Prevalence of FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD+) in de novo acute myeloid leukemia patients categorized according to cytogenetic risk

ABSTRACT CONTEXT AND OBJECTIVE: The mechanism involved in leukemogenesis remains unclear and more information about the disruption of the cell proliferation, cell differentiation and apoptosis of neoplastic cells is required. DESIGN AND SETTING: Cross-sectional prevalence study at the Discipline of Hematology, Hospital São Paulo, Universidade Federal de São Paulo. METHODS: We investigated FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD+) in 40 adult patients with de novo acute myeloid leukemia (AML), categorized according to cytogenetic results, from September 2001 to May 2005. RESULTS: Thirteen patients (32.5%) were classified as presenting the favorable karyotype, 11 patients (27.5%) as an intermediate group, 7 patients (17%) as an undefined group and 9 patients (22.5%) as the unfavorable group. FLT3/ITD+ was found in 10 patients (25%): 3 with FLT3/ITD+ and favorable karyotype; 4 with FLT3/ITD+ and intermediate karyotype; 2 with FLT3/ITD+ and undefined karyotype; and only 1 with FLT3/ITD+ and unfavorable karyotype. Among the patients without FLT3/ITD+, 10 presented favorable karyotype, 8 intermediate, 4 undefined and 8 unfavorable karyotype. The cytogenetic results showed no correlations between FLT3/ITD presence and the prognostic groups (P = 0.13). We found that 2 patients were still alive more than 24 months later, FLT3/ITD+ did not influence the patients’ survival rate. CONCLUSION: We found the same frequency of AML with FLT3/ITD+ in both the favorable and intermediate prognosis groups. Only one patient presented AML, FLT3/ITD+ and unfavorable karyotype (the hypothetical worst clinical situation). Therefore, the prognostic advantage of favorable cytogenetics among patients with FLT3/ITD+ remains to be elucidated, for it to be better understood.


INTRODUCTION
Acute myeloid leukemia (AML) is a clonal disorder that involves mutated hematopoietic precursors with inappropriate properties (e.g.uncontrolled proliferation, absence of apoptosis, abnormal cell differentiation, etc.). 1 The crucial question is which parts are defective in the multistep anomaly-generating process of genetic programming of AML cells.According to the "two hit" model of leukemogenesis, 2 class I mutations confer a proliferative and/or survival advantage to cells, while class II mutations give rise to impaired differentiation and abnormal apoptosis.Despite the important role of FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD+), a class I mutation, in providing a myeloproliferative signal and/or survival advantage to leukemia cells, this mutation does not affect differentiation by itself.Thus, for AML onset, additional class II mutations such as chromosome rearrangements are probably required, 3 e.g.promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARα), core binding factor/smooth muscle myosin heavy chain (CBFß/SMMHC), mixed-lineage leukemia (MLL) and acute myeloid leukemia-1 transcription factor/eight-twenty-one corepressor (AML1/ETO).These result in abnormalities relating to the CBF complex, MLL, Hox proteins and retinoic acid receptors. 1he FLT3 gene encodes an important tyrosine kinase receptor that is critical for normal hematopoiesis. 4FLT3/ITD + 5 promotes disruption of a repressor sequence in the juxtamembrane domain of the FLT3 gene, leading to constitutive autophosphorylation of the receptor, 6 thereby promoting proliferation and inhibiting apoptosis in leukemic cells. 6,7LT3/ITD + is found in 20% to 30% of de novo AML patients [7][8][9][10] and is the most mutated gene in leukemia, with a significant clinical impact on patients. 11e approached this subject by investigating FLT3/ITD + in de novo AML patients categorized according to cytogenetic results, focusing especially on the group with favorable prognosis and normal karyotype, and associating this with age and white blood cell (WBC) count.

