Pediatric Hematology and Oncology, 31:131–137, 2014 C Informa Healthcare USA, Inc. Copyright ISSN: 0888-0018 print / 1521-0669 online DOI: 10.3109/08880018.2013.870624
ORIGINAL ARTICLE Myeloid Leukemias and Myeloproliferative Disease
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FLT3-ITD Mutation in Relation to FLT3 Expression in Pediatric AML: A Prospective Study from India Surender Kumar Sharawat,1 Radhika Bakhshi,2 Sreenivas Vishnubhatla,3 Ritu Gupta,4 and Sameer Bakhshi5 1 Department of Medical Oncology, Dr. B. R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India; 2 Department of Biomedical Sciences, Shaheed Rajguru College of Applied Sciences, University of Delhi, New Delhi, India; 3 Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India; 4 Laboratory Oncology Unit, Dr. B. R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India; 5 Department of Medical Oncology, Dr. B. R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India
There is lack of data with regard to FLT3 expression in FLT3-ITD positive pediatric AML patients. Further, FLT3-ITD has not been systematically analyzed for outcome from Indian subcontinent. Amongst 64 consecutive pediatric AML patients, FLT3-ITD was present in 12 (19%) patients. All patients with FLT3-ITD achieved CR; those with FLT3-ITD mutation had inferior DFS (P = .029). FLT3 expression by flow-cytometry was observed in all FLT3-ITD positive patients, whereas 40/52 (77%) FLT3-ITD negative patients expressed FLT3 (P = .06). FLT3 expression in 12 FLT3-ITD positive patients was unable to show an association between FLT3 expression and outcome. In FLT3-ITD negative patients, higher surface expression of FLT3 significantly predicted poor EFS (P = .001) and OS (P = .007). Keywords acute myeloid leukemia, FLT3 expression, FLT3-ITD mutation, pediatric, prognosis
INTRODUCTION Fms like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase expressed on surface of hematopoietic progenitor cells. Role for FLT3 in leukemogenesis became evident when an unexpectedly long transcript in the juxtamembrane domain was noted in nearly 30% of the acute myeloid leukemia (AML) patients [1]. The duplications were always in-frame but varied considerably in length and exact location from patient to patient. FLT3 internal tandem duplication (ITD) creates an in-frame transcript, which is translated into a protein that is constitutively activated, and promotes ligand-independent proliferation and survival of myeloblasts [2]. FLT3-ITD has been shown to be associated with increased transcript level of FLT3 in adult AML [3]. Received 12 August 2013; accepted 26 November 2013. Surender Kumar Sharawat is thankful to Indian Council of Medical Research (ICMR), New Delhi, India for a Senior Research Fellowship. Address correspondence to Dr. Sameer Bakhshi, Additional Professor of Pediatric Oncology, Department of Medical Oncology, Dr. B. R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi 110 029, India. E-mail: [email protected]
S. K. Sharawat et al.
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In pediatric AML, prognostic significance of FLT3-ITD has been well documented [4–6]. Few studies from Indian subcontinent have shown that FLT3-ITD is observed in 17–20% of AML patients [7–9]. However, there is no data wherein FLT3-ITD has been systematically analyzed for outcome from India. Further, data for FLT3 surface expression in FLT3-ITD positive pediatric patients with AML are limited [10, 11]. In view of these lacunae, we conducted a prospective study in pediatric AML patients wherein we analyzed FLT3-ITD and its correlation with baseline patient characteristics, FLT3 expression and outcome. MATERIAL AND METHODS Patient Selection, Treatment, and Sampling Newly diagnosed consecutive de novo AML patients ≤ 18 years age were enrolled from March 2008-June 2010 prospectively. Acute promyelocytic leukemia (APL) was excluded. The study was approved by institute ethics committee, and prior to therapy an informed consent was taken for evaluation of peripheral blood and/or bone marrow. Bone marrow or peripheral blood (if peripheral blast count was more than 