Validation of a high-sensitivity assay for detection of chimeric antigen receptor T-cell vectors using low-partition digital PCR technology Journal Article
| Authors: | Arcila, M. E.; Patel, U.; Momeni-Boroujeni, A.; Yao, J.; Chan, R.; Chan, J.; Rijo, I.; Yu, W.; Chaves, N.; Patel, H.; Kakadiya, S.; Lachhander, S.; Senechal, B.; Riviere, I. C.; Wang, X.; Sadelain, M.; Nafa, K.; Salazar, P.; Palomba, L.; Curran, K. J.; Park, J. H.; Daniyan, A.; Borsu, L. |
| Article Title: | Validation of a high-sensitivity assay for detection of chimeric antigen receptor T-cell vectors using low-partition digital PCR technology |
| Abstract: | Although in vivo engraftment, expansion, and persistence of chimeric antigen receptor (CAR) T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital PCR assay for ultrasensitive detection of CAR constructs after treatment, circumventing known technical limitations of low-partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene, and Memorial Sloan Kettering CAR constructs, were employed to validate testing on the Bio-Rad digital PCR low-partitioning platform; results were compared with Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500 ng. Using dual-input reactions (20 and 500 ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1 × 10–5 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared with the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6 to 28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005% to 74% (vector versus reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (P < 0.005). Only three patients with undetectable constructs had disease progression at the time of sampling. © 2023 Association for Molecular Pathology and American Society for Investigative Pathology |
| Keywords: | genetics; polymerase chain reaction; t lymphocyte; t-lymphocytes; reproducibility; reproducibility of results; receptors, antigen, t-cell; chimeric antigen receptor; lymphocyte antigen receptor; technology; humans; human; receptors, chimeric antigen |
| Journal Title: | Journal of Molecular Diagnostics |
| Volume: | 25 |
| Issue: | 9 |
| ISSN: | 1525-1578 |
| Publisher: | Elsevier Science, Inc. |
| Date Published: | 2023-09-01 |
| Start Page: | 634 |
| End Page: | 645 |
| Language: | English |
| DOI: | 10.1016/j.jmoldx.2023.06.002 |
| PUBMED: | 37330049 |
| PROVIDER: | scopus |
| PMCID: | PMC10488325 |
| DOI/URL: | |
| Notes: | The MSK Cancer Center Support Grant (P30 CA008748) is acknowledged in the PDF -- Corresponding author is MSK author: Laetitia Borsu -- Source: Scopus |
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