Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry

qPCR assay for detection of Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Elements from CAR-T and TCR-T cells in fresh and formalin-fixed tissue

“…Antibodies specific for detection of CAR-T cells in IHC have been reported, but their sensitivity is unknown, and they are only useful for specific CAR-T constructs [ 29 ]. Prior studies using WPRE RNA ISH and unique CAR-specific sequences have also been reported by us and others [ 23 , 28 ]. The RNA-based WPRE ISH assay previously reported by our group, although broadly applicable and useful for providing spatial information, is inherently less reliable due to the lability of RNA.…”

“…We have interpreted this aggregate information as a positive result for patient 6. In one case (patient 1), the presence of infiltrating CAR-T cells detected by WPRE qPCR was corroborated by flow cytometry analysis of a paired fresh tumor biopsy at the time of tissue sampling (data not shown) and by in situ hybridization (ISH) staining of the same FFPE tissue biopsy [ 23 ].…”

“…The presence or absence of WPRE signal we observed was as expected for the patient samples based on the clinical history and timepoint of treatment with adoptive cellular therapy products. Additionally, the FFPE biopsy sample from patient 1 was selected for our assay validation based on confirmation in prior studies to have had CAR-T cells present using WPRE ISH [ 23 ]. This allowed for a comparison of our results with previously published imaging, to serve as orthogonal confirmation that this biopsy did contain infiltrating CAR-T cells.…”

“…There are several assays used to detect CAR-T/TCR-T cells in tissue reported in previous literature, which include detection by flow cytometry, ISH, and immunohistochemistry (IHC) [ 2 , 23 , 25 – 28 ]. Flow cytometric analysis allows for the identification and characterization of the CAR-T/TCR-T cell subpopulation and the patient’s own immune cells using peripheral blood samples.…”

93 PCR assay to identify woodchuck hepatitis virus posttranscriptional regulatory element in CAR-T and TCR-T cells for fresh and formalin-fixed samples

“…1 Producing CART and TCRT cells involves transduction with viral vectors that usually contain the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) to enhance transcription. 2 As adoptive cellular therapies become more widely used in cancer care, adverse events (AE) and safety monitoring questions have been raised requiring methods to quantify the amount of CART and TCRTs in patient tissues. We developed a PCR assay to target WPRE for use on archival formalin-fixed, paraffin embedded (FFPE) tissues with the goal of providing valuable information regarding CART/ TCRT localization and AEs as most engineered cell products used at our institution are transduced with a viral vector containing WPRE.…”