Archive for the ‘Droplet Digital PCR’ Category
Researchers at Fred Hutchinson Cancer Research Center have used Droplet Digital PCR (ddPCR™) to demonstrate for the first time the quantification of a special class of tumor-attacking immune cell known to improve cancer survival, a subpopulation of T-cells called tumor-infiltrating T-lymphocytes or TILs. The study, led by Dr. Jason Bielas, Associate Member of the Public Health Sciences Division at Fred Hutch, paves the way for further study of the role of TIL quantification in immunotherapy and as a cancer survival predictor.
“Now that we have the sensitivity and ability to reproducibly count TILs in tumors, we may be able to stratify and more effectively treat patients based on tumor TIL count, especially with immunotherapeutics coming to market,” said Dr. Bielas, one of the lead authors of a paper reporting the TIL quantification results in Science Translational Medicine.
Quantifying TILs Using ddPCR
TILs directly attack tumor cells in a variety of cancer types. While the presence and quantity of TILs strongly correlate with increased patient survival, current tests are semiquantitative at best. As a result, TILs cannot be used for clinical decision making.
According to Dr. Bielas, TILs have a “genomic signature that can be digitally exploited.” This signature, which exhibits a vast amount of diversity, determines the genetic identity, or clonality, of the T-cell receptors (TCR) expressed on the surface of each TIL. With the advent of digital PCR – and the generation of tens of thousands of data points produced by Droplet Digital PCR – it is now possible to quantify these signatures, enabling the determination of the number of TILs.
“There’s no way you could do this with any method other than digital PCR because of the numerous primer pairs and probes that we have (45 forward primers, 13 reverse primers, and 30 probes),” said Dr. Bielas. “Digital PCR partitions all the reactions so you can amplify these targets independently of PCR efficiency without any competing side reactions.”
Fred Hutch researchers developed the Droplet Digital PCR-based “QuanTILfy” assay using Bio-Rad Laboratories’ QX100 ddPCR system. They then used QuanTILfy to count TILs, determine their frequency, and develop a grouping system to classify “clonality,” which might be a marker of druggable targets.
Fred Hutch researchers performed the QuanTILfy assay on primary tumors from 30 ovarian carcinoma patients with known survival outcomes, ranging from 1 to 122 months. TIL frequency was approximately threefold higher in patients with a survival rate of more than five years compared with patients with survival rates of less than two years. These results show that higher TIL levels correlate positively with patient survival, consistent with the hypothesis that TILs play an active role in suppressing tumor formation.
The researchers also demonstrated that QuanTILfy can be used to accurately and reproducibly characterize T-cell clonality in patients with T-cell acute lymphoblastic leukemia. In each case, they saw a single QuanTILfy assay subgroup, indicative of clonal T-cell expansion. This finding was confirmed by deep sequencing.
The QuanTILfy assay proved to be both sensitive and accurate. In a mixture of human T-cells purified from blood and normal human lung fibroblasts, the assay demonstrated the ability to detect a single TCR rearrangement among 10,000 tumor cells. Importantly, it also demonstrated the ability of ddPCR technology to quantify a large number of markers simultaneously in a single reaction through multiplexing.
Bio-Rad Laboratories, Inc. announced the release of its PrimePCR assays for Droplet Digital PCR systems. This release expands Bio-Rad’s current offering by an additional 46 mutation detection assays and 323 copy number assays. In addition, the existing set of gene expression qPCR primer-only assays can now be used with the recently launched QX200™ Droplet Digital PCR system, featuring EvaGreen detection capabilities.
The new ddPCR assays are the only predesigned and fully wet-lab validated assays for digital PCR, alleviating the burden placed on researchers of design and experimental optimization and offering precision and sensitivity without a standard curve. The assays enable novel research strategies and accelerate discovery for inherited disorders, cancer, and infectious diseases.
Many methods for mutation analysis offer poor selectivity and fail to detect mutation events with abundances of less than one in 100 wild-type sequences. Bio-Rad’s ddPCR technology provides an absolute measure of target DNA molecules, and together with the PrimePCR mutation detection assays, can enable the detection of one mutant molecule in the background of 100,000 wild-type sequences (0.001%). Measuring these extremely low levels of mutation abundance can lead to dramatically more sensitive and less invasive diagnostics.
Current methods, including qPCR and next-generation sequencing, also lack the resolution needed to accurately analyze copy number variation (CNV). Due to their high precision and absolute measurement capability, ddPCR assays enable the quantitative discrimination required to resolve small-fold changes in gene copy numbers. ddPCR assays can also be used to detect subsequent changes in the expression of target genes.
