慢性淋巴细胞白血病（CLL）是一种临床和生物学上异质性的疾病，其结果有所不同。在过去的十年中，下一代测序技术的应用使得对CLL病因相关的疾病特异性基因组、表观遗传、免疫基因和转录因子标记的广泛映射成为可能。这些技术提高了我们对于肿瘤异质性和进化对疾病结果的影响的认识，尽管它们主要是在核酸的大量制备上进行的。作为进一步发展，近年来出现了新技术，可以在单个细胞水平上进行高分辨率的映射。这些包括单细胞RNA测序评估肿瘤微环境中淋巴细胞和非恶性细胞的转录组；B和T细胞受体重排的免疫基因组学分析；单细胞测序方法用于研究甲基化和染色质在基因组中的可访问性；以及针对拷贝数变异和单核苷酸变异的定向单细胞DNA测序进行分析。此外，基于蛋白质表达的细胞亚群的同时分析也可以通过各种抗体方法获得。在本综述中，我们讨论了不同的单细胞测序技术以及迄今为止它们如何应用于研究CLL的发病和进展，包括对治疗的反应。考虑到我们正在从化疗免疫疗法向靶向疗法转变，这后一方面尤为重要，因为它可能对克隆动力学产生不同的影响。我们还讨论了新的可能性，例如综合的多组学分析，以及不同单细胞技术的内在局限性，从样品制备到使用现有的生物信息学流程进行数据解释。最后，我们讨论了这个快速发展的领域的未来方向。版权所有©2023 Oder、Chatzidimitriou、Langerak、Rosenquist和Österholm。

Chronic lymphocytic leukemia (CLL) is a clinically and biologically heterogeneous disease with varying outcomes. In the last decade, the application of next-generation sequencing technologies has allowed extensive mapping of disease-specific genomic, epigenomic, immunogenetic, and transcriptomic signatures linked to CLL pathogenesis. These technologies have improved our understanding of the impact of tumor heterogeneity and evolution on disease outcome, although they have mostly been performed on bulk preparations of nucleic acids. As a further development, new technologies have emerged in recent years that allow high-resolution mapping at the single-cell level. These include single-cell RNA sequencing for assessment of the transcriptome, both of leukemic and non-malignant cells in the tumor microenvironment; immunogenetic profiling of B and T cell receptor rearrangements; single-cell sequencing methods for investigation of methylation and chromatin accessibility across the genome; and targeted single-cell DNA sequencing for analysis of copy-number alterations and single nucleotide variants. In addition, concomitant profiling of cellular subpopulations, based on protein expression, can also be obtained by various antibody-based approaches. In this review, we discuss different single-cell sequencing technologies and how they have been applied so far to study CLL onset and progression, also in response to treatment. This latter aspect is particularly relevant considering that we are moving away from chemoimmunotherapy to targeted therapies, with a potentially distinct impact on clonal dynamics. We also discuss new possibilities, such as integrative multi-omics analysis, as well as inherent limitations of the different single-cell technologies, from sample preparation to data interpretation using available bioinformatic pipelines. Finally, we discuss future directions in this rapidly evolving field.Copyright © 2023 Oder, Chatzidimitriou, Langerak, Rosenquist and Österholm.