30%) (5 ml) was collected in ethylenediaminetetraacetic acid (EDTA) coated sterile vacutainer (BD). Patients were categorized into good, intermediate and poor risk cytogenetic groups [12]. Induction 3+7 regimen (Daunorubicin and cytosine arabinoside) with Daunorubicin at 60 mg/m2 for three days were administered to all patients. Patients failed to achieve complete remission (CR) after first induction, received combination of cytosine arabinoside, daunorubicin, and etoposide (ADE) protocol. Patients who were not in CR after two inductions were declared refractory. Post remission, patients received three cycles of high dose cytarabine at 18 gm/m2 /cycle [13]. FLT3-ITD Mutation Detection FLT-3 ITD was screened by amplification of mutated gene by polymerase chain reaction (PCR) using specific primers. DNA was isolated from 5×106 cells using DNA isolation kit (Qiagen). DNA was subjected to qualitative and quantitative analysis by gel electrophoresis and spectrophotometry (Nano Drop). 150 mg was used for FLT3-ITD detection by PCR using following primers: Forward 5 -GTAAAACGACGGCCAGTGCAGAACTGCCTATTCCTA-3 Reverse 5 CAGGAAACAGCTATGACCTGTCCTTCCACTATACTGT-3 with 35 cycles and annealing temperature 53◦ C. PCR product was resolved on 3.5% agarose gel. Flow Cytometry Mononuclear cells (MNCs) were separated from peripheral blood or bone marrow using density gradient media (Ficoll hypaque; Sigma 1077, Sigma–Aldrich, Inc. USA). Isolated MNCs were washed twice and finally resuspended in phosphate buffered saline. Myeloblasts were characterized and gated by side scatter and CD 45 antibody conjugated with fluorochromes fluorescein isothiocyanate. Median-fluorescenceintensity (MFI) and percentage expression of CD135 was evaluated on dim CD45 gated myeloblasts by flow-cytometry [14]. Total 20,000 events were acquired on multi parameter FACS Calibur flowcytometer, and analysis was performed using cell quest pro software (Becton Dickinson, San Jose, CS, USA). Statistical Analysis Positive or negative surface expression for FLT3 was considered as expression greater than or less than 20%. However, median values of quantitative variables were used as a cut off point for categorization into high or low expression. Data was expressed as median (range) and mean ± SD. End point for survival data was 5th September 2012. Differences between values were determined using Kruskal Wallis test and Pediatric Hematology and Oncology
Clinical Significance of FLT3 Mutation and Expression in Pediatric AML
TABLE 1 Relationship of FLT3 ITD Mutation with Baseline Patient Characteristics and Survival FLT3 ITD positive (N = 12)
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Variables
Median age in years (Range) Sex (Male:Female) Median hemoglobin (g/dL) (Range) Median WBC (/mm3) (Range) Median platelets (/mm3) (Range) Median PB blast% (Range) Median BM blast% (Range) Cytogenetics (n = 50) Good risk Intermediate Poor risk FAB AML subtype M0 M1 M2 M4 M5 M7 CR EFS∗ ± S.E (CI) DFS∗ ± S.E (CI) OS∗ ± S.E (CI) Median EFS Median DFS Median OS
FLT3 ITD negative (N = 52)
P
A. Relationship FLT3 ITD status with baseline patient characteristics 7 (2–16) 10.0 (1–18) .268 3:1 8.0 (3.4–11.2)
3:1 6.65 (3.3–14.5)
.998 .575
17250 (3900–98000)
22700 (700–350000)
.500
28500 (3500–165000)
30000 (1900–272550)
.812
43.0 (2–91) 70.0 (15–100)
60.0 (0–95) 67.5 (20–100)
.851 .829
3 (30.0%) 4 (40.0%) 3 (30.0%)
11 (29%) 20 (53%) 7 (18%)
.601
3 (24%) 5 (42%) 2 (17%) 1 (8%) 1 (8%)
3 (6%) 6 (12%) 30 (58%) 8 (15%) 4 (8%) 1 (2%)
.727
B. Relationship of FLT3 ITD status with outcome 12(100%) 42(78%) 16.67 ± 10.76 (2.65–41.30) 33.44 ± 6.61 (21.00–46.34) 18.18 ± 11.63 (2.85–44.17) 51.03 ± 8.55 (33.42–66.16) 24.44 ± 13.92 (4.46–52.84) 39.70 ± 7.12 (25.88–53.19) 10.8 Months 11.0 Months 8.5 Months 17.0 Months 23 Months
.089 .660 .029 .777
∗
Survival at 54 months; WBC, White blood cells; PB, Peripheral Blood; BM, Bone Marrow; FAB, French-American-British; FLT 3ITD, FMS like tyrosine kinase 3 internal tandem duplication; CR, Complete Remission; EFS, Event Free Survival; DFS, Disease Free Survival; OS, overall survival; S.E, Standard Error; CI, 95% confidence interval.