PrimePCR ddPCR assays are available in multiple reaction sizes and are compatible with both the QX100 and QX200 platforms using the ddPCR supermix for probes.
For more information on Bio-Rad’s PrimePCR products, please visit www.bio-rad.com/PrimePCR.
Since its introduction in 2011, Bio-Rad Laboratory’s Droplet Digital PCR (ddPCR™) technology has demonstrated the potential to be a transformative technology, particularly in clinical applications.
In the past, tools developed for such applications have been limited by their inadequate precision and/or their lack of sensitivity for detecting rare species. But thanks to ddPCR technology, researchers can now focus on more of these “needle-in-a-haystack” problems. Less than two years since Bio-Rad brought ddPCR systems to the market, their application has resulted in nearly 50 peer-reviewed publications citing the technology.
The advantages of ddPCR technology have already had an important impact on medical research.
“The HIV community, for instance, has benefited from ddPCR’s ability to make more sensitive measurements, which can also depend on its attribute of increased precision,” said George Karlin-Neumann, the scientific affairs director at Bio-Rad’s Digital Biology Center. “To have greater sensitivity down to very, very low levels depends on being able to distinguish something from nothing. You need a system that inherently has very low noise.”
Droplet Digital PCR Is a Sensitive Tool for Detecting Residual HIV DNA
A great example of the clinical potential of ddPCR systems is the work of Matt Strain and Douglas Richman of the Center for AIDS Research at the University of California, San Diego School of Medicine, who validated the technology’s performance in HIV provirus detection. Subsequently, in collaboration with Deborah Persaud of Johns Hopkins Children’s Medical Center in Baltimore, the researchers used the technology to demonstrate that an infant born with HIV was functionally cured.
In a recent BioTechniques podcast, Dr. Strain said that ddPCR assays demonstrate an increase in precision and accuracy over their entire dynamic range relative to real-time PCR assays, particularly at low numbers. Additionally, the total cost per sample of Droplet Digital PCR assays is at least 10 to 100 times less than that of older chip-based digital PCR systems.
“When you’re talking about factors of a hundred or more in cost, there really isn’t any comparison,” said Dr. Strain.
Dr. Richman will present information on using ddPCR to detect latent HIV, including assaying rare events in a large number of cells and retrieving clinically relevant data.
A Glimpse at Droplet Digital PCR’s Future in Diagnostics
The research group headed by Hanlee Ji, an assistant professor at Stanford University School of Medicine, focuses on translational and clinical questions of cancer genetics that, once answered, have the potential to improve cancer patient care. The investigators have developed numerous methods for the accurate interrogation of cancer genomes that overcome challenges associated with clinical samples and the genetic variability resulting from tumor evolution. In this endeavor, Droplet Digital PCR is one of their chief tools.
“Droplet Digital PCR has accelerated our discoveries,” said Dr. Ji. “Given its ease of use, superior performance in terms of accuracy, and rapid development time for novel assays, Droplet Digital PCR has repeatedly demonstrated its vast utility and potential for future diagnostic application.”
Dr. Ji recently gave a talk on using ddPCR technology to track the presence, expansion, and disappearance of pathogenic genetic variants in cancer, infectious diseases, and other human diseases over time, and also discussed the technology’s potential for highly informative diagnostics. He now uses Bio-Rad’s recently launched second-generation ddPCR instrument, the QX200™ Droplet Digital PCR system, the only digital PCR system that works with both DNA-binding dye and TaqMan probe chemistries.
The QX100™ system boasts a lineup of prestigious users including:
- Jim Huggett,the author of the digital MIQE (dMIQE) guidelines and a scientist at LGC (the UK’s designated National Measurement Institute for chemical and bioanalytical measurement)
- David Dodd of the University of Texas, Southwestern
- Leonardo Pinheiro of Australia’s National Measurement Institute
- Ross Haynes of the U.S. National Institute of Standards and Technology
- Vicki Hwang of the University of California, Davis
- Alec Morley of Flinders University and co-author of the first paper to use digital PCR
- Keith Jerome of the University of Washington
- Gary Lee of Sangamo BioSciences
- Sabita Sankar of MolecularMD
- Donna Sullivan of the University of Mississippi Medical Center
There are also a number of emerging applications using ddPCR technology including microRNAs, single-cell gene expression, gene linkage, multiplexing, EvaGreen applications, and validating next-generation sequencing data.
For more information on the QX200 Droplet Digital PCR system, visit www.bio-rad.com/QX200.
To view Bio-Rad’s six-part webinar series on Droplet Digital PCR and the complete list of Droplet Digital PCR system publications, visit http://www.bio-rad.com/ddPCR-Webinars.