Despite recent advances and development of new therapeutic targets and personalized medicine, acute myeloid leukemia (AML) treatment remains defiant. Identification of residual leukemic cells (measurable residual disease -MRD) by Multiparametric Flow Cytometry has been a fast and effective tool, but disease heterogeneity makes MRD assessment a clinical challenge, especially in cases with uncommon features, needing clinical and technical expertise to be accurately performed and interpreted.

To emphasize how absent CD33 expression impacts on interpreting MDR in AML.

We report three clinical cases of AML with absent CD33 assessed for FLT3 and NPM1 mutation, RUNX1/RUNX1T1 and CBFB-MYH11 rearrangements, karyotype and an 8-color panel immunophenotyping (Table 1). Empty gating strategy was used to identify LAIPs (Leukemia Associated-Immunophenotype), different from normal and leukemic stem cells, compared to healthy and regenerating non-AML bone marrow (BM) from diagnosis and follow up (Figures 1 and 2). Assays were performed at the Hematology Laboratory of Ribeirão Preto the Medical School, University of São Paulo, in accordance to Local Ethical Boards.

Patient's demographic and clinical data are summarized on Table 2. MFC revealed completely negative CD33 from diagnosis to follow-ups. Treatment consisted of 2 induction cycles (cycle 1: 3 days of Daunorubicin 60 mg/m2 and 7 days of cytarabine 200 mg/m2; cycle 2: 6 days of cytarabine 1 g/m2 twice a day). Consolidation consisted of 1 or 2 cycles of 6 days of cytarabine 1 g/m2 twice a day. BMs were obtained at diagnosis, after 1st and 2nd induction, after 1st and 2nd Consolidation, and 3, 9, 12 and 15 months after consolidation of chemotherapy. Even with different number of follow-ups for each patient, time analysis was preserved. Complete remission (complete or incomplete) was achieved by day 30 after 1st induction and no change in the outcome was reported, even though an altered maturational phenotype persisted with complete absent CD33 expression, during and after treatment, suggesting a very rare polymorphism of CD33 receptor that mimicked MRD positivity. Since CD33 is a common myeloid antigen expressed on malignant blasts in AML, it is prominent evaluate and carefully analyze this marker, once is known that polymorphisms, for example, rs12459419 can affect CD33-antibody conjugated based therapy. CD33low blasts are associated to a more mature AML and its high expression is related to adverse landscapes in pediatric AML, highlighting the importance of CD33 evaluation. Regarding vulnerable steps of MRD assessment, Post –analytical phase must embrace a set of standards in order to prevent report errors. Here we present three cases of AML with absent CD33 from diagnosis till follow-ups visits. CD33 absence mimics positivity for MRD studies. This data emphasizes the importance of caution in MFC analysis and correlation of diagnostic and follow-ups results and interpretation, as constant training of the team.