Detection and Quantification of Chimeric Antigen Receptor Transgene Copy Number by Droplet Digital PCR versus Real-Time PCR.

Abstract

Chimeric antigen receptor (CAR) T-cell immunotherapy is a new strategy for the treatment of refractory B-cell malignancies; therefore, the rapid and accurate quantification of CAR transgene copy number is essential. Real-time PCR was used for quantifying the copy number of chimeric antigen receptor transgene. Droplet digital PCR (ddPCR) is an absolute quantification method that does not require a standard curve. In this study, key performance parameters of the ddPCR and real-time PCR methods were assessed, including linearity, detection range, the lower limit of detection, repeatability, reproducibility, and accuracy, using a series of gradient diluted standards and clinical peripheral blood samples from CAR T-cell patients. The two platforms showed a good correlation for the standards (Pearson R2 = 0.9966; P < 0.0001) and clinical samples (Pearson R2 = 0.8952; P < 0.0001), and both showed good linearity (R2 = 0.9996 for ddPCR; R2 = 0.9984 for real-time PCR) over the detection range. Compared with real-time PCR, ddPCR showed lower intra-assay and interassay CVs for the series of diluted standards, which indicated ddPCR has better repeatability and reproducibility. The limit of detection of ddPCR was lower compared with that of real-time PCR. The combined results suggest that ddPCR is a more promising tool for the detection and quantification of the chimeric antigen receptor transgene copy number.

• Authors: Yaoyao Lou, Caixia Chen, Xiaolu Long, Jia Gu, Min Xiao, Di Wang, Xiaoxi Zhou, Tongjuan Li, Zhenya Hong, Chunrui Li, Jianfeng Zhou, Liting Chen
• Journal: The Journal of molecular diagnostics : JMD (J Mol Diagn) Vol. 22 Issue 5 Pg. 699-707 (05 2020) ISSN: 1943-7811 [Electronic] United States
• PMID: 32409121 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2020 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Chemical References

• Receptors, Chimeric Antigen

Topics

• Flow Cytometry
• Gene Dosage
• Humans
• Immunotherapy, Adoptive
• Real-Time Polymerase Chain Reaction (methods, standards)
• Receptors, Chimeric Antigen (genetics, immunology)
• Reproducibility of Results
• Sensitivity and Specificity
• T-Lymphocytes (immunology, metabolism)
• Transgenes