Elyamany

FLT3 Internal Tandem Duplication and D835 Mutations in Patients with Acute Lymphoblastic Leukemia and its Clinical Significance

Ghaleb Elyamany1,2, Mohammed Awad2, Omar Alsuhaibani2, Kamal Fadalla3, Omer Al Sharif4, Mohammad Al Shahrani4, FahadAlabbas4 and Abdulaziz Al-Abulaaly3

1 Department of Hematology and Blood Bank, Theodor Bilharz Research Institute.
2 Dept. of Central Military Laboratory, Prince Sultan Military Medical City, Riyadh, Saudi Arabia.
3 Dept. of Adult Clinical Hematology and Stem cell Therapy, Prince Sultan Military Medical City, Riyadh, Saudi Arabia.
4 Dept. of Pediatric Hematology/Oncology, Prince Sultan Military Medical City, Riyadh, Saudi Arabia.

Corresponding author: Ghaleb Elyamany MD, PhD, FRCPC. Lecturer, Theodor Bilharz Research Institute, Egypt Consultant Hematopathology, Prince Sultan Military Medical City, SA. PO Box 7897, Riyadh 11159, Kingdom of Saudi Arabia. Tel: 001 966 1 4777714 extension 28140, FAX 01 4783033. E-mail: ghalebelyamany@yahoo.com

Published: June 1, 2014
Received: January 9, 2014
Accepted: May 9, 2014
Meditterr J Hematol Infect Dis 2014, 6(1): e2014038, DOI 10.4084/MJHID.2014.038
This article is available on PDF format at:

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The fms-like tyrosine kinase 3 (FLT3) gene is a member of the class III receptor tyrosine kinase family. Mutations of FLT3 were first described in 1997and account for the most frequent molecular mutations in acute myeloid leukemia.
Currently, there is no published data on FLT3 mutations in Saudi acute lymphoblastic leukemia (ALL) patients.
In this retrospective study, we have examined a cohort of 77 ALL patients to determine the prevalence of FLT3 mutations and the possible prognostic relevance of these mutations in ALL patients. Correlations to other biologic factors such as karyotype, molecular mutations, and leukocyte count were also considered.
FLT3 internal tandem duplication (ITD) mutations and point mutation in tyrosine kinase domain (D835) were analyzed in ALL patients, at diagnosis, by polymerase chain reaction (PCR).
Two cases (2.6%, 2/77) were positive for FLT3 mutations; one was found to have FLT3/ITD and the other FLT3/D835.
Our findings suggest that FLT3 mutations are not common in Saudi ALL and do not affect clinical outcome.

Introduction

The human fms-like tyrosine kinase 3 (FLT3) gene is located on chromosome 13q12 and encompasses 24 exons. It encodes a membrane-bound glycosylated protein of 993 amino acids with a molecular weight of 158-160kDa, as well as a non-glycosylated isoform of 130-143 kDa that is not associated with the plasma membrane.[1] The structure of FLT3 is shown in the figure 1.[2]


Figure 1 Figure 1. Structure of FLT3 receptor.

Genomic aberrations of FLT3, including internal tandem duplication (ITD) and point mutations, have been demonstrated in approximately 25–35% of adults with acute myeloid leukemia (AML).[3-7] ITD of the FLT3 gene is common in AML and is associated with a bad prognosis and poor response to chemotherapy. Single base mutations at the FLT3 tyrosine kinase domain (TKD), which frequently involves aspartic acid 835 of the kinase domain (D835), leads to a gain of function; however, due to its rarity, its prognostic significance is not well defined.[8]
FLT3 is rarely mutated in leukemic lymphoblasts in adult and pediatric ALL;[3,4,9,10] however, FLT3 mutations are relatively common among the cytogenetic subgroups of hyperdiploidy and mixed-lineage leukemia (MLL) translocation.[11]
Recent studies have indicated a low overall frequency in childhood ALL (in the 1-8% range) while consistently demonstrating a higher incidence among those with MLL gene rearrangement and high hyperdiploidy.[13-17] In adult ALL, FLT3 mutations are even rarer.[18]
While there have been several studies[19-27] describing activating mutations of the FLT3 gene in AML, there has been little work on these mutations in ALL.
In this study, we analyzed the prevalence of the two types of FLT3 activating mutations in 77 patients with ALL and its prognostic significance. No data currently exist regarding FLT3 mutations in Saudi ALL patients and this study is the first one conducted in Saudi Arabia describing FLT3 mutations in ALL patients.


