Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia.

Clonal evolution in acute myeloid leukemia (AML) originates long before diagnosis and is a dynamic process that may affect survival. However, it remains uninvestigated during routine diagnostic workups. We hypothesized that the mutational status of bone marrow dysplastic cells and leukemic blasts, analyzed at the onset of AML using integrated multidimensional flow cytometry (MFC) immunophenotyping and fluorescence-activated cell sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis. Dysplastic cells were detected by MFC in 285 of 348 (82%) newly diagnosed patients with AML. Presence of dysplasia according to MFC and World Health Organization criteria had no prognostic value in older adults. NGS of dysplastic cells and blasts isolated at diagnosis identified 3 evolutionary patterns: stable (n = 12 of 21), branching (n = 4 of 21), and clonal evolution (n = 5 of 21). In patients achieving complete response (CR), integrated MFC and FACS with NGS showed persistent measurable residual disease (MRD) in phenotypically normal cell types, as well as the acquisition of genetic traits associated with treatment resistance. Furthermore, whole-exome sequencing of dysplastic and leukemic cells at diagnosis and of MRD uncovered different clonal involvement in dysplastic myelo-erythropoiesis, leukemic transformation, and chemoresistance. Altogether, we showed that it is possible to reconstruct leukemogenesis in ∼80% of patients with newly diagnosed AML, using techniques other than single-cell multiomics.

Assessment of Minimal Residual Disease by Next Generation Sequencing in Peripheral Blood as a Complementary Tool for Personalized Transplant Monitoring in Myeloid Neoplasms.

Patients with myeloid neoplasms who relapsed after allogenic hematopoietic stem cell transplant (HSCT) have poor prognosis. Monitoring of chimerism and specific molecular markers as a surrogate measure of relapse is not always helpful; therefore, improved systems to detect early relapse are needed. We hypothesized that the use of next generation sequencing (NGS) could be a suitable approach for personalized follow-up post-HSCT. To validate our hypothesis, we analyzed by NGS, a retrospective set of peripheral blood (PB) DNA samples previously evaluated by high-sensitive quantitative PCR analysis using insertion/deletion polymorphisms (indel-qPCR) chimerism engraftment. Post-HCST allelic burdens assessed by NGS and chimerism status showed a similar time-course pattern. At time of clinical relapse in 8/12 patients, we detected positive NGS-based minimal residual disease (NGS-MRD). Importantly, in 6/8 patients, we were able to detect NGS-MRD at time points collected prior to clinical relapse. We also confirmed the disappearance of post-HCST allelic burden in non-relapsed patients, indicating true clinical specificity. This study highlights the clinical utility of NGS-based post-HCST monitoring in myeloid neoplasia as a complementary specific analysis to high-sensitive engraftment testing. Overall, NGS-MRD testing in PB is widely applicable for the evaluation of patients following HSCT and highly valuable to personalized early treatment intervention when mixed chimerism is detected.