FLT3 (CD135) and Interleukin‐2 receptor alpha‐chain (CD25) expression in acute myeloid leukemia: improving the correspondence to FLT3 – internal tandem duplication mutation

Sir, activating internal tandem duplication (ITD) is the FMS-like tyrosine kinase 3 (FLT3) gene mutation that was found to determine acute myeloid leukemia (AML) prognosis and predict poor outcome [1]. However, there is lack of agreement across studies upon the prognostic role of FLT3 protein, CD135, in AML [2, 3]. Acting differently, expression of CD25, the a-chain of interleukin-2 receptor, on AML myeloblasts was more uniformly related to FLT3-ITD mutation and was described to have a negative impact on achieving complete remission (CR) and subsequent time to relapse [4]. In the present study, we evaluated the FLT3-ITDrelated markers, CD135 and CD25, using flow cytometry, in de novo patients with AML. We aimed at defining their pattern of expression, and their correlation to FLT3ITD mutation, therapy response at day 28, and other prognostic determinants. Sixty two patients with AML, presented at Ain Shams University Hospitals, were enrolled in our study over a 10-month period. They were classified into 38 males and 24 females; 48 adult patients (39 of whom were of the <60-year subgroup, 9 patients of the >60-year subgroup); and 14 pediatric patients. After obtaining an informed consent from patients and controls, required samples were collected prior to commencing treatment. Flow cytometric immunophenotyping, and cytogenetic and molecular analyses were used for diagnosis and classification. PCR testing for FLT3-ITD mutation was done, and the ratio between mutant and WT-FLT3 was determined for positively mutated patients [1]. Day 28-bone marrow (BM) morphological remission was used to indicate CR. Treatment protocols followed the NCCN guidelines [5]. AML-related cytogenetic abnormalities were detected in 31 patients; prognostically, 11, 47, and 4 patients were categorized into favorable, intermediate, and unfavorable genetics groups, respectively. PCR testing for FLT3-ITD revealed positive mutation in 43.5% (27/62); allelic ratios of FLT3-ITD showed a median value of 0.53 (IQR: 0.32–0.83). Surface expression of CD135 and CD25 was assessed using carboxyfluorescein-conjugated anti-CD135 and phycoerythrin-conjugated anti-CD25 monoclonal antibodies using Coulter Epics XL 3-color flow cytometer (Coulter Electronics, Hialeah, FL, USA). Isotypic controls for FITCand PE-conjugated monoclonal antibodies were included in the analysis. We applied the following statistical tests: Mann– Whitney U-test, to compare medians of continuous variables between groups; Spearman’s rank test, to test correlations between different parameters; chi-square test, to compare proportions between two qualitative parameters; one-way ANOVA, to relate CD135 or CD25 levels to FAB subtypes; multivariate analysis, to evaluate prognostic markers in relation to CR; and receiver operating characteristics analysis, to calculate the best cutoff values of CD135 capable of identifying FLT3-ITD. Of the 62 patients, 37 (59.7%) were positive for CD135 (median: 25.8; IQR: 5.9–63.5); 21 of 62 (33.9%) were positive for CD25 (median: 2.3; IQR: 0.62–14.3); 18 of 62 (29.03%) showed positivity for both markers; 19 of 62 (30.6%) were CD135CD25 ; 3 of 62 (4.8%) were CD135 CD25; 22 of 62 (35.5%) were CD135 CD25 . Comparable values of CD135 and CD25 were found in males and females, and in adult and pediatric age groups, although CD25 values were higher in the <60 years adults compared with older patients (P < 0.001). We found no significant difference in CD135 or CD25 values across the genetic prognostic groups. On analyzing the expression of both immunophenotypes across the FAB subtypes, CD135 expression was significantly higher in AML-M5 than in either AML-M2 or AML-M4 (P < 0.05). CD25 showed insignificant difference across FAB subtypes (P = 0.09), although the highest mean value was found in AML-M3. Positive expression of CD135 was associated with lower platelet counts (P = 0.01), higher total leukocytic counts (TLC) (P = 0.042), and higher BM blast counts (P < 0.001); CD25 positivity was associated with lower platelet counts (P < 0.001) and higher BM blast counts (P = 0.03). Among the known prognostic