幼年型髓系增殖性疾病性白血病（JMML）是一种少见的早期儿童骨髓增生性疾病，其发展是由于RAS信号通路基因突变。下一代高通量测序（NGS）可以识别各种次生分子遗传事件，这些事件可以促进JMML的进展，并转化为次生急性髓系白血病（sAML）。单细胞DNA测序（scDNA-seq）的方法可以克服大规模NGS的局限性，探索个体细胞水平的遗传异质性，这有助于更好地理解导致JMML进展的机制，并提供评估白血病对治疗反应的机会。在本研究中，我们采用两步式液滴微流控方法，检测了数千个单个细胞中的DNA变异，并分析了两名JMML患者在造血干细胞移植（HSCT）前后sAML转化过程中的克隆动力学。在诊断时，我们的两名患者仅携带“典型的”RAS信号通路基因突变，该突变通过靶向DNA测序进行检测。对JMML转化为sAML的样本进行分析后，发现了额外的遗传事件，这些事件可能是患者疾病进展的潜在驱动因素。scDNA-seq能够测量嵌合水平，并在HSCT后在第二位患者中检测到残留肿瘤克隆（肿瘤细胞敏感性小于0.1%）。所获得的数据表明，scDNA-seq评估JMML到sAML的克隆进化、治疗反应和移植程度监测的价值。 ©2023. Springer Nature Limited.

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disease of early childhood that develops due to mutations in the genes of the RAS-signaling pathway. Next-generation high throughput sequencing (NGS) enables identification of various secondary molecular genetic events that can facilitate JMML progression and transformation into secondary acute myeloid leukemia (sAML). The methods of single-cell DNA sequencing (scDNA-seq) enable overcoming limitations of bulk NGS and exploring genetic heterogeneity at the level of individual cells, which can help in a better understanding of the mechanisms leading to JMML progression and provide an opportunity to evaluate the response of leukemia to therapy. In the present work, we applied a two-step droplet microfluidics approach to detect DNA alterations among thousands of single cells and to analyze clonal dynamics in two JMML patients with sAML transformation before and after hematopoietic stem cell transplantation (HSCT). At the time of diagnosis both of our patients harbored only "canonical" mutations in the RAS signaling pathway genes detected by targeted DNA sequencing. Analysis of samples from the time of transformation JMML to sAML revealed additional genetic events that are potential drivers for disease progression in both patients. ScDNA-seq was able to measure of chimerism level and detect a residual tumor clone in the second patient after HSCT (sensitivity of less than 0.1% tumor cells). The data obtained demonstrate the value of scDNA-seq to assess the clonal evolution of JMML to sAML, response to therapy and engraftment monitoring.© 2023. Springer Nature Limited.