Single-cell proteogenomic sequencing allows early detection of relapse clone with CN-LOH at FLT3-ITD locus from initial diagnosis in AML. Clinical relevance of single-cell proteogenomic sequencing in an FLT3-ITD+ AML case

Internal tandem duplication of the FMS-like tyrosine kinase 3 gene (FLT3-ITD) is one of the most clinically relevant mutations in acute myeloid leukemia (AML). A high FLT3-ITD allelic ratio (AR) (>0.5) is strongly associated with poor prognosis. FLT3-ITDs are heterogeneous mutations, varying in sizes and locations with some patients having multiple FLT3-ITDs. There are conflicting reports on whether these characteristics of FLT3-ITDs can be used to improve risk stratification. Unfortunately, conventional clinical techniques such as quantitative PCR are often not adequate in identifying these characteristics at sufficient resolution. Using single-cell proteogenomic sequencing, we characterized an AML case with hotspot mutations in NPM1 and FLT3 tyrosine kinase domain and two FLT3-ITDs at diagnosis, while only the NPM1 mutation and one FLT3-ITD were present at relapse. Through this approach, we identified the clonal architecture including two FLT3-ITDs at diagnosis (21bp and 39bp) and dynamics and evolution of identified subclones. Incorporating DNA mutations and cell surface phenotype from diagnosis and relapse, we mapped the evolution of the different clones over time and could identify a subclone at diagnosis that eventually gave rise to relapse, which harbors homozygous 21bp FLT3-ITD through copy neutral loss-of-heterozygosity at chr13q and shows immature myeloid cell signature. Overall, the current study demonstrates that proteogenomic single-cell sequencing is a promising approach that enables simultaneous analyses of complex FLT3-ITDs, their co-occurring genomic lesions, and cell surface phenotype at the single-cell level, and begins to explain why some AML cases with a low FLT3-ITD AR have an adverse outcome.

FMS样酪氨酸激酶3基因（FMS-like tyrosine kinase 3, FLT3）的内部串联重复（internal tandem duplication, ITD）是急性髓系白血病（acute myeloid leukemia, AML）中临床相关性最高的突变类型之一。高FLT3-ITD等位基因比率（allelic ratio, AR）（>0.5）与不良预后显著相关。FLT3-ITD属于异质性突变，其片段长度与插入位点存在差异，部分患者可携带多个FLT3-ITD。目前关于FLT3-ITD的上述特征能否用于优化风险分层的研究结论尚存争议。遗憾的是，常规临床检测技术如定量PCR（quantitative PCR）往往无法提供足够分辨率以识别这些特征。本研究通过单细胞蛋白质基因组测序（single-cell proteogenomic sequencing），对1例初诊时携带NPM1热点突变、FLT3酪氨酸激酶结构域突变以及2个FLT3-ITD的AML病例进行了表征；该患者复发时仅携带NPM1突变与1个FLT3-ITD。通过该技术手段，我们解析了其克隆结构（初诊时存在2个FLT3-ITD，分别为21bp与39bp）以及各亚克隆的动态演化过程。结合初诊与复发阶段的DNA突变信息与细胞表面表型，我们绘制了不同克隆的随时间演化轨迹，并鉴定出1个初诊时的亚克隆：该亚克隆最终导致复发，其携带来源于13号染色体长臂（chr13q）拷贝中性杂合性缺失的纯合21bp FLT3-ITD，且呈现未成熟髓系细胞特征。总体而言，本研究证实，蛋白质基因组单细胞测序是一种极具潜力的技术，可在单细胞层面同时分析复杂的FLT3-ITD、其伴随的基因组病变以及细胞表面表型，并初步阐明了部分FLT3-ITD等位基因比率较低的AML病例为何仍存在不良预后的原因。