CAR T-cell detection scoping review: an essential biomarker in critical need of standardization

Abstract

The expansion and persistence of chimeric antigen receptor (CAR) T-cells in patients are associated with response, toxicity, and long-term efficacy. As such, the tools used to detect CAR T-cells following infusion are fundamental for optimizing this therapeutic approach. Nevertheless, despite the critical value of this essential biomarker, there is significant variability in CAR T-cell detection methods as well as the frequency and intervals of testing. Furthermore, heterogeneity in the reporting of quantitative data adds layers of complexity that limit intertrial and interconstruct comparisons. We sought to assess the heterogeneity of CAR T-cell expansion and persistence data in a scoping review using the PRISMA-ScR checklist. Focusing on 21 clinical trials from the USA, featuring a Food and Drug Administration-approved CAR T-cell construct or one of its predecessors, 105 manuscripts were screened and 60 were selected for analysis, based on the inclusion of CAR T-cell expansion and persistence data. Across the array of CAR T-cell constructs, flow cytometry and quantitative PCR were identified as the two primary techniques for detecting CAR T-cells. However, despite apparent uniformity in detection techniques, the specific methods used were highly variable. Detection time points and the number of evaluated time points also ranged markedly and quantitative data were often not reported. To evaluate whether subsequent manuscripts from a trial resolved these issues, we analyzed all subsequent manuscripts reporting on the 21 clinical trials, recording all expansion and persistence data. While additional detection techniques–including droplet digital PCR, NanoString, and single-cell RNA sequencing–were reported in follow-up publications, inconsistencies with respect to detection time points and frequency remained, with a significant amount of quantitative data still not readily available. Our findings highlight the critical need to establish universal standards for reporting on CAR T-cell detection, especially in early phase studies. The current reporting of non-interconvertible metrics and limited provision of quantitative data make cross-trial and cross-CAR T-cell construct comparisons extremely challenging. Establishing a standardized approach for collecting and reporting data is urgently needed and would represent a substantial advancement in the ability to improve outcomes for patients receiving CAR T-cell therapies.

The Clinical Significance of Circulating Lymphocytes Morphology in Diffuse Large B-Cell Lymphoma As Determined by a Novel, Highly Sensitive Microscopy

“…Furthermore, our finding demonstrating a positive correlation between day 5 CAR T measurement and the achievement of CR corresponds with clinical trials based on molecular methods [6,7,[34][35][36]. It is worth mentioning that previous studies, evaluating the cellular kinetics of Tisa-cel in B-cell ALL and chronic lymphocytic leukemia (CLL) transfused patients, showed that most circulating lymphocytes in the first days after infusion are CAR Ts [27]. Furthermore, the kinetics of atypical lymphocytes post-Tisa-cel (in pediatric ALL patients) had morphological changes that were distinct from other cell lineage lymphopoiesis or maturation, thus inferred to be CAR T [17].…”

“…We found a weak correlation (n = 10, r = 0.67, p = 0.034) between the time to peak of CAR Ts in the PBSs to the CAR+ cells number as measured by flow cytometry on day 7 post-transfusion. A recent review article indicated that current methodologies of CAR T cells' quantification are extremely heterogeneous and difficult to compare and standardize [27].…”

A Panorama of Immune Fighters Armored with CARs in Acute Myeloid Leukemia

“…A good correlation of qPCR results has been shown for detection of the CAR-protein on the surface of T cells with flow cytometry [42][43][44]. In a comprehensive review of the literature from Turicek et al, the authors found that flow cytometry (used in around 43% of trials) and qPCR (used in around 57%) were the most commonly employed methods of CAR detection [45]. The authors also highlighted the need of universal approaches in detection techniques and time points as well as in the reporting of quantitative data [45].…”

“…In a comprehensive review of the literature from Turicek et al, the authors found that flow cytometry (used in around 43% of trials) and qPCR (used in around 57%) were the most commonly employed methods of CAR detection [45]. The authors also highlighted the need of universal approaches in detection techniques and time points as well as in the reporting of quantitative data [45]. While B-cell aplasia has been widely used to monitor the persistence of CD19-CAR-T cells, there is no such way to define "functional persistence" in AML, which necessitates the optimization of detection approaches for monitoring CAR-T cells in the blood.…”

Apheresis collection of mononuclear cells for chimeric‐antigen receptor therapies

“…Lack of standardization is readily apparent when considering the variety of culture systems currently used in the manufacturing process 62 . Similarly, there is heterogeneity in flow cytometric and polymerase chain reaction‐based methodologies used to detect CAR T cells post‐infusion and establish how frequently they are used 63 . However, the implementation of mass production steps seeking uniformity for therapies that are inherently examples of personalized precision medicine will represent a continuous challenge in the field 64 …”