Baseline immunophenotypic profile of bone marrow leukemia cells in acute myeloid leukemia with nucleophosmin-1 gene mutation: a EuroFlow study

Dear Editor, Molecular techniques are the gold standard method for the diagnosis of AML with mutated nucleophosmin gene ( NPM1 mut ). However, their worldwide availability is limited and they provide limited insight into disease heterogeneity. Hence, surrogate markers of NPM1 mut are used for fast diagnostic screening of the disease [1], including, among others, immunohistochemical detection of cytoplasmic NPM1 (NPM1c) [2], cup-like nuclear morphology [3], normal karyotype, and/or recurrent ﬂ ow cyto-metry pro ﬁ les -e.g., CD34 negativity, and/or a phenotype resembling acute promyelocytic leukemia (APL)- [4]. Nevertheless, some of these methods are also not widely available, they show limited sensitivity (e.g., low or absent NPM1c expression, particularly among monoblastic/monocytic AML-NPM1 mut ) [5], frequently lack standardized procedures [1], and they might also bring limited information about disease heterogeneity.


Dear Editor,
Molecular techniques are the gold standard method for the diagnosis of AML with mutated nucleophosmin gene (NPM1 mut ).However, their worldwide availability is limited and they provide limited insight into disease heterogeneity.Hence, surrogate markers of NPM1 mut are used for fast diagnostic screening of the disease [1], including, among others, immunohistochemical detection of cytoplasmic NPM1 (NPM1c) [2], cup-like nuclear morphology [3], normal karyotype, and/or recurrent flow cytometry profiles -e.g., CD34 negativity, and/or a phenotype resembling acute promyelocytic leukemia (APL)- [4].Nevertheless, some of these methods are also not widely available, they show limited sensitivity (e.g., low or absent NPM1c expression, particularly among monoblastic/monocytic AML-NPM1 mut ) [5], frequently lack standardized procedures [1], and they might also bring limited information about disease heterogeneity.
AML-NPM1 mut leukemia cells present with heterogeneous cytomorphology and immunophenotypic patterns of lineage commitment and antigen expression, including phenotypes associated with FLT3-internal tandem duplication (ITD) and a poor outcome [6].In parallel, neutrophil-lineage commitment of AML-NPM1 mut cells has been linked with underlying TET2 and IDH1/2 mutations, while the presence of monocytic lineage-committed and immature NPM1 mut cells has been related to DNMT3A mutations; indeed, these three patient subgroups show increasingly worse outcomes [4].Strikingly, NPM1 mut FLT3-ITD − patients displaying immature immunophenotypes show strong similarities with NPM1 mut FLT3-ITD + cases regarding leukemia cell transcriptomic profiles, response to therapy and poorer outcomes [7].Thus, baseline flow cytometric characterization of AML-NPM1 mut leukemia cell heterogeneity might contribute to guiding treatment decisions in these patients.Despite the above associations, specific immunophenotypic patterns of AML-NPM1 mut remain to be fully defined.
Herewith, we performed a detailed flow cytometric characterization of different subsets of BM leukemia cells from 377 AML patients, including 201 AML-NPM1 mut , 144 AML-NPM1 wt and 32 APL patients, based on the EuroFlow 8-color acute leukemia orientation tube (ALOT) and the AML/MDS antibody panel (Supplementary methods and Supplementary Table 1).FLT3-ITD was detected in 33% of AML-NPM1 mut cases, 19% of AML-NPM1 wt and 34% of APL patients.Our aim was to identify reliable phenotypic profiles for fast screening of NPM1 mut and/or FLT3-ITD to guide subsequent molecular diagnostic approaches that can be applied worldwide and provide a better understanding of disease heterogeneity.
The distribution of leukemia cell subsets was consistent with a lower maturation arrest of AML-NPM1 mut vs. NPM1 wt cells, associated with a lower frequency and size of immature leukemia cell expansions (p < 0.001), while depicting a higher prevalence of more differentiated AML cells committed to the neutrophil (p < 0.001) and/or the monocytic lineage (p = 0.02) (Supplementary Table 2 and Supplementary Fig. 1).These findings might contribute to explain the overall higher sensitivity to chemotherapy of AML-NPM1 mut [7].
