Abstract
An accurate T-cell quantification is prognostically and therapeutically relevant in various malignancies. We previously developed a digital PCR-based approach offering a precise T-cell enumeration in small amounts of DNA. However, it may be challenging to apply this method in malignant specimens, as genetic instability can disturb the underlying mathematical model. For example, approximately 24% of the tumors from The Cancer Genome Atlas pan-cancer data set carried a copy number alteration affecting the TRB gene T-cell marker, which would cause an underestimation or overestimation of the T-cell fraction. In this study, we introduce a multiplex digital PCR experimental setup to quantify T cells in copy number unstable DNA samples. By implementing a so-called regional corrector, genetic alterations involving the T-cell marker locus can be recognized and corrected for. This novel setup is evaluated mathematically in silico and validated invitro by measuring T-cell presence in various samples with a known T-cell fraction. The utility of the approach is further demonstrated in copy number altered cutaneous melanomas. Our novel multiplex setup provides a simple, but accurate, DNA-based T-cell quantification in both copy number stable and unstable specimens. This approach has potential clinical and diagnostic applications, as it does not depend on availability of T-cell epitopes, has low requirements for sample quantity and quality, and can be performed in a relatively easy experiment.
Highlights
T cells form a crucial part of the adaptive immune system, the most specified form of our body’s defense system
Approximately 24% of the tumors carried a copy number alterations (CNAs) affecting the TRB gene (DB locus) and approximately 17% affecting the TRD gene (DD locus), but large differences were observed between cancer types (Figure 1, A and B)
Further evaluation of the affected cases showed that these CNAs typically encompassed much larger genomic regions than the T-cell marker locus only
Summary
T cells form a crucial part of the adaptive immune system, the most specified form of our body’s defense system. By using duplex digital PCR, an absolute quantification of both targets could be established, thereby offering a DNA-based method to accurately quantify T cells.[4,5]. This experimental rationale, referred to as the classic model, allows us to measure T-cell presence under the assumption of genomic stability of the target and reference loci. This requirement is typically met when a DNA sample of nonmalignant origin is analyzed; benign samples, in general, do not present with copy number alterations (CNAs). It may be advantageous to perform a Tcell quantification in malignant samples, as the presence of tumor-infiltrating T cells is prognostically and therapeutically relevant in various types of cancer.[7,8] as tumor material (eg, small biopsies) is frequently scarce or of limited quality, golden standard methods like flow cytometry and immunohistochemistry may be unfeasible, whereas DNA-based analyses remain possible
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