independent Students t test. Outcome analysis was assessed based on event free survival (EFS), disease free survival (DFS), and overall survival (OS). EFS was defined as time between diagnosis and first event such as failure to achieve CR, relapse, or death. DFS was defined as time from CR until relapse or date of last follow-up. OS was defined as time between diagnosis and death or last follow-up. Kaplan Meier curves were obtained for survival analysis followed by log rank test. P value ≤ .05 was considered significant. All statistical analysis was done using STATA 11.0. RESULTS Baseline Characteristics, FLT3-ITD Status, and Outcome During the study period, total 68 pediatric patients were registered in our centre and after exclusion [Biphenotypic leukemia (n = 2), APML (n = 1), treatment not taken (n = 1)] total 64 patients were enrolled in the study with median age 10 years (range: 1–18). FLT3-ITD was present in 12 (19%) patients. None of the baseline patient characteristics correlated with FLT3-ITD (Table IA). C Informa Healthcare USA, Inc. Copyright
S. K. Sharawat et al.
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CR was achieved in 53 (82.8%) patients. At median follow-up of 18.3 (range: .5–53.4) months, EFS was 30.2 ± 5.8% (19.3–41.7), DFS 43.03 ± 7.3% (28.4–56.7) and OS 37.1 ± 6.3% (24.8–49.4). All patients with FLT3-ITD mutation achieved CR (Table 2); FLT3-ITD mutated patients were associated with inferior EFS, DFS, and OS, but difference was significant only for DFS (Table IB). Correlation of FLT3-ITD with FLT3 Expression FLT3 surface expression was positive (>20% expression) in 52 (81%) patients. Positive FLT3 expression was observed in all patients who were positive for FLT3-ITD, whereas 40/52 (77%) patients without FLT3-ITD expressed FLT3 (P = .06). Mean FLT3 expression was 46% in FLT3-ITD positive patients versus 49% in those who were FLT3-ITD negative (P = .798). FLT3 surface expression in 12 FLT3-ITD positive patients was unable to show an association between FLT3 expression and outcome (Figure 1A-C). In FLT3-ITD negative patients, there was no significant difference in the cytogenetic risk groups between those with high FLT3 surface expression versus those with low FLT3 surface expression (Table 3). Notably, in FLT3 ITD negative patients, CR was observed in 17/28 (60.7%) patients in those with high FLT3 expression, versus 24/24 (100%) in those with low FLT3 expression (P = .001); of the 11 induction failures in this subgroup, eight were refractory to treatment while three died of infection-related complications. Additionally, in FLT3 ITD negative patients, higher surface expression of FLT3 on the blasts significantly predicted poor EFS and OS, but no difference in DFS was observed when compared with the cohort with low FLT3 expression (Figure 1D-F).
DISCUSSION In pediatric AML, incidence of FLT3-ITD has been reported to vary from 6.6–16.5% in Western countries [4, 15], whereas the only study from India analyzing pediatric AML patients had 20% incidence of FLT3-ITD mutation [8] as compared to 18.7% in the current study. The prognosis of patients with FLT3-ITD is poor in both adult and pediatric AML patients. Interestingly, in the current study, the impact of mutation was minimal at initial response to therapy, as evidenced by the fact that all FLT3-ITD positive patients went into remission. However, on follow-up, there was a significantly higher relapse rate in those who were positive for FLT3-ITD as evidenced by poor DFS, although a significant difference was not observed for EFS and OS. The increased early events in the group of patients who were FLT3 ITD negative but with high expression of FLT3, represented refractory AML and induction deaths, and the events resulted in a significantly inferior EFS and OS in this subgroup when compared with patients who were FLT3 ITD negative, but had low expression of FLT3. The difference in EFS and OS was most likely due to the difference in CR rate and deaths due to refractory disease rather than relapses as there was no difference in DFS. FLT3 high expression predicted poor EFS and OS in the subgroup of patients who were negative for FLT3-ITD, whereas FLT3 expression did not predict survival in the subgroup of FLT3-ITD positive patients. The limitation of the study is the small sample size and thus the results need to be validated in larger cohorts. The strength of the study is that this is the only study that has evaluated FLT3-ITD mutation along with FLT3 expression in pediatric AML. This is the first study from the Indian subcontinent that has explored the effect of FLT3-ITD on outcome. We observed a relatively higher incidence of FLT3-ITD mutation in our cohort as compared to Western studies. FLT3 expression did not affect outcome in the FLT3-ITD positive Pediatric Hematology and Oncology
∗
Not available Poor risk Poor risk Good risk Good risk Poor risk Intermediate risk Intermediate risk Good risk Not available Intermediate risk Intermediate risk
Cytogenetics
39.97 (Low expression) 41.61 (Low expression) 82.84 (High expression) 67.07 (High expression) 34.91 (Low expression) 27.32 (Low expression) 35.02 (Low expression) 21.57 (Low expression) 31.96 (Low expression) 21.47 (Low expression) 99.91 (High expression) 53.29(High expression)
Median FLT3 expression (High/Low)∗ Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Remission 4.9 8.4 7.7 9.6 8.1 11.9 10.8 15.8 12.6 13.9 46.3 52.8
EFS (Months)
Median value of entire cohort was used as cut off for high and low expression of FLT3; # LFU lost to follow up
1 2 3 4 5 6 7 8 9 10 11 12
S.No
TABLE 2 Detailed Presentation of Patients with FLT3 ITD Mutation (N = 12)
1 7 5.7 8.5 6.3 9.5 7.4 14.7 11.4 12.8 39.3 45.6
DFS (Months)
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4.9 8.6 10.9 11.1 12.5 17.4 18.7 26.5 35 35.9 46.3 52.8
OS (Months)
Died Relapse LFU# Died (Relapsed) Died (Relapsed) Died (Relapsed) Died (Relapsed) Died (Relapsed) CR-Relapse-CR CR-Relapse-CR Died (Relapsed) CR CR
Status
FIGURE 1 (A-F): Survival of FLT3 ITD positive and negative patients with FLT3 surface expression (percentage). Median values of FLT3 surface expression were used as cut off point for categorization into high or low expression.
