This prospective study included all the cases of AML in children aged 18 years or less whose samples were submitted to the Molecular Biology Laboratory of the Hospital del Salvador for analysis. The study subjects presented to 17 public hospitals across Chile between January 2011 and December 2017. The study was approved by the Ethics Committee of the Metropolitan Orient Area of Santiago. Each patient or parent gave written consent to participate. The study was performed according to the Helsinski Declaration and was funded in part by a Grant from the Fundación Nuestros Hijos.

Bone marrow or peripheral blood samples were obtained at diagnosis. Diagnostic analyses were centralized and conducted at the Hematology Lab, Hospital del Salvador. Analyses consisted of a morphologic review, the cases being categorized according to the French-American-British (FAB) classification system, and subsequently confirmed by flow cytometry (FACSCalibur, Becton & Dickinson, B&D), using monoclonal antibodies: anti CD2, CD7, CD11b, CD13, CD14, CD15, CD19, CD33, CD34, CD64, cCD71a, CD117, HLA-DR., MPO and anti-lisozima.

Cytogenetics and FISH were performed at two reference laboratories, Hospital Luis Calvo Mackenna and del Salvador, using culture and G banded, and clonal abnormality defined according to the International Nomenclature of Human Cytogenetics System.18

Molecular analyses were performed at the Molecular Biology Laboratory, Hospital del Salvador, using the real-time polymerase chain reaction (RT-PCR) (Rotor Gene Q/Quiagen).

Patients were stratified into 3 risk groups:15 favorable: t(15;17), t(8;21) and inv16; intermediate: NK, or other types that were neither favorable or unfavorable, including 11q23, and; unfavorable: −7, −5, or structural alterations of 5p or 5q, t(6;9), complex karyoype (≥ 3 alterations) or 12p abnormalities.3–5 Cases without cytogenetic information and with no recurrent molecular abnormalities were designated as intermediate risk.

Patients received treatment at their referring center. Those under 15 years old received the PINDA-LMA 2006 Protocol.19 Cases under one year old received the same protocol, but with ARA-C doses adjusted for age. Those with Down Syndrome also received PINDA-LMA 2006, but with a reduced antracycline dose. Patients with Acute Promyelocytic Leukemia (APL) received the Internacional APL Study protocol. Patients between 15 and 18 years of age, received the PANDA-LMA protocol20 and those with APL were treated using the LPA 2000 Protocol. Patients who did not acheive complete remission (CR) during induction were treated with HAM (citarabine 3 g/m2 every 12 h/day iv for 3 days and mitoxantrone 10 mg/m2/day iv days 3 and 4). The allogeneic stem cell transplant (all-SCT) was performed at the Hospital Luis Calvo Mackenna.

Complete remission (CR) was defined as < 5% blasts and no extramedullary disease. Refractory disease was defined as > 15% blasts post-induction or post-reinduction. Overall survival (OS) was defined as the date of diagnosis to date of death from any cause and event-free suvival (EFS), as the date of diagnosis to the first event (relapse or death from any cause without relapse).

Non-promyelocytic AML and APL were evaluated separately, with respect to the prognostic significance of the FLT3/ITD and NPHM1 and their correlation to the white cell count (WCC) at diagnosis, risk of relapse and risk of death.

Continuous variables were expressed as mean (±standard deviation) if they were distributed normally, or otherwise, as median (percentile 25 - 75). The Student's t-test was used to evaluate quantitative variables with normal distribution and the Mann-Whitney U test was used for variables that were not normally distributed. For categorical variables, the data were expresed as a percentage and the independent test based on χ² was used to determine significant differences.

To evaluate the correlation between the OS or EFS and FLT3/ITD positivity, the logistic regression model was used, with adjustment for univariate variables, such as age, sex, WCC at diagnosis and normal karyotype. The association between the FLT3/ITD mutated or non-mutated with the OS and EFS was reported as an Odds ratio (OR) and was considered significant if the 95% Confidence Interval (CI95%) did not exceed 1. Kaplan-Meier survival curves were generated. A p-value of < 0.05 was considered to be statistically significant. The data were analyzed using the STATA software (serial no. 301,406,367,260).

Clinical characteristics are shown in Table 1. As a whole, 71% (164/232) were non- promyelocytic AML and 29% (68/232), APL. Cytogenetic information was obtained for 207 cases (89%) and the karyotype was normal in 49 (24%). Cytogenetic alterations are presented in Table 2. Risk stratification showed that the percentage of patients with low and intermediate risk was similar and that only 4% fell into the high-risk category. Six out of 24 cases without cytogenetic data were classified according to recurrent molecular alterations and the rest, with no informative cytogenetic/molecular information, were allocated to the intermediate-risk group.

All patients, except three who died at diagnosis, were treated. A hundred and ninety-five of 229 cases (85%) achieved CR, which in three cases was after reinduction with HAM. Twenty patients proceeded to all-SCT: seven low-risk (in the second CR), 12 intermediate-risk and 1 high-risk (in the first CR). Fifty-five patients died of leukemia, 32 due to treatment complications and four during transplant, and one patient committed suicide.

The clinical and laboratory characteristics of non-APL AML and ALP patients with mutated and unmutated FLT3 are shown in Tables 3 and 4.

The FLT3/ITD mutation was detected in 10% (14/133) of non-promyelocitic AML and 25.4% (16/63) of APL cases (p < 0.007). The FLT3/TKD was studied in only 66 cases and was mutated in 3% of all those with non-promyelocitic AML. The FLT3/ITD was mutated in 21% (8/38) of the patients with NK, excluding children with Down's Syndrome. The FLT3/TKD could not be detected in 18 children.

There was an association between the FLT3 mutation and FAB classification, as it was present in every microgranular (M3v) subtype case (Table 4).

In multivariate analysis, the FLT3/ITD mutated was a significant predictor of outcome, with ORs of 5.1 and 3.7 for risk of death and risk of relapse, respectively, compared to those not mutated.

On the other hand, in the multivariate analysis of APL, none of the factors, including the FLT3/ITD, were predictive of death and, in the univariate analysis, only the WCC at diagnosis was an adverse factor.

The NPM1 mutation was observed in 3.2% (5/155) of the cases (median age 14), all of whom had non-promyelocytic AML and an NK. Three also had FLT3/ITD mutated and only one of these is alive. One relapsed and the other died due to SCT complications. There were two NPM1 mutated FLT3/ITD non-mutated patients, of whom one is alive and the other died during induction.

The CR rate was similar for the FLT3 mutated and non-mutated leukemia. (Tables 3 and 4). With a median follow-up of 35.3 months (0 - 116), the 5-year OS for children with non-promyelocytic AML, FLT3/ITD non-mutated vs mutated, was 62.7% (CI95%: 53.3 −70.7%) vs. 21.4% (CI95%: 5.2 - 44.8%) (p < 0.001) (Figure 1). The five-year EFS for patients with FLT3/ITD non-mutated or mutated was 79.5% (CI95%: 70.9- 85.8%) vs. 50% (CI95%: 22.9–72.2%), respectively (p < 0.01) (Figure 2). However, survival differences in APL for non-mutated and mutated cases did not reach significance.

Figure 1.

Overall survival of 133 children with non-promyelocytic AML with non-mutated and mutated FLT3/ITD.

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Figure 2.

Event-free survival of 133 children with non-promyelocytic AML with non-mutated and mutated FLT3/ITD.

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