Altered Immunophenotypes on Leukemic and/or Monocytic Cells from Acute Myeloid Leukemia Highly Predict for Nucleophosmin Gene Mutation

Introduction. Nucleophosmin gene mutation (NPM1^mut) occurs in around 30% of acute myeloid leukemia (AML) patients, frequently linked with favourable prognosis in the absence of FLT3-ITD^mut, which occurs in around 40% of NPM1^mutAML. Therefore, more expeditious diagnostic approaches may contribute to early diagnosis and prognostic stratification of these patients. Herein, we investigated the association of immunophenotypic features of leukemic and monocytic cells with the presence of NPM1^mut in AML.

Methods. A total of 404 bone marrow (BM) samples from newly-diagnosed AML patients according to WHO 2017 classification were retrospectively studied by 8-color flow cytometry, including 225 AML with NPM1^mut and 179 cases wild type gene (NPM1^wt). Information on FLT3-ITD could be obtained from 397/404 cases. Thus, FLT3-ITD^mut was present in 85/397 (21%), being concomitant with NPM1^mutin 62/85 AML cases (73%). Logistic regression analysis was used to identify predictive phenotypes for the presence of NPM1^mut.

Results. Overall, blast cell with immunophenotypic features of monocytic differentiation (corresponding to FAB M4 and M5 AML subtypes) were observed among 135/225 (60%) and 69/179 (38.5%) AML patients with NPM1^mutand NPM1^wt, respectively (p<0.001). In the remaining AML cases without monocytic differentiation (FAB M0, M1 and M2; n=200), both immature/myeloid blasts and remaining monocytic cells were immunophenotypically characterized.

Among AML with monocytic blast cell differentiation, altered monocytic phenotypes were more frequent among NPM1^mut vs. NPM1^wtcases (98% vs. 36%) (p<0.001). In detail, these phenotypic alterations consisted of asynchronous expression of CD300e prior CD14 (79% vs. 5% NPM1^wtcases, respectively; p<0.001) and/or CD35 prior CD14 (84% vs. 30%), which were observed independently of FLT3-ITD. Of note, coexistence of the latter two asynchronous monocytic patterns (CD300e prior CD14 and CD35 prior CD14) on monocytic blast cells was specific for NPM1^mut (64% vs. 0% NPM1^wtpatients; p<0.001).

Noteworthy, in AML cases without blast cell monocytic differentiation, remaining monocytic cells showed similar asynchronous phenotypic patterns, which were also more frequent among NPM1^mut cases (78% vs. 23% of NPM1^wtcases, respectively; p <0.001). Thus, remaining monocytic cells from NPM1^mut AML cases also showed a higher frequency of asynchronous CD300e prior CD14 (64% vs. 8% of NPM1^wt cases; p<0.001) and CD35 prior CD14 expression (58% vs. 20% of NPM1^wt cases, respectively; p<0.001).

In addition, aberrant CD9 blast cell expression was found in a significant proportion of all AML cases studied (124/222, 56%). However, altered CD9 was more frequent on (either monocytic or immature/myeloid) blast cells from AML cases with NPM1^mut (76% vs. 46% NPM1^wtcases; p<0.001), but not in AML with only-FLT3-ITD^mut(39% vs. 46% of NPM1^wtFLT3-ITD^wt; p>0.05). In turn, aberrant CD25 expression on blast cells was otherwise linked to FLT3-ITD^mut (61% vs. 20% of FLT3-ITD^wtcases; p<0.001), being more frequent in AML with FLT3-ITD^mutNPM1^wt and double mutated AML cases vs. AML with FLT3-ITD^wtNPM1^mut and FLT3-ITD^wtNPM1^wt (79% and 47% vs. 20% and 20% of cases, respectively; p<0.001). In addition, overall, FLT3-ITD^mut was associated with a significantly higher proportion of immature (i.e. CD34⁺) blasts, as compared to FLT3-ITD^wt cases (median of 10% vs. 0.3% CD34⁺ blasts; p<0.001).

In multivariate analysis, baseline detection of monocytic-lineage blast cells with asynchronous expression of CD300 prior CD14 -C-index= 0.954, odds ratio (OR), 78.8; 95% confidence interval (CI), 13.1-471; p<0.001- and CD35 prior CD14 (OR, 24.5; 95% CI, 4.8-123; p<0.001) and detection of these asynchronous patterns on remaining monocytic cells from AML without monocytic differentiation (C-index= 0.816, OR, 14.7; 95% CI, 6.4-34; p<0.001 and, OR, 2.5; 95% CI, 1.2-5.3; p=0.01, respectively) showed the highest predictive value for NPM1^mut in AML. In turn, CD25 aberrant blast cell expression was the only immunophenotypic parameter with predictive value for FLT3-ITD^mut (OR, 6.4; 95% CI, 2.9-14.5; p<0.001).

Conclusions. Detection of specific aberrant immunophenotypic patterns among blast cells and/or remaining monocytic cells from AML patients is highly predictive for NPM1^mut, which may contribute to early diagnosis and follow-up of these patients.