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Clinical Significance of FLT3 Mutation and Expression in Pediatric AML
TABLE 3 Cytogenetic Risk Group of Patients Without FLT3 ITD Mutation (N = 52) Cytogenetic risk group Not available Good risk Intermediate risk Poor risk
High FLT3 expression (N = 28)
Low FLT3 Expression (N = 24)
8 (28.6%) 6 (21.4%) 10 (35.7%) 4 (14.3%)
6 (25%) 5 (20.8%) 10 (41.7%) 3 (12.5%)
P .932
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patients. Despite all FLT3-ITD positive patients achieving CR, they had a significantly inferior DFS. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. Source of funding: Institute Research Grant from All India Institute of Medical Sciences, New Delhi, India. REFERENCES [1] Nakao M, Yokota S, Iwai T, et al. Internal tandem duplication of the FLT3 gene found in acute myeloid leukemia. Leukemia. 1996;10(12):1911–1918. [2] Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood. 2002;100(5):1532–1542. [3] Ozeki K, Kiyoi H, Hirose Y, et al. Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia. Blood. 2004;103(5):1901–1908. [4] Kang HJ, Hong SH, Kim IH, et al. Prognostic significance of FLT3 mutations in pediatric nonpromyelocytic acute myeloid leukemia. Leuk Res. 2005;29(6):617–623. [5] Meshinchi S, Alonzo TA, Stirewalt DL, et al. Clinical implications of FLT3 mutations in pediatric AML. Blood. 2006;108(12):3654–3661. [6] Shimada A, Taki T, Tabuchi K, et al. Tandem duplications of MLL and FLT3 are correlated with poor prognoses in pediatric acute myeloid leukemia: a study of the Japanese childhood AML Cooperative Study Group. Pediatr Blood Cancer. 2008;50(2):264–269. [7] Ahmad F, Mandava S, Das BR. Analysis of FLT3-ITD and FLT3-Asp835 mutations in de novo acute myeloid leukemia: evaluation of incidence, distribution pattern, correlation with cytogenetics and characterization of internal tandem duplication from Indian population. Cancer Invest. 2010;28(1):63–73. [8] Ghosh K, Swaminathan S, Madkaikar M, et al. FLT3 and NPM1 mutations in a cohort of AML patients and detection of a novel mutation in tyrosine kinase domain of FLT3 gene from Western India. Ann Hematol. 2012;91(11):1703–1712. [9] Sazawal S, Kumar B, Hasan SK, et al. Haematological & molecular profile of acute myelogenous leukaemia in India. Ind J Med Res. 2009;129(3):256–261. [10] Vora HH, Shukla SN, Brahambhatt BV, et al. Clinical relevance of FLT3 receptor protein expression in Indian patients with acute leukemia. Asia-Pacific J Clin Oncol. 2010:6(4):306–319. [11] Brown P, Alonzo TA, Gerbling RB, et al. High level expression of wild type FLT3 is associated with poor outcome and selective sensitivity to FLT3 inhibitors in childhood acute myeloid leukemia: a children’s oncology group study. ASH Ann Meeting Abst. 2008;112:147. [12] Grimwade D, Walker H, Oliver F, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood. 1998:92(7):2322–2333. [13] Sharawat SK, Bakhshi R, Vishnubhatla S, et al. BAX/BCL2 RMFI ratio predicts better induction response in pediatric patients with acute myeloid leukemia. Pediatr Blood Cancer. 2013:60(8):E63-66. doi: 10.1002/pbc.24518. [14] Sharawat SK, Gupta R, Raina V, et al. Increased co-expression of c-KIT and FLT-3 receptors on myeloblasts: independent predictor of poor outcome in pediatric acute myeloid leukemia. Cytometry B. 2013 (in press). [15] Meshinchi S, Woods WG, Stirewalt DL, et al. Prevalence and prognostic significance of FLT3 internal tandem duplication in pediatric acute myeloid leukemia. Blood. 2001;97(1):89–94. C Informa Healthcare USA, Inc. Copyright
ORIGINAL ARTICLE Myeloid Leukemias and Myeloproliferative Disease
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FLT3-ITD Mutation in Relation to FLT3 Expression in Pediatric AML: A Prospective Study from India Surender Kumar Sharawat,1 Radhika Bakhshi,2 Sreenivas Vishnubhatla,3 Ritu Gupta,4 and Sameer Bakhshi5 1 Department of Medical Oncology, Dr. B. R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India; 2 Department of Biomedical Sciences, Shaheed Rajguru College of Applied Sciences, University of Delhi, New Delhi, India; 3 Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India; 4 Laboratory Oncology Unit, Dr. B. R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India; 5 Department of Medical Oncology, Dr. B. R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India
