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

Abstract

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.

Moving toward a conceptualization of measurable residual disease in myelodysplastic syndromes

“… 3 , 10 MFC-MRD has a limit of detection (LOD) from 0.1% to 0.01% (10 –3 -10 −4 ), although higher sensitivities (10 −5 -10 −6 ) are reported for leukemic stem cell (LSC) detection using immunophenotypic aberrant hematopoietic stem cell (HSC) populations. 11 MFC assessment of different from normal dysplastic maturation 12 , 13 , 14 could supplement MFC-MRD quantitation of aberrant blast or stem cells ( Figure 1 ). 15 However, interpretation of MFC-MRD in MDS may be limited by residual CH-related changes of hematopoietic cells as well as the challenge of discriminating between lower-risk dysplastic clones and leukemic blasts.…”

Subclones with variants of uncertain clinical significance might contribute to ineffective hemopoiesis and leukemia predisposition

“…On the other hand, more than 25% of AML cases do not display somatic mutations in any myeloid neoplasm‐related genes, 11 therefore, additional pathogenetic mechanisms are required for dysplastic hemopoiesis, such as immune dysregulation 12 . However, clinical significance of clonal hemopoiesis without evident cytopenia(s) or marrow dysplasia is still unclear and is categorized under the definitions of clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenia of unknown significance (CCUS) when somatic mutations have a variant allele frequency (VAF) between 2% and 30% 13–15 . Moreover, molecular alterations can be classified in five categories, according to American College of Genetics and Genomics and their roles in a specific disease: benign; likely benign; variant of uncertain significance (VUS); likely pathogenic; and pathogenic 16 .…”

“…12 However, clinical significance of clonal hemopoiesis without evident cytopenia(s) or marrow dysplasia is still unclear and is categorized under the definitions of clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenia of unknown significance (CCUS) when somatic mutations have a variant allele frequency (VAF) between 2% and 30%. [13][14][15] Moreover, molecular alterations can be classified in five categories, according to American College of Genetics and Genomics and their roles in a specific disease: benign; likely benign; variant of uncertain significance (VUS); likely pathogenic; and pathogenic. 16 In particular, a VUS is a variant with insufficient or conflicting evidence on its pathogenic role in a specific disease.…”