Generic Multiplex Digital PCR for Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples

Abstract

An accurate T cell quantification is prognostically and therapeutically relevant in various clinical applications, including oncology care and research. In this chapter, we describe how T cell quantifications can be obtained from bulk DNA samples with a multiplex digital PCR experiment. The experimental setup includes the concurrent quantification of three different DNA targets within one reaction: a unique T cell DNA marker, a regional corrector, and a reference DNA marker. The T cell marker is biallelically absent in T cells due to VDJ rearrangements, while the reference is diploid in all cells. The so-called regional corrector allows to correct for possible copy number alterations at the T cell marker locus in cancer cells. By mathematically integrating the measurements of all three markers, T cells can be accurately quantified in both copy number stable and unstable DNA samples.

Vitreous fluid-isolated DNA for the genetic analysis of primary uveal melanoma: a proof-of-concept study

“…All digital PCR experiments were carried out using the QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, USA) using the assays and following the protocols described earlier [16][17][18][19][20]22]. Typically, 3.5 uL isolated vfDNA was analysed in a single 22 uL experiment, using 11 uL ddPCR™ Supermix for Probes (No dUTP, Bio-Rad) and primers and FAM-or HEX-labelled probes in final concentrations of 900 and 250 nM for duplex experiments, or optimised concentrations for multiplex experiments.…”

“…CNAs were measured via two different approaches [19]. Following the classic approach, copy number values were calculated based on the measured concentrations of a DNA target on chromosome 3p (PPARG) and 8q (PTK2), and a reference gene on chromosome 5p (TERT) or 14q (TTC5) assumed to be stable:…”

“…To evaluate the statistical power of detecting CNAs in the vfDNA+/UM+ samples, we performed in silico digital PCR simulations using our R library digitalPCRsimulations (available via https://github.com/rjnell/digitalPCRsimulations), as described recently [19]. In short, for each individual sample, we determined the sensitivity (% of simulated experiments) of detecting a chromosomal loss (copy number = 1) or gain (copy number = 3), assuming that this alteration is clonally present in the entire vfDNA melanoma-cell population.…”

“…CNAs are usually studied by comparing the absolute abundance of a target of interest to a stable genomic reference (classic approach, Figure 5A) [19]. As an alternative, CNAs may be measured by evaluating the allelic (im)balance between the two variants of a heterozygous SNP on the chromosome of interest (SNP-based approach, Figure 5A).…”

“…Additionally, knowledge about the molecular profile of the primary tumour has been positively associated with various aspects of psychosocial well-being [13] and might be of (future) clinical relevance with regard to novel (neo-)adjuvant therapies [5,14,15]. Previously, we introduced digital PCR assays to analyse the key genetic alterations in uveal melanoma to quantify the intratumour heterogeneity in bulk tumour specimens [16][17][18][19][20].…”