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].

重建克隆进化范式有助于我们了解谱系转换的过程 [6]。因此，我们通过单细胞靶向 DNA 测序描述了患者 1 （P01） 的克隆进化模式（补充表 3）。通过单细胞基因组测序和质量控制，我们在克隆结构推断的四个时间点共获得了 6566 个具有相关基因突变 （FLT3 、 BCORL1 和 STAG2） 的高质量细胞 （图 1B，补充表 4）。Tapestri 见解分析（图 1B）显示，FLT3-ITD 突变始终持续在四个不同的时间点 （83.3%、69.4%、98.8%、9.0%）。髓系复发开始后，先前存在的 BCORL1 突变迅速扩大，而 STAG2 突变发生在 BCORL1 突变的存在 （16.1% 、 4.3% 、 96.1% 、 0.0% 。与T1_Pre_CART时间点相比，我们发现 BCORL1 突变负荷 （图 1C） 在 T3_Relapse 时间点增加。此外，单细胞膜蛋白数据（补充图 1A-D，补充表 5）显示 B-ALL 原始细胞表达典型的 B-ALL 相关标志物 CD19 和 CD10，以及髓系相关标志物 CD33 和 CD123 的共表达T1_Pre_CART时间点，原始细胞在复发时失去了淋巴标志物 CD19 的表达，证实了谱系转换现象。

此外，通过全外显子测序和靶向测序重建患者 2 （P02） 的克隆进化结构 （图 1D，补充表 6）。在 P02 的整个治疗过程中，融合基因 EP300：：ZNF384 的表达持续存在，IKZF2 突变克隆在 CD19 CAR-T 治疗后消失，BCOR 基因突变克隆在髓系复发时出现。通过 BCR 测序，我们观察到T2_Relapse时间点存在与 T1_Pre_CART 时间点相同的免疫球蛋白序列（图 1E，补充表 7），我们还可以观察到免疫球蛋白序列的克隆频率在复发期间增加，表明它们在骨髓重编程过程中富集。这一结果表明，髓系祖细胞的起源是从 B-ALL 细胞重新编程的 [7]。