CTC, there are 2 copies of the genomic DNA, which amounts to 6 picrograms of material. As a sense of reference, next generation sequencing (NGS) platforms require inputs of ~30 ng of DNA. Therefore, extensive levels of amplification must be undertaken. This can create significant problems. For example, due to the high G/C content of the human genome and high numbers of repetitive elements, the genome is biased in terms of its representation following amplification.
Also, there is a big push now to detect epigenetic modifications in the genome and even in RNA molecules. Unfortunately, extensive levels of amplification can mask these modifications making them in some cases impossible to detect. Even the droplet digital PCR techniques can have difficulties in detecting small mass inputs secured from liquid biopsy markers. This will become particularly problematic as efforts move into the regime of early detection, where the mass secured from the marker becomes even less.
In the case of proteins, this can be even more problematic due to the complexity associated with protein structure (20 different amino acid residues) and the post-translational modifications they can undergo. As a case example, for extracellular vesicles in early stage cancer disease, a mL of plasma may contain ~1-100 target protein molecules that require detection.
When did liquid biopsy start playing a role in precision medicine and how big of a role is liquid biopsy as a companion diagnostic tool?
Liquid biopsies have been around much before the initiation of the Precision Medicine Initiative as launched by ex-President Obama several years ago. Thus, since this initiative started, liquid biopsy markers have played an integral role in precision medicine. Why? One of the main components of precision medicine is securing molecular characteristics of a person’s disease and the panel of liquid biopsy markers can provide molecular information that can serve as surrogates for the tumor tissue. While several groups have found disconcordance between the primary tumor and the liquid biopsy marker, this is not too surprising given the fact that solid tissue biopsies of the primary tumor do not, for the most part, sample metastatic sites. In addition, CTCs can undergo transitions once released from the primary tumor, for example epithelial-mesenchymal transitions, EMT.
Liquid biopsy markers can serve as attractive companion diagnostic markers for drug discovery due to their ease of acquisition. This will be particularly important in organ-specific cancer diseases where solid tissue procurement is difficult, a good example being non-small cell lung cancer. Recently, the FDA approves Roche's cobas EGFR Mutation Test as a companion diagnostic test for AstraZeneca's IRESSA (gefitinib) in first-line non-small cell lung cancer (NSCLC), offering patients who are not eligible for tissue biopsy a non-invasive option via a blood draw. This particular test (cobas) uses cell free DNA as the liquid biopsy marker and performs a special type of PCR that can screen for all of the possible mutations in the EGFR gene. However, there is still a lot of room for expansion of liquid biopsy based markers serving as companion diagnostic tests.
What are the challenges you see for companion diagnostic test development?
There are a number of challenges with the expansion of liquid biopsy markers into the drug discovery and drug testing field, mostly due to the rather evolutionary nature. This is predicated on the fact that a constant flux of new technologies has and continues to be evolving in this field and as a result, there are a lack of FDA approved tests that are based on the use of liquid biopsy markers. There are two layers of technology, one for the isolation and enrichment of the liquid biopsy marker from clinical samples, and the second is the analysis platform – looking at the molecular content of the marker. Some of the later technologies exist, for example NGS, and droplet digital PCR. However, the mass limits generated by the liquid biopsy markers makes the marriage of the liquid biopsy marker with the platform challenging. In the case of the former, the analytical figures of merit of many isolation platforms are not sufficient for interfacing to the downstream molecular analysis platform. Thus, in many new cases new molecular analysis technologies may need to be generated to accommodate the significant mass limits imposed by the use of a liquid biopsy marker.
Do you have any advice for young scientists who are entering the field of liquid biopsy?
This is a very, very exciting field with a lot of opportunities to change the way physicians/clinicians manage the oncology diseases as well as other diseases. As noted above, there is still a lot of innovation that is needed on the hardware (isolation technologies) and software (molecular assays) side of things. This is compounded by the keen interest in liquid biopsies by the medical community. However, this is a crowded field with a lot of academic and private-sector interests.
One does need to keep in mind fatigue. For example, efforts focused on CTC based projects is becoming challenged by the fact that the only FDA approved test is the Veridex CellSearch platform for securing prognostic information on breast, prostate, and colorectal cancers. With the large investment in research and finances, no technologies that have cleared the FDA has evolved in the last ~20 years. Funding and interest at the federal and private sectors have waivered at this point in terms of CTCs. EVs are fairly new and research in this area as such is premature and still needs significant progress to move forward as well as clinical trials to support EVs in a clinical diagnostic or prognostic test. Finally, cell free DNA, especially with its success in marrying with NGS has had some success in the oncology field, as well as others, such as prenatal diagnostics.