Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

Abstract

Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.

Single-cell multiomics dissection of basal and antigen-specific activation states of CD19-targeted CAR T cells

“…With the advent of revolutionized single-cell mRNA sequencing (scRNA-seq), it is now possible to characterize the full spectrum of immune cell functional states and gene programs in a comprehensive and unbiased manner. Although not fully used, several reports have leveraged scRNA-seq to compare the transcriptional states of 4-1BB and CD28 CAR T cells, 11 to understand the clonal kinetics and transcriptional programs preinfusion and postinfusion, 12 to identify pre-existing CD19-negative subclones, 13 to reveal subpopulations, stimulation, and exhaustion signatures, 14 and to track dynamic development of CAR T cell dysfunction in clinical samples. 15 Yet despite some transcriptomic features have been identified associated with the clinical observations, 16 17 how the constitutional properties of these cellular products mediate therapeutic activities is incompletely understood.…”

Epigenetic regulator genes direct lineage switching in MLL-AF4 leukaemia

“…Our finding that frontline chemotherapy itself may contribute to relapse highlights the urgent need to find alternative therapies for this high-risk leukaemia. Equally, however, the associated loss of B cell surface markers (e.g., CD19) provides an alternative mechanism for relapse following CAR-T cell or blinatumomab therapy (Gardner et al, 2016;Rayes, McMasters and O'Brien, 2016) in addition to mutations, alternative splicing (Sotillo et al, 2015;Orlando et al, 2018;Rabilloud et al, 2021) and T cells trogocytosis (Hamieh et al, 2019). Whilst these therapies target lineage specific surface markers, lineage-switched (pre-)leukaemic progenitor populations escape epitope recognition and provide a potential clonal source for the relapse.…”

Exploiting Single-Cell Tools in Gene and Cell Therapy

“…While a number of groups have begun to use single-cell approaches to dissect various aspects of CAR T cell-based therapy (129,157,158), the gene therapy field has not explored this to the same extent. That said, a handful of studies have used bulk sequencing approaches to examine post-transplantation clonal dynamics in a small number of patients (159)(160)(161).…”

“…While groups reported acquired CD19 loss-of-function mutations ( 126 ) and abnormal splicing events leading to loss of CD19 expression ( 127 , 128 ), the specific origin of CD19 - cancer cells was not clear. A recent paper using single-cell techniques provides evidence that in at least some patients, treatment-resistant CD19 - cancer cells exist prior to treatment ( 129 ), underscoring the vital role of single-cell approaches pinpointing the mechanisms by which cancer cells escape treatment and informing strategies targeting refractory disease.…”

“…This finding demonstrates the value of single-cell approaches in generating essential information that can then be fed back into clinical practice. Another recent publication applying single-cell technologies reported that the disease-driving clone observed in one patient’s relapsed B cell acute lymphoblastic leukemia existed prior to anti-CD19 CAR T treatment ( 129 ). Taken together, these studies clearly illustrate how single cell-based datasets can provide clinically relevant insights into various aspects of the cell therapy process ( Figure 2 ).…”