Integrated flow cytometry and sequencing to reconstruct evolutionary patterns from dysplasia to acute myeloid leukemia
Catia Simoes 1 , Maria Carmen Chillon 2 , David Martínez-Cuadrón 3 , Maria José Calasanz 4 , María-Belén Vidriales 5 , Iria Vazquez 6 , Montserrat Hernández-Ruano 7 , Beñat Ariceta 8 , Paula Aguirre-Ruiz 9 , Leire Burgos 6 , Diego Alignani 10 , Sarai Sarvide 11 , Sara Villar 8 , Ana Alfonso 12 , Felipe Prosper 13 , Rosa Ayala 14 , Joaquin Martínez-López 15 , Juan Miguel Bergua Burgues 16 , Susana Vives 17 , Jose Antonio Perez-Simon 18 , María García-Fortes 19 , Teresa Bernal Del Castillo 20 , Mercedes Colorado 21 , Mayte Olave 22 , Juan I Rodríguez-Gutiérrez 23 , Jorge Labrador 24 , Marcos González 25 , Jesus San-Miguel 26 , Miguel A Sanz 27 , Pau Montesinos 3 , Bruno Paiva 28
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 workup.
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 sorting (FACS) with next-generation sequencing (NGS), could reconstruct leukemogenesis.
Dysplastic cells were detected by MFC in 285 of 348 (82%) newly-diagnosed AML patients. Presence of dysplasia according to MFC and WHO criteria had no prognostic value in the elderly. NGS of dysplastic cells and blasts isolated at diagnosis identified three evolutionary patterns: stable (n=12/21), branching (n=4/21) and clonal evolution (n=5/21). In patients achieving complete response, 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 approximately 80% of newly diagnosed AML patients, using techniques other than single-cell multiomics.