Novel Multiplex dPCR Assay Strengthens CAR-T Therapy Monitoring

Research study background

Chimeric antigen receptor T (CAR-T) cell therapies have transformed the treatment landscape for B-cell malignancies and are now being studied in early phase clinical trials for other hematologic and solid tumors. At Children’s Hospital Colorado, physician-scientists in the Center for Cancer and Blood Disorders are driving these advances from bench to bedside, with novel discoveries that continue to shape CAR-T therapeutic strategies.

In a brief report published in Molecular Therapy Oncology, pediatric hematology and oncology experts at Children’s Colorado describe their work to improve and streamline monitoring of CAR-T persistence after infusion — a key predictor of short-term efficacy and long-term survival. Digital polymerase chain reaction (dPCR) detects the CAR-T transgene more accurately and with greater ease than quantitative PCR, but it provides an incomplete picture of a patient’s immune status.

Variations in test sensitivity, accuracy and reagents across CAR-T designs further complicate study comparisons, especially given rapid post-infusion changes. These methods also burden pediatric patients, who face repeated, potentially invasive large-volume blood draws. To address these challenges, the team developed a novel T cell receptor (TCR) dPCR assay that multiplexes with CAR-T-specific and control gene dPCR assays and enables simultaneous quantitation of CAR-T cells, total T cells and total nucleated cells from a small blood sample.

“With this new assay, not only can clinicians measure how many CAR-T cells are circulating in a patient's blood but also they can measure total T cells with the exact same sample and the exact same test,” says Amanda C. Winters, MD, PhD, pediatric hematologist-oncologist at Children’s Colorado and lead study author. “This saves money and helps patients who have very low white blood cell counts, which often happens early after CAR-T infusion.”

Researchers tested the new assay across mixed samples of T cells, including cell-line dilutions, peripheral samples from healthy donors and enriched T cells, demonstrating a strong correlation between reference measurements and measured results. When tested against blood samples from patients treated on clinical trials with the CAR-T construct UCD19 for total T cells and CAR-T cells, assay results strongly correlated with CF. These experiments proved the TCR dPCR assay was not only accurate, but compatible across multiple CAR-T constructs and enabled integrated readouts with minimal sample input. While there were some study limitations, none meaningfully impacted assay performance. Results demonstrated that TCR dPCR allows for a minimal volume of blood to provide maximal information about immune status and CAR-T numbers.

Clinical implications

The TCR dPCR assay is already being used to monitor pediatric and adult CAR-T clinical trials supported by the Gates Institute at the University of Colorado Anschutz Medical Campus as part of an ongoing cross-campus collaboration with Children’s Colorado. With the study’s publication, this real-time testing method is also available for integration into other CAR-T trial sites.