BTK抑制剂、Bcl-2抑制剂以及其他靶向治疗药物显著改善了慢性淋巴细胞白血病（CLL）患者的预后。随着存活率的增加，监测疾病和解析对这些药物的潜在耐药机制对于制定有效的治疗策略至关重要。我们采用双重测序技术，该技术可以检测到超低等位基因频率下的突变，以鉴定与CLL药物耐药有关的五个基因中的突变，并跟踪这些突变在接受多种靶向治疗且最终在哌替布替尼治疗过程中出现疾病进展的两名患者中的演变情况。在这两名患者中，我们检测到扩增并达到显著癌细胞分数（CCF）的变异。在R001患者中，尽管在曾接受过扎奴布替尼（BTK p.L528W、p.C481S和PLCG2 S707F、L845F、R665W以及D993H）治疗后出现进展，我们仍检测到多个已知的耐药突变同时发生在BTK和PLCG2基因中。相比之下，R002患者在阿卡布鲁替尼治疗后出现了多个BTK突变，包括已知的耐药突变p.C481R、p.T474I和p.C481S。我们发现哌替布替尼可以抑制，但无法完全根除这两名患者的BTK p.C481S突变，但在接受哌替布替尼治疗期间出现了PLCG2的其他耐药突变以及新的BTK突变。例如，R001患者的BTK p.L528W的频率增加了1000倍以上（从CCF 0.02%到35%），R002患者的BTK p.T474I的CCF从0.03%增加到4.2%（超过100倍）。我们的数据揭示了在不同靶向治疗的选择性压力下，耐药克隆在患者疾病过程中的进化动态。

BTK inhibitors, Bcl-2 inhibitors, and other targeted therapies have significantly improved outcomes for patients with chronic lymphocytic leukemia (CLL). With increased survivorship, monitoring disease and deciphering potential mechanisms of resistance to these agents are critical for devising effective treatment strategies. We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib. In both patients, we detected variants that expanded and reached significant cancer cell fraction (CCF). In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S, PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment including known resistance mutations p.C481R, p.T474I and p.C481S. We found that pirtobrutinib was able to suppress, but not completely eradicate, BTK p.C481S mutations in both patients, but other resistance mutations such as mutations in PLCG2 and new BTK mutations increased while receiving pirtobrutinib. For example, BTK p.L528W in patient R001 increased in frequency more than 1000-fold (from CCF 0.02% to 35%), and p.T474I in patient R002 increased in CCF from 0.03% to 4.2% (more than 100-fold). Our data illuminates the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.