Next-Generation Sequencing of Minimal Residual Disease for Predicting Relapse after Tisagenlecleucel in Children and Young Adults with Acute Lymphoblastic Leukemia.

We assessed minimal residual disease (MRD) detection and B-cell aplasia after tisagenlecleucel therapy for acute lymphoblastic leukemia (ALL) to define biomarkers predictive of relapse (N = 143). Next-generation sequencing (NGS) MRD detection >0 in bone marrow (BM) was highly associated with relapse. B-cell recovery [signifying loss of functional chimeric antigen receptor (CAR) T cells] within the first year of treatment was associated with a hazard ratio (HR) for relapse of 4.5 [95% confidence interval (CI), 2.03-9.97; P < 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD >0 (HR = 4.87; 95% CI, 2.18-10.8; P < 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44-7.69; P = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87-50; P < 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33-4.79; P = 0.7). Relapses occurring with persistence of B-cell aplasia were largely CD19- (23/25: 88%). Detectable BMNGS-MRD reliably predicts risk with sufficient time to consider approaches to relapse prevention such as hematopoietic cell transplantation (HCT) or second CAR-T cell infusion. SIGNIFICANCE: Detectable disease by BMNGS-MRD with or without B-cell aplasia is highly predictive of relapse after tisagenlecleucel therapy for ALL. Clonotypic rearrangements used to follow NGS-MRD did not change after loss of CD19 or lineage switch. High-risk patients identified by these biomarkers may benefit from HCT or investigational cell therapies.See related commentary by Ghorashian and Bartram, p. 2.This article is highlighted in the In This Issue feature, p. 1.

Monitoring of tisagenlecleucel transgene DNA using a quantitative polymerase chain reaction assay.

Chimeric antigen receptor (CAR)-T cell therapies reprogram T cells to engage and eliminate cancer cells. Patients' T cells are transduced in vitro using lentiviral or retroviral vectors containing a CAR transgene. Following infusion, CAR-T cells expand in vivo and may persist in the peripheral blood and bone marrow for years. Therefore, monitoring in vivo copies of the CAR transgene requires highly sensitive, validated analytical methods. Herein, we describe the validation of a qPCR assay to detect tisagenlecleucel transgene in patient samples. The limit of detection and lower limit of quantitation were 3.1 and 10 copies/200 ng genomic DNA, respectively, equivalent to ∼50 copies/µg genomic DNA and in alignment with US Food and Drug Administration guidance on bioanalytical method validation. The assay allowed quantitation of the tisagenlecleucel transgene over a wide dynamic range with a high degree of linearity, that is, 101-106 copies/200 ng genomic DNA (R2 ≥ 0.9988). Coefficients of variation of measured transgene copies ranged from 0.2% to 12.8%. A droplet digital PCR assay was performed as a method of validation and showed a strong correlation with the qPCR assay (R2 = 0.9980, p < 0.0001). This qPCR assay is being utilized to monitor tisagenlecleucel expansion and persistence in clinical trials.

Monitoring CAR-T cell kinetics in clinical trials by multiparametric flow cytometry: Benefits and challenges.

Exceptional clinical responses produced by the first chimeric antigen receptor T [CAR-T] cell therapies, and their entry into commercial markets prompted a logarithmic increase in the number of next generation CAR-T clinical trials. As a result, there is a growing interest in understanding the analytical approaches utilized for reliable monitoring of these "living" drugs, and the challenges encountered during their clinical development. Multiparametric flow cytometry (MFC) assays have played a crucial role in understanding the phenotype and function of first approved CAR-T therapies. Herein, three main areas for monitoring CAR-T therapies in clinical trials are discussed: (1) analytical considerations critical for development of MFC assays for the reliable enumeration of CAR-T levels, (2) operational challenges associated with clinical trial sampling and transportation, and (3) differential cellular kinetics observed by MFC and qPCR analyses and their relationship with efficacy (measurable residual disease levels). Initial experiences described here may enable design of fit-for-purpose tools and help to more rapidly advance the development of next generation CAR-T therapies.