Introduction

Material and Methods

Study Group: A retrospective review of both adult and pediatric (ages 1 to 15) cases of ALL was performed. Data was obtained from the files of the Department of Hematopathology, Prince Sultan Military Medical City, Saudi Arabia from 2005 to 2013. Leukemia samples were obtained from either bone marrow (BM) or peripheral blood (PB), at diagnosis, from patients with ALL (70 BM samples and 7 PB samples). The peripheral blood samples all had more than 15% blasts at diagnosis. Five samples obtained from normal bone marrow healthy donors were screened for FLT3 mutations as a reference group. Among the 77 patients, with an established diagnosis by cell morphology and flow cytometric immunophenotyping, 48 were pediatric (62.3%), 29 were adult (37.7%), in total, 45 of the patients were male (58.4%) and 32 female (41.6%) Table 1.


Table 1 Table 1. Clinical characteristics and cytogenetic findings of the patients included in the study.

Samples were evaluated in addition to cytomorphology, and multiparameter flow cytometry, by cytogenetics, fluorescence in situ hybridization (FISH), and molecular genetics in parallel.
Pediatric patients were treated according to the UKALL 2003 chemotherapy protocol. Initially, eligible pediatric patients were stratified into three risk groups, standard risk (22 patients), intermediate risk (14 patients) and high risk (12 patients) based on age, WBC at presentation, immunophenotype and cytogenetic abnormalities.
Treatment of adult ALL patients was divided into two age groups. Patients at 20 years of age or less were treated according to the Dana-Farber Cancer Institute All Consortium Protocol 00-01[28] and patients over 20 years of age were treated with Hyper-CVAD chemotherapy.[29] At first, risk groups at diagnosis were categorized into a standard risk (15 patients) and high risk (14 patients) to determine the intensity of therapy. This study was approved by the Research and Ethics Committee at this institution.

Morphologic Analysis: For each case in this study, Wright-Giemsa-stained peripheral blood and bone marrow aspirate smears were reviewed. Aspirate clot and biopsy specimens were fixed in formalin, routinely processed and the histologic sections were stained with Hematoxylin and Eosin and reviewed.

Flow CytometricImmunophenotypic Methods: All samples were assessed by multicolor flow cytometry using a large panel of antibodies, including CD2, cytoplasmic and surface CD3, CD4,CD5, CD7, CD8, CD10, CD19,CD20, CD22, cytoplasmic CD79a, CD13, CD15, CD33, CD11c, CD14, CD64, CD38, CD34, CD117, cytoplasmic terminal deoxynucleotidyltransferase (TdT), and myeloperoxidase (MPO). Antigens were scored as positive using a cutoff of 20% or more leukemic blasts staining brighter than an isotype-matched negative control.

Molecular Methods

  1. Analysis of FLT3-ITD mutation. DNA was extracted using a QIAamp DNA Kit (Qiagen) according to the manufacturer’s recommendations.PCR amplification was composed of 200ng of DNA, 50mM KCL, 10mM Tris-HCL, pH8.3, 1.5mM MgCL2, 0.001%(wt/vol) gelatin, 200 µM dNTPs, 0.4µM of each primer (5’-GCAATTTAGGTATGAAAGCCAGC-3’ and 5’-CTTTCAGCATTTTGACGGCAACC-3’), and 1U of gold Taq polymerase, in a volume of 50µl.[23]
    The PCR consisted of an initial incubation step at 95°C for 10 minutes followed by 35 cycles at 94°C for 30 seconds, 57°C for 60 seconds, and 72°C for 90 seconds. The final extension step was at 72°C for 10 minutes on a GeneAmp PCR system 9700(Applied Biosystems). PCR products were analyzed on standard 3% agarose gels. Normal amplification generates a 330bp product; whereas, FLT3 ITD mutations (FLT3/ITD+) show longer PCR products. 

