Chimeric antigen receptor T-cell (CART) and T-cell receptor (TCRT) therapy are immunotherapies developed by modifying immune cells to target cancer. Producing CART and TCRT cells involves transduction with viral vectors that usually contain the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). As adoptive cellular therapies become more widely used in cancer care, adverse events (AE) and safety monitoring questions have been raised. We developed a PCR assay to target WPRE for use on archival formalin-fixed, paraffin embedded (FFPE) tissues with the goal evaluating CART/TCRT localization in patient tissues which are taken for routine clinical care. We designed WPRE primers to generate short amplicon for use on FFPE tissues. Limit-of-detection experiments confirmed that our PCR protocol detects CART/TCRT constructs for an institutionally-manufactured CD19 CART, and HA-1, and MAGEA1 TCRT products. The limit-of-detection concentration for the CD19 CART was 1000 copies of WPRE DNA/10uL and MAGEA1 at 100 copies/10uL. In fresh samples, we detected CART DNA at 0.0001% dilution (CAR-T cells into PBMC), and TCRT DNA at 0.001%. For extracted FFPE DNA from cells, we detected WPRE in all four product samples of various CART/TCRT admixtures confirmed by in-situ hybridization. We have also detected WPRE sequences in the FFPE DNA of a pre-clinical humanized mouse sample with MAGEA1 TCRT and a clinical lymphoma sample containing CD19 CART. We are in the process of testing our assay on archival patient samples. Detecting CART/TCRT cells in FFPE samples using a WPRE PCR assay is a novel yet simple technique suitable for clinical application in understanding AEs of immunotherapy. In detecting CART and TCRT in FFPE, we are now able to study trafficking of these immune effector cells when AEs occur or when post-treatment biopsies are available.

Cutaneous graft-versus-host disease (cGVHD) is the leading cause of morbidity and mortality post-allogeneic hematopoietic cell transplantation (HCT). There is general uncertainty about the utility and safety of skin biopsy for diagnosing cGVHD in the early post-transplant period. In collaboration with UW Medicine and Mayo Clinic, I conducted a retrospective analysis of skin biopsies performed within 100 days post-HCT between 2000 and 2014. 740 biopsies from 602 patients (356 male, 256 female) were included in analysis. 87.1% (n=525) were white. The age range was 19-75y, with a mean of 50.3y. 456 (61.6%) skin biopsies were performed on inpatients, and 284 (38.3%) on outpatients. Only 8 (1.3%) patients had documented biopsy complications. The primary complication was excessive bleeding, which resolved after application of pressure bandage. Under the guidance of dermatopathologist Dr. Michi Shinohara, I conducted analyses to explore demographic and hematological features that may influence patient care or increase risk for biopsy complications. For example, on average, Hispanic patients received biopsies 8 days later following rash onset compared to their non-Hispanic counterparts. I am currently looking into clinical approach differences between Hispanic and non-Hispanic patients, such as complication documentation/assessment variability and presence of additional consultations. With respect to blood features, the mean neutrophil count was 0.228 K/µL and 2.98 K/µL and the mean platelet count was 18.7 K/µL and 100.6 K/µL for patients with and without biopsy complications, respectively. The complication rate for patients with either extremely low neutrophils (<0.11 K/µL) or platelets (<20 K/µL) was 5.3%. We conclude that skin biopsies performed in the immediate post-HCT period have a very low serious complication rate, even in patients with low cell counts. When skin biopsies are otherwise medically indicated in this patient population, concern regarding skin biopsy safety should not deter performance of this procedure in this patient population.