Patient samples
The eligible cases consisted of adult patients referred to Hospital São Paulo, Universidade Federal de São Paulo -Escola Paulista de Medicina (Unifesp-EPM) from September 2001 to May 2005 (Table 1), with de novo AML according to World Health Organization diagnostic criteria. 12Immunophenotypic cell characterization was performed using a FACScalibur flow cytometer (Becton Dickinson, California, United States) with standardized monoclonal antibodies, in accordance with previously published protocols.The Unifesp Ethics Committee approved this study.

Cytogenetics
Chromosome analyses were performed on short-term non-stimulated cultures from bone marrow cells.These were harvested after 24 hours and slides from these cultures were banded using the Trypsin-Giemsa technique (GTG). 13The metaphases were analyzed using an image capture system (Olympus BX microscope and Cytovision data process-ing through Applied Biosystems).The abnormalities were described in accordance with the International System for Human Cytogenetic Nomenclature (ISCN). 14The karyotype results were categorized as the following prognostic groups: favorable, with t(8;21), t (15;17) or inv (16); intermediate, with normal karyotype or structural or numerical changes not encompassed by the favorable or adverse risk groups; undefined, with metaphases unavailable; and unfavorable, with complex karyotype, monosomy of chromosome 5 or 7, deletions of the long arm of chromosome 5 or 3q abnormalities. 15lymerase chain reaction (PCR) Deoxyribonucleic acid (DNA) from bone marrow cells was obtained by means of proteinase K digestion, phenol-chloroform extraction and ethanol precipitation.This was then analyzed for the presence of FLT3/ITD + by means of the polymerase chain reaction using published primers (ITD/Dialab) for exons 14 and 15. 16 The amplification products were analyzed on 2% agarose gel and stained with ethidium bromide.

RESULTS
Forty eligible patients were enrolled: 18 men and 22 women (male/ female ratio of 0.82).The median age was 52 years (range 21-91 years).Karyotyping was successful in the cases of 33 patients (83%), while seven samples were unavailable (17%).According to the karyotyping results, the patients were classified into prognostic groups, stratified as shown in Table 1.
The median age of the patients with favorable karyotype was 38 years, while it was 54 years in the unfavorable group.Patients with FLT3/ITD + had a median age of 54 years (range 22-87 years), while patients without FLT3/ITD + had a median age of 53 years (range 21-91 years) (P = 0.97).
Patients with FLT3/ITD + had a median white blood cell (WBC) count of 9.2 x 10 9 /l (range: 0.6 x 10 9 /l to 202 x 10 9 /l) and patients without FLT3/ITD + had a median WBC count of 12.1 x 10 9 /l (range: 1.9 x 10 9 /l to 240 x 10 9 /l) (P = 0.22).Two patients with FLT3/ITD + had a WBC count of up to 50 x 10 9 /l, while seven patients without FLT3/ ITD + had a WBC count of up to 50 x 10 9 /l.The median WBC count of the patients with unfavorable karyotype was 21 x 10 9 /l, while it was 9.7 x 10 9 /l in the intermediate group and 9.3 x 10 9 /l in the group with favorable karyotype.Patients with FLT3/ITD + had a mean WBC count of 30.4 x 10 9 /l, while patients without FLT3/ITD + had a mean WBC count of 45.3 x 10 9 /l.