  2. Analysis of the FLT3- D835 mutation. PCR amplification was set up as above using specific primers[4] for exon 20 (5’-CCGCCAGGAACGTGCTTG-3’ and 5’-GCAGCCTCACATTGCCCC-3’). PCR product was digested with EcoRV (Promega), at 37°C for 2h. The digestion products were separated on a 3% agarose gel, and incomplete digestion indicated the presence of mutant.

Statistical analysis: The Kaplan-Meier technique was used to analyze the probability of overall survival (OS). OS was calculated from time of diagnosis to death. Continuous variables, such as white blood cell count and hemoglobin, were compared using the Kruskal-Wallis test. Differences between means were considered as significant at P < 0.05.
Complete remission (CR) was defined by less than 5% blast cells in a normocellular marrow and peripheral blood neutrophil count equal to or greater than 1.5 x 109/liter with a platelet count of more than 100 x 109/liter. Normalization of cytogenetic abnormalities was not a prerequisite for CR.

Results

Clinical characteristics at presentation and cytogenetic analysis of the patients in the studied group are summarized in table 1.
In total, the patient age range was 1 to 81 years with a median of 15 years. Pediatric patients were defined as less than 15 years of age. Of the 77 patients, 48 were pediatric (62.3%), all with de novo ALL (42 cases B-ALL and 6 cases T-ALL). 29 were adult patients (37.7%) in which there were 27 cases of de novo ALL (22 cases B-ALL and 5 cases T-ALL) and 2 cases of CML transforming to ALL. Two cases from the pediatric group were diagnosed as biphenotypic leukemia. In total, 45 of the patients were male (58.4%) and 32 female (41.6%).
Two of the 77 ALL patients examined showed FLT3 mutations (2/77) with an overall prevalence of 2.6%. Positive FLT3 mutation patients were both pediatric, one male and one female. One was found to have FLT3/ITD and the other was positive for FLT3/D835 (Figure 2). None of the adult ALL patients was positive for FLT3 mutations. None of the T-ALL (T-ALL - 11/77) and CML patients transforming to ALL showed FLT3/ITD or D835 mutations. None of the patients had a combination of FLT3/ITD and D835 mutation in the FLT3 gene.


Figure 2 Figure 2. PCR analysis of FLT3-ITD and D835 mutations. A) Agrose gel shows patients positive for ITD lanes 4, patients negative for ITD lanes 2, 3, 5, 6, positive control lane 7, no template control lane 8, Marker lane 1, 9. B) Agrose gel shows digested sample for D835 lanes 2-7, positive patients lane 4 (114bp, 68 bp, 49bp), negative patients lanes 2, 3, 5, 6 (68bp, 49bp), positive control lane 7, Undigested sample lane 8 (114bp), no template control lane 9, Marker lanes 1, 10.

The FLT3/ITD positive patient (male, 14 years old) showed a WBC of 91 x 109/liter with 70% of the peripheral blasts. The bone marrow (BM) blasts were 95% and showed a good response to chemotherapy resulting in CR. The FLT3/D835 positive patient (female, 4 years old) showed a WBC of 4.6 x 109/liter with 32% of peripheral blasts. The bone marrow (BM) blasts were 95%, and continuous complete remission (CCR) was achieved after chemotherapy.
Cytogenetic and molecular studies revealed that the FLT3/D835 positive patient was hyperdiploidy and reported as 54,XX,+2,+4,+8,+14,+16,+20,21,+21.nuc ish (ABL1,BCR) x2[100]/ (ETV6,RUNX1)x3[95/100]/(5'MLL,3'MLL,5'MLL con 3'MLL)x2[100] /(5'MYC.3'MYC,5'MY con 3'MYC)x3 [95/100]/ (TCF3,PBX1)x2[100]. An extra copy of chromosome 2, 4, 8, 12, 14, 16, 20 and 21 was detected in 95% of the studied cells. Cytogenetic analysis could not be performed due to an insufficient number of metaphases.

