Mutations in myeloid transcription factors and activated signaling genes predict chronic myeloid leukemia outcomes.

Advancements in genomics are transforming the clinical management of chronic myeloid leukemia (CML) towards precision medicine. The impact of somatic mutations on treatment outcomes is still under debate. We studied the association of somatic mutations in epigenetic modifiers genes and activated signaling/myeloid transcription factor (AS/MTF), with disease progression and treatment failure in CML patients following tyrosine kinase inhibitor (TKI) therapy. A total of 394 CML samples were sequenced, including 254 samples collected at initial diagnosis, and 140 samples taken during follow-up. Single-molecule molecular inversion probe (smMIP)-based next-generation sequencing (NGS) was conducted targeting recurrently mutated loci in 40 genes with a limit of detection of 0.2%. A total of 70 mutations were detected in 57 (22.4%) diagnostic samples, while 64 mutations were detected in 39 (27.9%) of the follow-up samples. Carrying any mutation at initial diagnosis was associated with worse outcomes following TKI therapy, particularly in AS/MTF genes. Patients having these mutations at initial diagnosis and treated with Imatinib showed higher risks of treatment failure (HR 2.53, 95% CI [1.13-5.66], p=0.0239). The adverse prognostic impact of the mutations was not clear for patients treated with second-generation TKIs (2G-TKI). The multivariate analysis affirmed that mutations in AS/MTF genes independently serve as adverse prognostic factors for molecular response, failure-free survival (FFS), and progression risk. Additionally, there was an observable non-significant trend indicating a heightened risk of progression to advanced disease and worse overall survival (OS). Conclusion: Mutations in the AS/MTF genes using smMIP-based NGS can help identify patients with a potential risk of both treatment failure and progression, even from initial diagnosis, and may help upfront TKI selection.

Single cell proteogenomic sequencing identifies a relapse-fated AML subclone carrying FLT3-ITD with CN-LOH at chr13q.

Internal tandem duplication of the Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FLT3-ITD) is one of the most clinically relevant mutations in acute myeloid leukemia (AML), with a high FLT3-ITD allelic ratio (AR) (≥0.5) being strongly associated with poor prognosis. FLT3-ITDs are heterogeneous, varying in size and location, with some patients having multiple FLT3-ITDs. Bulk cell-based approaches are limited in their ability to reveal the clonal structure in such cases. Using single-cell proteogenomic sequencing (ScPGseq), we attempted to identify a relapse-fated subclone in an AML case with mutations in WT1, NPM1, and FLT3 tyrosine kinase domain and two FLT3-ITDs (21 bp and 39 bp) (low AR) at presentation, then relapsed only with WT1 and NPM1 mutations and one FLT3-ITD (high AR). This relapse-fated subclone at presentation (∼2.1% of sequenced cells) was characterized by the presence of a homozygous 21 bp FLT3-ITD resulting from copy neutral loss of heterozygosity (CN-LOH) of chr13q and an aberrant, immature myeloid cell surface signature, contrast to the cell surface phenotype at presentation. In contrast to results from multicolor flow-cytometry, ScPGseq not only enabled the early detection of rare relapse-fated subclone showing immature myeloid signature but also highlighted the presence of homozygous 21 bp FLT3-ITDs in the clone at presentation.