There is lack of data with regard to FLT3 expression in FLT3-ITD positive pediatric AML patients. Further, FLT3-ITD has not been systematically analyzed for outcome from Indian subcontinent. Amongst 64 consecutive pediatric AML patients, FLT3-ITD was present in 12 (19%) patients. All patients with FLT3-ITD achieved CR; those with FLT3-ITD mutation had inferior DFS (P = .029). FLT3 expression by flow-cytometry was observed in all FLT3-ITD positive patients, whereas 40/52 (77%) FLT3-ITD negative patients expressed FLT3 (P = .06). FLT3 expression in 12 FLT3-ITD positive patients was unable to show an association between FLT3 expression and outcome. In FLT3-ITD negative patients, higher surface expression of FLT3 significantly predicted poor EFS (P = .001) and OS (P = .007). Keywords acute myeloid leukemia, FLT3 expression, FLT3-ITD mutation, pediatric, prognosis
INTRODUCTION Fms like tyrosine kinase 3 (FLT3) is a type III receptor tyrosine kinase expressed on surface of hematopoietic progenitor cells. Role for FLT3 in leukemogenesis became evident when an unexpectedly long transcript in the juxtamembrane domain was noted in nearly 30% of the acute myeloid leukemia (AML) patients [1]. The duplications were always in-frame but varied considerably in length and exact location from patient to patient. FLT3 internal tandem duplication (ITD) creates an in-frame transcript, which is translated into a protein that is constitutively activated, and promotes ligand-independent proliferation and survival of myeloblasts [2]. FLT3-ITD has been shown to be associated with increased transcript level of FLT3 in adult AML [3]. Received 12 August 2013; accepted 26 November 2013. Surender Kumar Sharawat is thankful to Indian Council of Medical Research (ICMR), New Delhi, India for a Senior Research Fellowship. Address correspondence to Dr. Sameer Bakhshi, Additional Professor of Pediatric Oncology, Department of Medical Oncology, Dr. B. R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi 110 029, India. E-mail: [email protected]
S. K. Sharawat et al.
Pediatr Hematol Oncol Downloaded from informahealthcare.com by University of Bath on 10/04/14 For personal use only.
In pediatric AML, prognostic significance of FLT3-ITD has been well documented [4–6]. Few studies from Indian subcontinent have shown that FLT3-ITD is observed in 17–20% of AML patients [7–9]. However, there is no data wherein FLT3-ITD has been systematically analyzed for outcome from India. Further, data for FLT3 surface expression in FLT3-ITD positive pediatric patients with AML are limited [10, 11]. In view of these lacunae, we conducted a prospective study in pediatric AML patients wherein we analyzed FLT3-ITD and its correlation with baseline patient characteristics, FLT3 expression and outcome. MATERIAL AND METHODS Patient Selection, Treatment, and Sampling Newly diagnosed consecutive de novo AML patients ≤ 18 years age were enrolled from March 2008-June 2010 prospectively. Acute promyelocytic leukemia (APL) was excluded. The study was approved by institute ethics committee, and prior to therapy an informed consent was taken for evaluation of peripheral blood and/or bone marrow. Bone marrow or peripheral blood (if peripheral blast count was more than 30%) (5 ml) was collected in ethylenediaminetetraacetic acid (EDTA) coated sterile vacutainer (BD). Patients were categorized into good, intermediate and poor risk cytogenetic groups [12]. Induction 3+7 regimen (Daunorubicin and cytosine arabinoside) with Daunorubicin at 60 mg/m2 for three days were administered to all patients. Patients failed to achieve complete remission (CR) after first induction, received combination of cytosine arabinoside, daunorubicin, and etoposide (ADE) protocol. Patients who were not in CR after two inductions were declared refractory. Post remission, patients received three cycles of high dose cytarabine at 18 gm/m2 /cycle [13]. FLT3-ITD Mutation Detection FLT-3 ITD was screened by amplification of mutated gene by polymerase chain reaction (PCR) using specific primers. DNA was isolated from 5×106 cells using DNA isolation kit (Qiagen). DNA was subjected to qualitative and quantitative analysis by gel electrophoresis and spectrophotometry (Nano Drop). 