Discussion

FLT3 gene mutations, particularly ITD in AML, are well established as the most frequent somatic alterations in AML. A poor prognosis is associated with FTL3 gene mutations in AML and they are found in approximately 5-15% of children and 25-35% of adults with AML.[4,7,19,20,23,31] FLT3 mutations are also found in adult and pediatric ALL, but are much rarer than in AML.[3,4,9,10] FLT3 is overexpressed at the level of RNA and protein in most B lineage and acute myeloid leukemias. It is also overexpressed in a smaller subset of T-ALL and chronic myeloid leukemias (CML) in blast crisis.[32]

This study is the first to report FLT3 mutations on ALL patients in Saudi Arabia. The overall rate of patients with ALL and FLT3 mutations, in this study, was 2.6% which is comparable with previously published reports.[4,14,33,34,35]

The incidence of FLT3 mutations in pediatric ALL of this study was 4.2% (2/48). The results are within the range of similar studies conducted in different geographic regions (see table 2).


Table 2 Table 2. Frequency of FLT3 mutations in ALL patients in the current study and in previous studies.

The incidence of FLT3 mutations in pediatric leukemia is of particular interest due to the several promising FLT3 inhibitors currently under development.[45]
The frequency of both ITD and D835 mutations among adult ALL patients in this study was 0% (0/29); other studies have reported similar results;[19,43,44] however, a few studies have reported FLT3 mutations among adult ALL patients at a very low frequency.[18] As this is a rare occurrence, the limited sample size of this study is the most likely cause of the difference. Similarly, neither FLT3/ITD nor FLT3/D835 mutations were evident in any case of CML transforming to ALL or in T-All (0/11) which is consistent with the study by Andersson et al. 2008 (0% in 15 T-ALL patients).[13] Some larger studies have reported a low frequency of FLT3/ITD and/or FLT3/D835 mutations ranging from 3.3% to 5.5% among T-ALL patients.[18,39,46]
In this study, one patient (male, 14 years old) was found to have the FLT3/ITD mutation. He showed leukocytosis and a high blast cell count in PB and BM. This patient exhibited a good response to chemotherapy and achieved complete remission, which is in-line with results of previous studies.[35,37,39] This patient was also positive for myeloid antigen expression and was diagnosed as acute mixed-lineage leukemia (biphenotypic leukemia). This observation was reported in other studies[17,34,35] which indicates a higher frequency among the biphenotypic group. Cytogenetic analysis could not be performed due to the low number of metaphases.
The other FLT3 mutation patient in this study (female, 4 years old), was found to the FLT3/D835 mutation with no leukocytosis; however, she did show a high blast count in PB and BM. Cytogenetic and molecular studies revealed no association with balanced translocations or MLL gene rearrangement; however, hyperdiploidy (extra copy of chromosome 2, 4, 8, 12, 14, 16, 20 and 21) was detected in 95% of the studied cells. This finding represents a 16.7% incidence of hyperdiploidy in ALL (1/6) cases. Our results reinforced previous observations, Armstrong et al. 2004,[12] in that the presence of the FLT3 mutation in hyperdiploid ALL did not affect the clinical outcome as the patient responded well to chemotherapy and achieved continuous complete remission (CCR).
In both cases, molecular studies using polymerase chain reaction (PCR) were negative for Philadelphia chromosome [t(9;22)(q34.1;q11.2)].
The only notable difference between this study and previous studies[11,12,36] is the lack of association between the high frequency of FLT3 mutation and MLL gene rearrangement. The small sample size (5 ALL patients) is the most likely cause of the difference.
Neither ITD’s or D835 mutations were detected in the healthy donors of this study, and this is consistent with previously reported data.[4,47]

Conclusion

FLT3 mutations exist in a small proportion of Saudi ALL patients. Regarding clinical outcomes, there was no prognostic significance in ALL patients with or without FLT3 mutations. The observation of high frequency in hyperdiploid ALL is in agreement with similar studies from other geographical regions; however, there is a lack of association between MLL gene rearrangement and FLT3 mutations among ALL patient that may be due to the limited sample size. Further studies are needed to confirm and establish these results.

Acknowledgment

We thank Jon Johnston, Principal Clinical Scientist from Histopathology lab for editing this research article. We thank all the Hematology, Cytogenetic and Molecular Laboratories Staffs, especially Mr. Mohamed Asiri and Dalal Alkhammash from Hematology Lab for data collection and Ms Nadia Halawani from Molecular Lab for the great support in Molecular work of this study. We also thank Ms Khawla Al-Fayez from Cytogenetic Lab for excellent support and data collection in cytogenetic aspect of this study.

