An Adapted Gating Strategy Integrating a Myelomonocytic Window Is Necessary For Correct Flow Cytometric Diagnosis In a Large Proportion Of AML With Mutated NPM1
Authors: Kull Miriam , Langer Christian , Tassara Michela , Schoepflin Christine , Bayer Olena , Morlok Carina , Kündgen Andrea , Salih Helmut R. , Kindler Thomas , Horst Heinz-August , Lübbert Michael , Salwender Hans , Götze Katharina , Thol Felicitas , Ganser Arnold , Paschka Peter , Gaidzik Verena I. , Döhner Konstanze , Döhner Hartmut , Schlenk Richard F. , Feuring-Buske Michaela 
Published: October 2019

Abstract

Background
Previously, it has been reported, that AML with mutated NPM1 is associated with a distinctive immunophenotype. In particular, low or absent expression of CD34 accompanied by high expression of CD33, and – at least in part of the cases - absence of HLA-DR expression was reported. CD45/side scatter (SSC) gating is widely used for the identification of blasts by flow cytometry (FC). Blast cell gates typically are defined by a low SSC and moderate CD45 expression. However, in a number of patients with NPM1mutation this typical blast cell gate comprises significantly lower blast percentages when compared to the morphological evaluation. In these patient samples a second population is present, which is characterized by a higher expression of CD45 and a brighter SSC signal (myelomonocytic region). Here we provide evidence, that the implementation of an adapted gating procedure integrating cells with higher CD45 expression and moderate SSC improves diagnostic accuracy in these cases.

Aim
To evaluate differential gating strategies in diagnostic multicolor flow cytometry in NPM1mutated AML.

Methods
After informed consent diagnostic work-up of patient samples with newly diagnosed AML within the AMLSG BiO Study (clinicaltrials.gov NCT01252485) was initiated and included rapid molecular screening for NPM1 and FLT3 mutations, and CBFß-MYH11, RUNX1-RUNX1T1 and PML-RARA fusions, conventional karyotyping, multicolor flow cytometry and centralized morphological assessment. Multicolor flow cytometry was performed using a Becton Dickinson FACS Canto-II and a comprehensive antibody panel (cytoplasmic staining: TdT, MPO, CD3, CD34, CD45, CD79a; surface staining: HLA-DR, NG2, CD3, CD7, CD10, CD11b, CD11c, CD13, CD14, CD15, CD19, CD33, CD34, CD41, CD42b, CD45, CD56, CD61, CD64, CD117, CD235) according to ELN-recommendation (Döhner et al. Blood 2010).

Results
Between October 2011 and July 2013 n=2117 patients were included into the AMLSG BiO protocol. In the current study immunophenotypic data of a total of n=263 pretreatment bone marrow samples of patients with NPM1 mutated AML (age 18 to 60 years) were included for further analyses. In n=175 patients only one blast population was present, which was characterized by the CD45 low/SSC low blast cell gate (group-1), whereas in n=87 two populations were detected and gated (group-2). Concurrent activating FLT3 mutations were present in 48% and 39% in group-1 and group-2 (p=0.19), respectively. In a first attempt, the immunophenotypically determined blast infiltration rate was correlated with morphological assessment. In group-1 the morphological blast count correlated well with the immunophenotypically determined blast infiltration (r=0.62, p=0.0001), whereas in group-2 the two methods did not correlate (r=0.34, p=0.07), when only the blast gate was taken into account. However, by applying the two-gate strategy including the myelomonocytic window the correlation could be restored (r=0.67, p<0.0001). Of note, a similar distribution of morphological subtypes (pure myeloid vs. myelomonocytic) was observed in the two groups (p=0.82). The expression profile in the blast gate in group-1 compared to group-2 did not differ with regard to cytoplasmic CD34 expression (median positive events, 9.0% vs. 6.0%, p=0.40). In contrast, the comparison of the blast gate in group-1 with the myelomonocytic window in group-2 revealed a significant difference (median positive events, 9.0% vs. 0.3%, p<0.0001). A differential pattern was identified for CD117 with similar expression levels in the blast gates of group-1 and group-2 (median positive events, 41% vs. 43%, p=0.94) but markedly different levels comparing blast gate group-1 with the myelomonocytic gate in group-2 (median positive events, 41% vs. 2%, p<0.0001). The expression levels of the myelomonocytic markers CD14 and CD64 as well as the T-cell marker CD7 were low in the blast gates of group-1 and significantly higher in group-2 (CD64, p<0.0001; CD14, p=0.03; CD7, p=0.0001) but very high in the myelomonocytic gate of group-2 compared to group-1 blast gate (CD64, p<0.0001; CD14, p<0.0001; CD7, p<0.0001). The expression profile of AML with mutated NPM1 was not affected by additional activating FLT3mutations independent of the gating strategy.

Conclusion
The diagnostic accuracy of multicolor flow cytometry can be markedly improved in AML with mutated NPM1 by applying an adapted gating strategy.

Disclosures:
Kindler: Novartis: Membership on an entity’s Board of Directors or advisory committees. Salwender:Janssen and Celgene: Honoraria. Götze:Celgene Corp: Honoraria. Schlenk:Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Chugai: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Ambit: Honoraria.