150 mg was used for FLT3-ITD detection by PCR using following primers: Forward 5 -GTAAAACGACGGCCAGTGCAGAACTGCCTATTCCTA-3 Reverse 5 CAGGAAACAGCTATGACCTGTCCTTCCACTATACTGT-3 with 35 cycles and annealing temperature 53◦ C. PCR product was resolved on 3.5% agarose gel. Flow Cytometry Mononuclear cells (MNCs) were separated from peripheral blood or bone marrow using density gradient media (Ficoll hypaque; Sigma 1077, Sigma–Aldrich, Inc. USA). Isolated MNCs were washed twice and finally resuspended in phosphate buffered saline. Myeloblasts were characterized and gated by side scatter and CD 45 antibody conjugated with fluorochromes fluorescein isothiocyanate. Median-fluorescenceintensity (MFI) and percentage expression of CD135 was evaluated on dim CD45 gated myeloblasts by flow-cytometry [14]. Total 20,000 events were acquired on multi parameter FACS Calibur flowcytometer, and analysis was performed using cell quest pro software (Becton Dickinson, San Jose, CS, USA). Statistical Analysis Positive or negative surface expression for FLT3 was considered as expression greater than or less than 20%. However, median values of quantitative variables were used as a cut off point for categorization into high or low expression. Data was expressed as median (range) and mean ± SD. End point for survival data was 5th September 2012. Differences between values were determined using Kruskal Wallis test and Pediatric Hematology and Oncology
Clinical Significance of FLT3 Mutation and Expression in Pediatric AML
TABLE 1 Relationship of FLT3 ITD Mutation with Baseline Patient Characteristics and Survival FLT3 ITD positive (N = 12)
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Variables
Median age in years (Range) Sex (Male:Female) Median hemoglobin (g/dL) (Range) Median WBC (/mm3) (Range) Median platelets (/mm3) (Range) Median PB blast% (Range) Median BM blast% (Range) Cytogenetics (n = 50) Good risk Intermediate Poor risk FAB AML subtype M0 M1 M2 M4 M5 M7 CR EFS∗ ± S.E (CI) DFS∗ ± S.E (CI) OS∗ ± S.E (CI) Median EFS Median DFS Median OS
FLT3 ITD negative (N = 52)
P
A. Relationship FLT3 ITD status with baseline patient characteristics 7 (2–16) 10.0 (1–18) .268 3:1 8.0 (3.4–11.2)
3:1 6.65 (3.3–14.5)
.998 .575
17250 (3900–98000)
22700 (700–350000)
.500
28500 (3500–165000)
30000 (1900–272550)
.812
43.0 (2–91) 70.0 (15–100)
60.0 (0–95) 67.5 (20–100)
.851 .829
3 (30.0%) 4 (40.0%) 3 (30.0%)
11 (29%) 20 (53%) 7 (18%)
.601
3 (24%) 5 (42%) 2 (17%) 1 (8%) 1 (8%)
3 (6%) 6 (12%) 30 (58%) 8 (15%) 4 (8%) 1 (2%)
.727
B. Relationship of FLT3 ITD status with outcome 12(100%) 42(78%) 16.67 ± 10.76 (2.65–41.30) 33.44 ± 6.61 (21.00–46.34) 18.18 ± 11.63 (2.85–44.17) 51.03 ± 8.55 (33.42–66.16) 24.44 ± 13.92 (4.46–52.84) 39.70 ± 7.12 (25.88–53.19) 10.8 Months 11.0 Months 8.5 Months 17.0 Months 23 Months
.089 .660 .029 .777
∗
Survival at 54 months; WBC, White blood cells; PB, Peripheral Blood; BM, Bone Marrow; FAB, French-American-British; FLT 3ITD, FMS like tyrosine kinase 3 internal tandem duplication; CR, Complete Remission; EFS, Event Free Survival; DFS, Disease Free Survival; OS, overall survival; S.E, Standard Error; CI, 95% confidence interval.
independent Students t test. Outcome analysis was assessed based on event free survival (EFS), disease free survival (DFS), and overall survival (OS). EFS was defined as time between diagnosis and first event such as failure to achieve CR, relapse, or death. DFS was defined as time from CR until relapse or date of last follow-up. OS was defined as time between diagnosis and death or last follow-up. Kaplan Meier curves were obtained for survival analysis followed by log rank test. P value ≤ .05 was considered significant. All statistical analysis was done using STATA 11.0. RESULTS Baseline Characteristics, FLT3-ITD Status, and Outcome During the study period, total 68 pediatric patients were registered in our centre and after exclusion [Biphenotypic leukemia (n = 2), APML (n = 1), treatment not taken (n = 1)] total 64 patients were enrolled in the study with median age 10 years (range: 1–18). FLT3-ITD was present in 12 (19%) patients. None of the baseline patient characteristics correlated with FLT3-ITD (Table IA). C Informa Healthcare USA, Inc. Copyright
