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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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A Comprehensive ddPCR Strategy for Sensitive and Reliable Monitoring of CAR-T Cell Kinetics in Clinical Applications.

Gertrud Wiedemann1,2, Ulrike Bacher1, Raphael Joncourt1

  • 1Department of Hematology and Central Hematological Laboratory, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.

International Journal of Molecular Sciences
|August 29, 2024
PubMed
Summary

Digital droplet PCR (ddPCR) offers a sensitive and precise method for monitoring chimeric antigen receptor T-cell (CAR-T) expansion in B-cell malignancies. This validated assay enables robust CAR-T cell quantification across various sample types, aiding clinical management.

Keywords:
B-cell lymphomaCAR-T cell therapiesacute lymphatic leukemia (ALL)droplet digital PCR (ddPCR)molecular monitoring

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Immunotherapy

Background:

  • Chimeric antigen receptor T-cell (CAR-T) therapy is a promising treatment for B-cell malignancies.
  • Accurate monitoring of CAR-T cell expansion and persistence is crucial for assessing treatment efficacy and patient outcomes.
  • Existing monitoring methods may lack the sensitivity and precision required for comprehensive CAR-T cell quantification.

Purpose of the Study:

  • To design, implement, and validate a digital droplet PCR (ddPCR) assay for monitoring CAR-T cell expansion.
  • To compare the performance of the developed ddPCR assay with quantitative real-time PCR (qPCR).
  • To assess the utility of ddPCR for quantifying CAR-T cells across different products and sample types in a clinical setting.

Main Methods:

  • Development of specific ddPCR assays targeting tisa-cel, axi-cel, and brexu-cel CAR-T products, normalized with the RPP30 gene.
  • Validation of ddPCR assays using blood samples from treated patients and comparison with a published qPCR assay.
  • Quantification of CAR-T DNA copy numbers and CAR-T mRNA expression in peripheral blood, bone marrow, and lymph node biopsies from 141 patients.

Main Results:

  • The developed ddPCR assays demonstrated high specificity and sensitivity, with a limit of detection of 20 copies/µg DNA.
  • ddPCR provided reliable and reproducible quantification of CAR-T cells across multiple sample types, including solid tissues.
  • Longitudinal monitoring revealed significant CAR-T cell expansion and long-term persistence, with peak expansion correlating with clinical outcomes.
  • CAR-T mRNA expression levels strongly correlated with DNA copy numbers, confirming active transgene expression.

Conclusions:

  • ddPCR is a highly sensitive, precise, and reproducible method for monitoring CAR-T cell expansion and persistence in patients with B-cell malignancies.
  • The developed ddPCR assays are suitable for clinical applications and can be adapted for future CAR-T products.
  • This monitoring approach supports the management and optimization of CAR-T cell therapies.