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Acute myeloid leukemia (AML) with FLT3 -ITD mutations ( FLT3 -ITD mut ) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3 -ITD-positivity. The aim of this study was to characterize the distribution of FLT3 -ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3 -ITD mut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3 -ITD mut AML ( n = 12). A higher FLT3 -ITD mut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/−,CD25−,CD99low/−) ( p = 0.0005) and mononuclear cells (MNCs) ( p < 0.0001). This was associated with significantly increased CD99 mean fluorescence intensity in LPCs. Significantly higher FLT3 -ITD mut burden was also observed in LPCs of AML patients with a small FLT3 -ITD mut clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows that FLT3- ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target in FLT3 -ITD mut AML.
Vitamin C has been shown to play a significant role in suppressing progression of leukemia through epigenetic mechanisms. We aimed to study the role of vitamin C in acute myeloid leukemia (AML) biology and clinical course. To this purpose, the plasma levels of vitamin C at diagnosis in 62 patients with AML (including 5 cases with acute promyelocytic leukemia, APL),7 with myelodysplastic syndrome (MDS), and in 15 healthy donors (HDs) were studied. As controls, vitamins A and E levels were analysed. Expression of the main vitamin C transporters and of the TET2 enzyme were investigated by a specific RQ-PCR while cytoplasmic vitamin C concentration and its uptake were studied in mononuclear cells (MNCs), lymphocytes and blast cells purified from AML samples, and MNCs isolated from HDs. There were no significant differences in vitamin A and E serum levels between patients and HDs. Conversely, vitamin C concentration was significantly lower in AML as compared to HDs (p<0.0001), inversely correlated with peripheral blast‐counts (p=0.029), significantly increased at the time of complete remission (CR) (p=0.04) and further decreased in resistant disease (p=0.002). Expression of the main vitamin C transporters SLC23A2, SLC2A1 and SLC2A3 was also significantly reduced in AML compared to HDs. In this line, cytoplasmic vitamin C levels were also significantly lower in AML-MNCs versus HDs, and in sorted blasts compared to normal lymphocytes in individual patients. No association was found between vitamin C plasma levels and the mutation profile of AML patients, as well as when considering cytogenetics or 2017 ELN risk stratification groups. Finally, vitamin C levels did not play a predictive role for overall or relapse-free survival. In conclusion, our study shows that vitamin C levels are significantly decreased in patients with AML at the time of initial diagnosis, further decrease during disease progression and return to normal upon achievement of CR. Correspondingly, low intracellular levels may mirror increased vitamin C metabolic consumption in proliferating AML cells.
Introduction Persistence of leukemia stem cells (LSCs) in patients with Acute Myeloid Leukemia (AML) achieving complete remission (CR) after chemotherapy leads to disease recurrence and poor outcome. Therefore, the identification of LSCs driving resistance to therapy represents an important challenge. LSCs reside within the CD34+/CD38- cells and it has been recently demonstrated that co-expression of CD99 allows for separation of LSCs from functionally normal hematopoietic stem cells in AML. We showed that the presence of a CD123/CD99/CD25+ population within CD34+ cells strongly correlates with FLT3-ITD-positivity. The aim of this study was to deeply characterize the molecular profile of CD34+/CD123+/CD99+/CD25+ LSCs to shed light on the subclonal architecture of FLT3-ITD+ AML and to track the expansion of mutated clones. Methods Molecular status of FLT3-ITD and NPM1 were investigated at the DNA level in a cohort of 150 de novo AML patients at diagnosis and at relapse, using standard procedures. Clonal evolution of FLT3-ITD mutations was studied at diagnosis in 14 FLT3-ITD mutated AML on sorted bone marrow populations, purified by the Cytoflex High-Speed Cell Sorter using a sequential gating strategy, to separate CD34/CD123/CD99/CD25+ LSCs-, CD34+ precursors (CD123/CD99/CD25-) and T-lymphocytes. Moreover, to characterize the genetic profile of FLT3-ITD-mutated clones, DNA targeted sequencing was performed on 22 cell populations isolated from 7 AML patients, using the Oncomine™ Myeloid Research Assay panel on the Ion Torrent™ S5 sequencer. Results 39 of 150 AML cases were FLT3-ITD+, at a median allelic ratio (AR) of 0.36 (range 0.05-7.9). The FLT3-ITD AR was significantly higher within the rare LSCs compartment as compared to MNC (median AR: 0.78, range 0.42-19.3, vs 0.09, range 0.05-0.6m p<0.0001, n=14), and to CD34+/CD123+/-/CD99low/-/CD25- precursors (Ctrl) (median AR=0.02, range 0-0.59) (p=<0.0001) (Fig 1A). In particular, a FLT3-ITD mutated minor subclone was present (median AR=0.05, range 0.05-0.08) in the MNC of 6 patients, and was highly enriched in the corresponding CD34+/CD123+/CD99+/CD25+ sorted LSCs (median AR=0.5, range 0.4-0.8). FLT3-ITD positive clones may also persist in sorted LSCs at the time of complete remission, being undetectable using standard techniques and driving later relapse (UPN1, Fig 1B). Furthermore, FLT3-ITD LOH may occur during disease progression, leading to AR >1, while the number of LSCs do not seem to expand (Fig 1B). These data confirm that LSCs may persist at rare frequency during progression, still representing the treatment-resistant FLT3-ITD reservoir, driving disease relapse with expansion of more mature, FLT3-ITD positive populations. Aiming at shedding light on the molecular heterogeneity and subclonal structure of AMLs, using targeted NGS we compared the mutational profiles of MNCs to that of highly purified LSCs and/or to the CD34+/CD123-/CD25-/CD99- counterpart (n=7 patients). In 4 cases the same mutation pattern was present in both MNCs and LSCs. In 3 cases we found additional mutations in NRAS, BCOR and KRAS in MNCs, indicating acquisition of other transforming events during maturation (Fig 2A). Furthermore, the variant allele frequency (VAF) of mutations was similar in MNCs and LSCs (p=0.12), while FLT3-ITD and TKD burden was enriched in LSCs (p=0.03) (Fig 2B). Conversely, the NPM1 mutation burden remained almost stable in the different cell subsets (Fig 2C). In conclusion, our study shows that FLT3 mutations occur in early LSCs characterized by the CD34+/CD123+/CD99+/CD25+ immunophenotype, which represent the leukemic reservoir. These data may be the rational for CD99-targeted treatments in FLT3-ITD mutated AML to achieve durable disease eradication. Disclosures Venditti: Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Buccisano:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees.
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