SP12 | MOLECULAR ALTERATIONS AND LEUKEMIC SUBCLONES BY FLOW CYTOMETRY: WHAT IS THE MEANING OF SUBCLONAL HEMATOPOIESIS?

Background. Clonal evolution is mainly defined based on appearance or expansion of clones harboring certain somatic mutations and/or cytogenetic abnormalities, while few studies have also investigated flow cytometry immunophenotyping variations associated with disease progression. In this study, flow cytometry immunophenotyping of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) with increased blast-2, and leukemic cells were characterized to identify the presence of subclones based on marker expression below the cut-off. Once identified, these clones were monitored by flow cytometry during follow-up, and these clone frequencies were correlated with variant allele frequency (VAF) of somatic mutations found by NGS at the same time point. Methods. Bone marrow (BM) specimens were collected in ethylenediaminetetraacetic acid tubes for immunophenotyping obtained from patients followed at the Hematology and Transplant Center, University Hospital “San Giovanni di Dio e Ruggi d’Aragona”, Salerno, Italy, after informed consent obtained in accordance with the Declaration of Helsinki and protocols approved by our local Ethic Committee. Next-generation sequencing (NGS) will be performed using SOPHIA Genetics Myeloid Solution panel (Sophia Genetics, Saint Sulpice, Switzerland), covering 30 genes, following manufacturer’s instructions, and analysis will be carried out as previously described. For immunophenotyping, 50 μL of fresh heparinized whole peripheral blood or bone marrow specimen will be stained with specific antibodies for characterization, as previously described. Results and Conclusions. A total of 38 consecutive patients diagnosed with AML or MDS with increased blast-2 followed at the Hematology and Transplant Center of Salerno from January 2024 to December 2024 were included. For 19 of them, flow cytometry was also available at disease re-evaluation and/or relapse. We showed that the immunophenotype of leukemic cells might greatly vary at disease relapse and certain phenotypic signatures might suggest a more aggressive disease.