By Syamala Ariyanchira,
November 4, 2015
The latest hype in clinical research circles is liquid biopsy. A quick search on PubMed indicated close to 1,000 publications on liquid biopsy this year, from January through October 2015.
The possibility to detect cancer during its early stages through a routine blood test can offer substantial commercial potential.
Current diagnostic practices based on tissue biopsies present numerous challenges due to the heterogeneity of tissue samples and the difficulty in accessing some of the affected organs, even through invasive procedures. The potential to detect cancer non-invasively is the major driver of liquid biopsy. It offers hope in other areas as well, including diabetes, organ transplantation and infectious diseases.
The advent of highly sensitive and advanced technology platforms such as next generation sequencing (NGS) has given the push to the commercial potential of the field by making fast sequencing of circulating tumour DNA a reality.
Even though commercial products are yet to be launched for clinical use, dPCR is receiving the recognition of researchers as an extremely sensitive and precise platform that can detect circulating nucleic acids in plasma and serum.
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dPCR platforms in the market include chip-based microfluidic platforms and droplet-based platforms. Both are capable of absolute quantification of nucleic acids in dilute samples. While companies such as Fluidigm use microfluidic chips, Bio-Rad and Rain Dance Technologies offer emulsion-based dPCR platforms, which are also called droplet digital PCR (ddPCR).
In liquid biopsy research, ddPCR is gaining more popularity over chip-based platforms. In addition to absolute quantification, ddPCR also offers reproducibility and unprecedented degrees of sensitivity and precision, which are extremely valuable for accurate diagnosis during the early disease stages.
The ddPCR companies have been promoting their platforms in research circles aggressively. The majority of the ddPCR publications have been focusing on the potential for early diagnosis, treatment monitoring, and relapse detection of cancer.
In one of its latest compilation of publications using its ddPCR platform, Bio-Rad listed almost 25 publications on liquid biopsy, just during the first six months in 2015. In 2014, for the full year, the number was only 10.
In terms of technological robustness, a study comparing the ddPCR platforms of Bio-Rad and Rain Dance Technologies in clinical settings is yet to come. Rain Dace Technologies offer higher multiplexing potential, with up to 10 channels and 10 million droplets, which may offer potentially better rare event detection. Though Bio-Rad’s platform offers a much lower number of droplets, the company is scoring high in research circles through the company’s effective promotion strategies.
Enabling the miRNA Promise
Application of micro RNAs (miRNAs) as biomarkers has been the focus of clinical research for many years. Identification of miRNAs offering high specificity related to various cancer types is a major area of research activity even now. These biomarkers were expected to have direct correlation with an advancement of the cancer type. The miRNA hypothesis received significant attention in early cancer detection research.
The ability to detect extremely small quantities of miRNA in plasma and serum samples is imperative to develop miRNA-based clinical diagnostic tools. This posed a major technological challenge a few years back. Quantitative PCR (qPCR), which is the gold standard, could not offer the required sensitivity to accurately quantify cell-free miRNAs circulating in the blood. Not surprisingly, the miRNA potential hit a dead end.
This new limit of sensitivity, precision, and reproducibility is creating significant excitement in research circles. Several research groups are currently exploring the liquid biopsy potentials of the miRNA-ddPCR combo approach in clinical settings.
The renewed optimism in the field can be spotted as early as 2013. One of the first publications that explored the head-to-head comparison of ddPCR and qPCR was by Feng Jiang’s research group at the University of Maryland School of Medicine. Their results emphasized the higher effectiveness of ddPCR over qPCR in clinical settings as liquid biopsy tool while targeting low concentrations of miRNA biomarkers.
The study was based on diluted plasma samples from 36 lung cancer patients and 38 controls. Bio-Rad’s QX 100 droplet generator and reader were the ddPCR platforms used by the team. qPCR was conducted on Applied Biosystems 7900HT thermocycler[i].
Based on their results, the authors suggested qPCR for miRNAs for clinical samples where higher concentrations are expected and ddPCR for lower concentrations. This was based on the lower accuracy demonstrated by ddPCR in samples with higher concentrations.
Another study published in the same year by Muneesh Tewari, et. al at the University of Michigan also showed greater precision and reproducibility of ddPCR over qPCR[ii].
Since then, several research groups have used ddPCR for absolute quantification of miRNAs in various clinical research contexts. One of the latest published in July 2015 by Wang, P. et. al once again confirmed the robustness of ddPCR against qPCR for lung cancer-related miRNA quantification[iii].
The Over Diagnosis Concern
It is a well-known fact that certain cancers remain asymptomatic for a long period. A clinical intervention may not be needed in several cases. The biological mechanisms that trigger advancement of cancer are yet to be understood.
One of the main concerns of liquid biopsy is the potential for over diagnosis. This is the concern for miRNA-ddPCR combo approach as well. As ddPCR removes the detection limits, it has to be acknowledged that the presence of miRNA biomarkers may not be directly correlated to the stage of cancer. Responsible development recognising the complexities associated with cancer advancement is critical.
The good news is, research can now progress faster-taking advantage of the unprecedented sensitivity of ddPCR. It gives hope that early detection and staging of cancer with a simple blood test may finally be possible with miRNA liquid biopsy.
[i] Jie Ma, Ning Li, Maria Guarnera and Feng Jiang Biomarker Insights, 2013:8 127-136
[ii] Christopher M Hindson, John R Chevillet, Hilary A Briggs, Emily N Gallichotte, Ingrid K Ruf, Benjamin J Hindson, Robert L Vessella, and Muneesh Tewari, Nature Methods, 2013:10, 1003–1005
[iii] Wang, P. et. al. Biosens Bioelectron, 2015:836-42