S. K. Sharawat et al.
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CR was achieved in 53 (82.8%) patients. At median follow-up of 18.3 (range: .5–53.4) months, EFS was 30.2 ± 5.8% (19.3–41.7), DFS 43.03 ± 7.3% (28.4–56.7) and OS 37.1 ± 6.3% (24.8–49.4). All patients with FLT3-ITD mutation achieved CR (Table 2); FLT3-ITD mutated patients were associated with inferior EFS, DFS, and OS, but difference was significant only for DFS (Table IB). Correlation of FLT3-ITD with FLT3 Expression FLT3 surface expression was positive (>20% expression) in 52 (81%) patients. Positive FLT3 expression was observed in all patients who were positive for FLT3-ITD, whereas 40/52 (77%) patients without FLT3-ITD expressed FLT3 (P = .06). Mean FLT3 expression was 46% in FLT3-ITD positive patients versus 49% in those who were FLT3-ITD negative (P = .798). FLT3 surface expression in 12 FLT3-ITD positive patients was unable to show an association between FLT3 expression and outcome (Figure 1A-C). In FLT3-ITD negative patients, there was no significant difference in the cytogenetic risk groups between those with high FLT3 surface expression versus those with low FLT3 surface expression (Table 3). Notably, in FLT3 ITD negative patients, CR was observed in 17/28 (60.7%) patients in those with high FLT3 expression, versus 24/24 (100%) in those with low FLT3 expression (P = .001); of the 11 induction failures in this subgroup, eight were refractory to treatment while three died of infection-related complications. Additionally, in FLT3 ITD negative patients, higher surface expression of FLT3 on the blasts significantly predicted poor EFS and OS, but no difference in DFS was observed when compared with the cohort with low FLT3 expression (Figure 1D-F).
DISCUSSION In pediatric AML, incidence of FLT3-ITD has been reported to vary from 6.6–16.5% in Western countries [4, 15], whereas the only study from India analyzing pediatric AML patients had 20% incidence of FLT3-ITD mutation [8] as compared to 18.7% in the current study. The prognosis of patients with FLT3-ITD is poor in both adult and pediatric AML patients. Interestingly, in the current study, the impact of mutation was minimal at initial response to therapy, as evidenced by the fact that all FLT3-ITD positive patients went into remission. However, on follow-up, there was a significantly higher relapse rate in those who were positive for FLT3-ITD as evidenced by poor DFS, although a significant difference was not observed for EFS and OS. The increased early events in the group of patients who were FLT3 ITD negative but with high expression of FLT3, represented refractory AML and induction deaths, and the events resulted in a significantly inferior EFS and OS in this subgroup when compared with patients who were FLT3 ITD negative, but had low expression of FLT3. The difference in EFS and OS was most likely due to the difference in CR rate and deaths due to refractory disease rather than relapses as there was no difference in DFS. FLT3 high expression predicted poor EFS and OS in the subgroup of patients who were negative for FLT3-ITD, whereas FLT3 expression did not predict survival in the subgroup of FLT3-ITD positive patients. The limitation of the study is the small sample size and thus the results need to be validated in larger cohorts. The strength of the study is that this is the only study that has evaluated FLT3-ITD mutation along with FLT3 expression in pediatric AML. This is the first study from the Indian subcontinent that has explored the effect of FLT3-ITD on outcome. We observed a relatively higher incidence of FLT3-ITD mutation in our cohort as compared to Western studies. FLT3 expression did not affect outcome in the FLT3-ITD positive Pediatric Hematology and Oncology
∗
Not available Poor risk Poor risk Good risk Good risk Poor risk Intermediate risk Intermediate risk Good risk Not available Intermediate risk Intermediate risk
Cytogenetics
39.97 (Low expression) 41.61 (Low expression) 82.84 (High expression) 67.07 (High expression) 34.91 (Low expression) 27.32 (Low expression) 35.02 (Low expression) 21.57 (Low expression) 31.96 (Low expression) 21.47 (Low expression) 99.91 (High expression) 53.29(High expression)
Median FLT3 expression (High/Low)∗ Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
Remission 4.9 8.4 7.7 9.6 8.1 11.9 10.8 15.8 12.6 13.9 46.3 52.8
EFS (Months)
Median value of entire cohort was used as cut off for high and low expression of FLT3; # LFU lost to follow up
1 2 3 4 5 6 7 8 9 10 11 12
S.No
TABLE 2 Detailed Presentation of Patients with FLT3 ITD Mutation (N = 12)
1 7 5.7 8.5 6.3 9.5 7.4 14.7 11.4 12.8 39.3 45.6
DFS (Months)
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4.9 8.6 10.9 11.1 12.5 17.4 18.7 26.5 35 35.9 46.3 52.8
OS (Months)
Died Relapse LFU# Died (Relapsed) Died (Relapsed) Died (Relapsed) Died (Relapsed) Died (Relapsed) CR-Relapse-CR CR-Relapse-CR Died (Relapsed) CR CR
Status
FIGURE 1 (A-F): Survival of FLT3 ITD positive and negative patients with FLT3 surface expression (percentage). Median values of FLT3 surface expression were used as cut off point for categorization into high or low expression.