Despite AML-NPM1 mut may originate from CD34 + hematopoietic progenitor cells (HPC) [8], most frequently they lack CD34 (7% vs. 94% NPM1 wt CD34 + cells).These cells may expand in BM due to consistent expression of HOX genes [9], which has been directly associated with NPM1 cytoplasmic dislocation [10].However, CD34 lo expression is not specific to AML-NPM1 mut , and it has been found to be independent of NPM1 dislocation [9].In line with these observations, we also found CD34 lo expression among NPM1 wt immature, neutrophil and monocytic lineage-committed AML cells, and thereby, this phenotype is of limited specificity for AML-NPM1 mut .
Beyond CD34 lo expression, other immunophenotypic features of AML-NPM1 mut cells supported a less pronounced maturation blockade vs. other AML patients.Hence, AML-NPM1 mut immature cells retained a higher capability for neutrophil lineage maturation OR odds ratio, CI confidence interval.
Hundreds of neutrophil and monocytic differentiationassociated genes are repressed in AML-NPM1 mut , which might be related to NPM1 haploinsufficiency and/or the cytoplasmic relocation and functional blockade of myeloid transcription factors interacting with NPM1c [12,13].For instance, the functional reduction of PU.1 represses the PU.1/CEBPA/RUNX1 myeloid transcriptional hub regulating terminal monocytic and neutrophil differentiation [13].Conversely, other nuclear transcriptional regulators inhibited by NPM1 under physiological conditions are not translocated to the cytoplasm, leading to abnormally high activation of their target genes [14].Therefore, asynchronous neutrophil and/or monocytic differentiation profiles of AML-NPM1 mut cells are consistent with abnormal activation vs. repression of distinct sets of myeloid gene promoters regulated by NPM1.
Expectedly, (non-APL) AML cases with FLT3-ITD showed greater BM leukemia cell infiltration and increased proportions of immature CD34 + leukemia cells, frequently in association with monocytic AML cells, independently of NPM1 comutation (Supplementary Fig. 1I and Supplementary Table 2).Such specific expansion of immature AML cells might be related to the physiological restriction of FLT3 gene expression to BM hematopoietic CD34 + HPC [15].
Noteworthy, FLT3-ITD promoted distinct immunophenotypic profiles in NPM1 mut and NPM1 wt AML (Supplementary results and Supplementary Fig. 3).Although CD34 and/or CD25 expression has been associated with FLT3-ITD [6], we show that both markers are more frequent among immature NPM1 mut FLT3-ITD + cells.Hence, CD25 positivity and heterogeneous CD34 expression on immature AML cells emerged as the best combination of predictors for FLT3-ITD among AML-NPM1 wt cases.Conversely, in AML-NPM1 mut cases, FLT3-ITD was strongly associated with a CD7 hi CD38 lo profile on immature leukemia cells and/or a CD117 het CD123 hi phenotype among neutrophil lineage leukemia cells (Table 1).
In summary, the mutational status of NPM1 and FLT3 is associated with unique BM leukemia cell distribution and immunophenotypic profiles, even when only cases with a normal karyotype were considered (data not shown), which might contribute to a fast diagnostic screening of NPM1 mut and/or FLT3-ITD in AML, and an improved classification of AML-NPM1 mut patients.Further prospective studies are needed to confirm these findings.

Fig. 1
Fig. 1 Monocytic maturation pathways in normal and AML bone marrow.Maturation pathways of monocytic cells in normal bone marrow (A, B, blue dots), and asynchronous AML-NPM1 mut patterns of expression of CD300e + CD14 − (C), CD35 + CD14 − (D).E and F depict normal patterns of acquisition of CD14 vs. CD300e in a patient with AML-NPM1 wt while showing an asynchronous CD14 + CD35 − phenotype (E, F) among monocytic cells (red dots).Arrows represent the normal (blue) and leukemia (red) maturation pathways of monocytic lineagecommitted (gated) CD64 hi cells.

Table 1 .
Univariate and multivariate logistic regression analysis of those immunophenotypic features of BM leukemia cells associated with NPM1 mutation (A) and FLT3-ITD (B) from AML patients (n = 377).