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

Nell, Rogier J.; Zoutman, Willem H.; Calbet-Llopart, Neus; García, Adriana; Menger, Nino V.; Versluis, Mieke; Puig, Susana; Gruis, Nelleke A.; Velden, Pieter A. van der

doi:10.1016/j.jmoldx.2021.10.007

Cited by 2 publications

(10 citation statements)

References 39 publications

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“…3. We recommend to construct confidence intervals for each obtained T cell fraction (see Tables 2 and 3 for all formulas [19,20]). These intervals, usually with a confidence level of 95%, indicate the precision of the calculated fractions: the wider the interval, the more uncertain the quantification is.…”

Section: Interpretation Of Resultsmentioning

confidence: 99%

“…Therefore, it should measure a DNA target located in close genomic proximity to the T cell marker, but its abundance should not be altered due to TR rearrangements. For ΔB, we recently validated a regional corrector targeting TRBC2, the secondary constant domain and last region of the TRB gene complex [19]. As TRBC2 is not deleted as part of VDJ rearrangements, it can be considered the closest genomic locus functioning as regional corrector for ΔB.…”

Section: Choosing An Experimental Setupmentioning

confidence: 99%

“…The strict genomic locations of the T cell markers, however, prevent from freely switching to another chromosome to overcome this problem. Based on copy number profiles of more than 10,000 cases spanning 31 tumor types from the TCGA pan-cancer dataset [16,18], we previously showed that CNAs involving the ΔB and ΔD marker loci are present in ~24% and ~17% of the tumors [19]. These frequencies indicate that our original methodology (referred to as the classic model) gives incorrect T cell fractions in on average one out of four (ΔB) or one out of five (ΔD) of the cancer specimens.…”

Section: Introductionmentioning

confidence: 99%

“…In contrast to the T cell marker, the regional corrector should not be deleted by TR rearrangements and is therefore biallelically present in all T cells. When a CNA in non-T cells involves the T cell marker region, the regional corrector will be affected likewise, allowing for a mathematical correction of the disrupted T cell quantification [19].…”

Section: Introductionmentioning

confidence: 99%

“…The experimental setup includes the concurrent quantification of three different DNA targets within one reaction: one of the unique T cell DNA markers (ΔB or ΔD), a regional corrector, and an independent reference DNA marker. By mathematically integrating the measurements of all three markers, T cells can be accurately quantified in both copy number stable and unstable DNA samples, as we previously validated [12,19]. (a) A T cell marker, here ΔB (HEX-labelled).…”

Section: Introductionmentioning

confidence: 99%

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

Nell

Zoutman

Versluis

et al. 2022

Methods in Molecular Biology

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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.

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Section: Interpretation Of Resultsmentioning

confidence: 99%

Section: Choosing An Experimental Setupmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Nell

Zoutman

Versluis

et al. 2022

Methods in Molecular Biology

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Allele-specific digital PCR enhances precision and sensitivity in the detection and quantification of copy number alterations in heterogeneous DNA samples: anin silicoandin vitrovalidation study

Nell,

Versluis,

Zoutman

et al. 2023

Preprint

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The analysis of genetic variation is of crucial importance in cancer care. Measuring copy number alterations, however, remains challenging in heterogenous DNA samples, such as (liquid) biopsies. Using digital PCR, these alterations are classically studied by comparing the abundances of the target of interest to a stable genomic reference. Alternatively, copy numbers may be quantified based on the allelic (im)balance of a heterozygous common single-nucleotide polymorphism (SNP). In this study, the accuracy, practicability, precision and sensitivity of both approaches are evaluatedin silicousing a newly introduced R library ‘digitalPCRsimulations’, andin vitroby analysing control samples and uveal melanoma specimens in duplex and multiplex experimental setups.Our analyses show that both methodologies are equally effective, as long as a stable reference is identified (classic approach) and the allelic imbalance is caused by the loss or gain/amplification of only one of both alleles (SNP-based approach). Though, heterogeneous copy number alterations can be measured with more precision and sensitivity using the SNP-based approach. In DNA from formalin-fixed and paraffin-embedded samples, the latter approach can also overcome technical artefacts causing inconsistencies between DNA amplicons.In conclusion, the limits of detecting copy number alterations in heterogeneous DNA can be improved using the SNP-based approach. Consequently, an increasing number of clinical samples may be successfully analysed, providing novel potential for the identification, prognostication and management of cancer.

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

Cited by 2 publications

References 39 publications

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

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

Allele-specific digital PCR enhances precision and sensitivity in the detection and quantification of copy number alterations in heterogeneous DNA samples: anin silicoandin vitrovalidation study

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