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Clinical Significance of FLT3 Mutation and Expression in Pediatric AML
TABLE 3 Cytogenetic Risk Group of Patients Without FLT3 ITD Mutation (N = 52) Cytogenetic risk group Not available Good risk Intermediate risk Poor risk
High FLT3 expression (N = 28)
Low FLT3 Expression (N = 24)
8 (28.6%) 6 (21.4%) 10 (35.7%) 4 (14.3%)
6 (25%) 5 (20.8%) 10 (41.7%) 3 (12.5%)
P .932
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patients. Despite all FLT3-ITD positive patients achieving CR, they had a significantly inferior DFS. Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article. Source of funding: Institute Research Grant from All India Institute of Medical Sciences, New Delhi, India. REFERENCES [1] Nakao M, Yokota S, Iwai T, et al. Internal tandem duplication of the FLT3 gene found in acute myeloid leukemia. Leukemia. 1996;10(12):1911–1918. [2] Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood. 2002;100(5):1532–1542. [3] Ozeki K, Kiyoi H, Hirose Y, et al. Biologic and clinical significance of the FLT3 transcript level in acute myeloid leukemia. Blood. 2004;103(5):1901–1908. [4] Kang HJ, Hong SH, Kim IH, et al. Prognostic significance of FLT3 mutations in pediatric nonpromyelocytic acute myeloid leukemia. Leuk Res. 2005;29(6):617–623. [5] Meshinchi S, Alonzo TA, Stirewalt DL, et al. Clinical implications of FLT3 mutations in pediatric AML. Blood. 2006;108(12):3654–3661. [6] Shimada A, Taki T, Tabuchi K, et al. Tandem duplications of MLL and FLT3 are correlated with poor prognoses in pediatric acute myeloid leukemia: a study of the Japanese childhood AML Cooperative Study Group. Pediatr Blood Cancer. 2008;50(2):264–269. [7] Ahmad F, Mandava S, Das BR. Analysis of FLT3-ITD and FLT3-Asp835 mutations in de novo acute myeloid leukemia: evaluation of incidence, distribution pattern, correlation with cytogenetics and characterization of internal tandem duplication from Indian population. Cancer Invest. 2010;28(1):63–73. [8] Ghosh K, Swaminathan S, Madkaikar M, et al. FLT3 and NPM1 mutations in a cohort of AML patients and detection of a novel mutation in tyrosine kinase domain of FLT3 gene from Western India. Ann Hematol. 2012;91(11):1703–1712. [9] Sazawal S, Kumar B, Hasan SK, et al. Haematological & molecular profile of acute myelogenous leukaemia in India. Ind J Med Res. 2009;129(3):256–261. [10] Vora HH, Shukla SN, Brahambhatt BV, et al. Clinical relevance of FLT3 receptor protein expression in Indian patients with acute leukemia. Asia-Pacific J Clin Oncol. 2010:6(4):306–319. [11] Brown P, Alonzo TA, Gerbling RB, et al. High level expression of wild type FLT3 is associated with poor outcome and selective sensitivity to FLT3 inhibitors in childhood acute myeloid leukemia: a children’s oncology group study. ASH Ann Meeting Abst. 2008;112:147. [12] Grimwade D, Walker H, Oliver F, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood. 1998:92(7):2322–2333. [13] Sharawat SK, Bakhshi R, Vishnubhatla S, et al. BAX/BCL2 RMFI ratio predicts better induction response in pediatric patients with acute myeloid leukemia. Pediatr Blood Cancer. 2013:60(8):E63-66. doi: 10.1002/pbc.24518. [14] Sharawat SK, Gupta R, Raina V, et al. Increased co-expression of c-KIT and FLT-3 receptors on myeloblasts: independent predictor of poor outcome in pediatric acute myeloid leukemia. Cytometry B. 2013 (in press). [15] Meshinchi S, Woods WG, Stirewalt DL, et al. Prevalence and prognostic significance of FLT3 internal tandem duplication in pediatric acute myeloid leukemia. Blood. 2001;97(1):89–94. C Informa Healthcare USA, Inc. Copyright
