Droplet Digital PCR (ddPCR™) enables accurate, precise, and sensitive quantification of specific nucleic acid sequences. In addition to the standard detection of two targets using two different fluorophores, it is possible to increase the number of targets detected by varying parameters that affect PCR efficiency and end-point fluorescence. In this case, we describe a method to multiplex assays by varying the concentrations of primers and probes or the type of fluorophores used. This allows users to expand the number of simultaneously detected targets up to four. Increasing the number of potential targets per test is a significant improvement for ddPCR, dramatically augmenting the information output of each sample.
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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.