ena-STUDY-UNIVERSITY OF TORONTO-28-07-2021-21:06:16:314-693
Single-cell proteogenomic sequencing allows early detection of relapse clone with CN-LOH at FLT3-ITD locus from initial diagnosis in AML
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.
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.