The dynamic evolution of lineage switch under CD19 CAR-T treatment in non-KMT2A rearranged B-ALL patients

Reconstructing the clonal evolution paradigm helps us understand the process of lineage switching [6]. Therefore, we depicted the clonal evolution pattern of patient 1 (P01) through single-cell targeted DNA sequencing (Supplementary Table 3). Through single-cell genomic sequencing and quality control, we obtained a total of 6566 high-quality cells with related gene mutations (FLT3, BCORL1, and STAG2) at the four time points for clone structure inference (Fig. 1B, Supplementary Table 4). The Tapestri insights analysis (Fig. 1B) revealed that the FLT3-ITD mutation consistently persisted at four different time points (83.3%, 69.4%, 98.8%, 9.0%). Pre-existing BCORL1 mutation rapidly expanded after the initiation of myeloid relapse, while STAG2 mutation occurred with the presence of BCORL1 mutation (16.1%, 4.3%, 96.1%, 0.0%). Compared with the T1_Pre_CART time point, we found that the BCORL1 mutation burden (Fig. 1C) increased at the T3_Relapse time point. Moreover, the single-cell membrane protein data (Supplementary Fig. 1A–D, Supplementary Table 5) showed that B-ALL blast cells expressed typical B-ALL-associated markers CD19 and CD10, along with co-expression of myeloid-associated markers CD33 and CD123 at the T1_Pre_CART time point and the blast cells lost the expression of lymphoid marker CD19 at relapse, confirming the phenomenon of lineage switch.

Moreover, the clonal evolution structure of patient 2 (P02) was reconstructed through whole exon sequencing and targeted sequencing (Fig. 1D, Supplementary Table 6). Throughout the treatment course of P02, the expression of fusion gene EP300::ZNF384 persisted, the IKZF2 mutated clone disappeared after CD19 CAR-T therapy, and the BCOR gene mutated clone emerged upon myeloid lineage relapse. Through BCR sequencing, we observed the presence of identical immunoglobulin sequences at the T2_Relapse time point as those in the T1_Pre_CART time point (Fig. 1E, Supplementary Table 7), and we could also observe that the clonal frequency of immunoglobulin sequences increased during relapse, suggesting their enrichment in the myeloid reprogramming process. This result suggested that the origin of myeloid progenitor cells was reprogrammed from B-ALL cells [7].