DISCUSSION
The real mechanism involved in the transformation from normal to leukemic cells remains unclear, and therefore some further light on this issue is needed.The presence of an abnormal karyotype in association with FLT3/ITD + might be one factor implicated in leukemogenesis, although not the only one.There is much evidence that chromosomal abnormalities and alterations to genomic DNA are implicated in deregulated proliferation, escape of apoptosis or defective apoptosis and subsequent excessive survival of neoplastic cells.
In the present study, we found that 25% of the patients presented FLT3/ITD + .According to the chromosomal abnormality classification, 13 patients had a favorable karyotype: three (23%) with FLT3/ITD + and 10 without mutations.Other  A = acute myeloid leukemia with recurrent chromosomal abnormalities; B = acute myeloid leukemia with multilineage dysplasia; C = acute myeloid leukemia after therapy (chemo or radio); D = acute myeloid leukemia not categorized; M = male; F = female.
Table 1.Cytogenetic findings, World Health Organization (WHO) criteria, prognostic group stratification and presence of FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD) with FLT3/ITD + had a poor prognosis, but in our study, we found that cases 11 and 21 remained alive for 31 and 27 months respectively, while only one patient (case 10) had a short survival (one month) after diagnosis.In fact, FLT3/ITD + did not influence the patients' survival rate.The favorable chromosome rearrangement may have prevailed over the FLT3 gene mutation.
Considering the importance and frequency of recurrent chromosomal abnormalities, AML with translocation t(8;21) results in an AML1/ETO fusion gene involved in blocking AML-1 dependent transcription activation.It also blocks the activating effects of CCAAT/enhancer-binding protein-alpha (C/EBPα), which is responsible for deregulated cell differentiation. 17,18We found AML with t(8;21) in 14% of our patients and in one with FLT3/ITD + (2.5%).This demonstrates that fusion rearrangement is not fundamental but contributes towards other genetic alterations such as mutations like FLT3/ITD + , 19 and towards leukemic events in some situations that are not fully understood.In fact, we recently reported on one case of myelodysplastic syndrome (MDS) in a follow-up study.This patient underwent a transformation to leukemia with AML/ETO fusion and acquisition of FLT3/ITD + , thereby corroborating Gilliland's "two hit" hypothesis for leukemogenesis. 20he CBF complex acts on genes that are crucial for myeloid proliferation.According to Reilly, 21 potential leukemic cells have CBF fusion genes but another hit is required for AML development.In cases of inversion of chromosome 16, the fusion protein CBFß/SMMHC is produced, which is a CBF inhibitor resulting in abnormal transcription of proliferative signals. 6In AML cases with inversion of chromosome 16, the consequence is inactivation of the CBFß protein, abnormal transcription of the proliferative signal and blocking of cell maturation. 6,17e observed AML with inv(16) in 7.5% of our patients and none with FLT3/ITD + .
In leukemia with t(15;17), blocking of cell differentiation is a consequence from the PML/RAR-α fusion protein, which leads to abnormal functioning of the retinoic acid receptor alpha. 22AML with t(15;17) was found in four of our patients (10% of the cases), and two of them had FLT3/ITD + .23,24 The incidence of FLT3/ITD + and normal karyotype in the present study was 40%, while Whitman et al. 25 reported this in 28% of their patients.Among the seven patients in our study without metaphases available, we found that three had FLT3/ITD + (42.8%), thus showing the need to apply several methods in order to comprehensively identify the disease, using other resources such as the PCR technique to characterize AML patients.26 In our study, the data are conflicting.We did not find any statistical difference in mean WBC counts between FLT3/ITD + patients and patients without FLT3/ITD + (P = 0.224).The median WBC count of patients with an unfavorable karyotype was higher than in cases with an intermediate or favorable karyotype.This shows that the higher count for the unfavorable group is a further factor for a poor prognosis.There was no age difference between AML patients with FLT3/ITD + and those without FLT3/ITD + , in agreement with a recent report by Levis and Small. 11 Altogether, for patients with de novo AML, the best approach includes cytogenetic analysis and subsequent categorization into favorable, intermediate, undefined or unfavorable groups and investigation of FLT3/ITD + .24

CONCLUSION
In conclusion, we found the same frequency of AML with FLT3/ ITD + in both the favorable and intermediate prognosis groups (3/10 and 4/10 patients were positive, respectively).There was only one patient with AML, FLT3/ITD + and unfavorable karyotype (i.e. the hypothetical worst clinical situation).The prognostic advantage of favorable cytogenetics among patients with FLT3/ITD + remains to be elucidated and the clinical significance is a matter of controversy.Thus, further investigation is warranted.
studies have reported that patients