Materials and Methods

Umbilical cord blood collection. Between August 2010 and July 2011, 177 mothers delivering their babies at Al-Isra'a hospital, Amman, Jordan, were approached to participate in this prospective study. One hundred and twenty-four mothers (70%) agreed to participate and signed a consent form. The UCB was collected exclusively from term (gestation period 37-42 weeks) single-birth babies born through normal vaginal delivery. Cord blood was collected after the baby was delivered but before the delivery of the placenta. A regular blood-donor set was used for UCB collection containing 28 ml citrate phosphate dextrose-adenine (CPD-A) anticoagulant. The collection was performed by the obstetrician delivering the baby and not by a trained technician. The umbilical cord (UC) was sterilized with povidone iodine in a unidirectional move, and 16-gauge needle of the prepared blood-donor set was inserted into the umbilical vein. Blood was allowed to flow by gravity, and the needle was removed when blood flow ceased as has been previously described.[6,10] The study design and UCB collection procedure was approved by the Hashemite University, and Al-Isra'a general hospital Institute Review Boards. 
Evaluation of umbilical cord blood parameters. For the current study, UCB units were deemed unacceptable if the total volume collected was less than 30 ml and/or if the unit was delivered for analysis past 24 hours of collection. The UCB units were processed and analyzed in the biology laboratory at the Hashemite University, Jordan. The UCB was incubated with FITC-conjugated anti-CD45 fluorescein (MACS, Germany) and PE-conjugated anti-CD34 PE (MACS, Germany) for 30 min at room temperature in the dark. After incubation, RBCs were lysed with the lysis solution (Coulter, France) and then washed twice with 10% bovine serum albumin (BSA) in phosphate buffer saline (PBS). For each tube, 20,000 live events were counted in a flowcytometer counter (Partec, Germany). CD34+ cells were selected based on their forward- and 90o-scatter properties and dim CD45 expression.[13] The clonogenic assay (CFU-GM assay) was performed as described previously.[14] Briefly, mononucleated cells (MNCs) were cultured at 1.0 X 105/ml in RBMI-1640 medium (MACS, Germany) containing 0.8% methylcellulose (Sigma Aldrich, USA), 20% fetal bovine serum (FBS; Lonza, Belgium), 450 µg/ml human transferrin, 10 ng/ml GM-CSF, 10 ng/ml IL-3 (Stem Cell Technologies), and 1% BSA. Cells were incubated at 37oC in a humidified atmosphere of 5% CO2 for 14 days. Colonies (clusters containing at least 50 cells) were counted using an inverted microscope (Leica, Germany). Viability was determined using trypan blue dye exclusion method, where the non-viable cells stain deep blue.
Maternal and neonatal data collection. Data regarding maternal age, the number of previous pregnancies and live births were collected from the medical files. Neonatal data such as the weight of the baby and the placenta, baby’s gender, and UC length were collected from the obstetric staff clinical notes at Al-Isra'a general hospital. A standard questionnaire was prepared and used for data collection.
Statistical analysis. Statistical analysis was carried out using STATISTICA 7 analysis program (StatSoft Inc., OK, USA). Results were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to test for a significant difference between mean values of all. Spearman's correlation was used to assess the association between the different variables. A p value of ≤ 0.05 was considered statistically significant.

Results

Characteristics of the study population. A total of 177 prospective mothers were approached to participate in the current study, 53 (30%) of them refused to participate due to cultural and/or lack of knowledge regarding  benefits of UCB and safety of the collection procedure. 124 units were prospectively collected for this study. In 17 (13.7%) UCB units the net volume of cord blood was less than 30 ml, in 23 (18.5%) units some maternal and/or neonatal data were missing, and in 9 (7.3%) units the samples were not delivered for the laboratory within 24 hour of collection. A total of 75 UCB units (60.5% of the total collected units) were included and analyzed in this study. The characteristics of the donating mothers and babies are shown in Table 1. The mean maternal age was 28 years (range 19-43). Thirty three percent of the donating mothers were delivering their first babies, 21.3% second, 20% third and 25.3% fourth or more. Fifty one percent of the delivered babies were males, and 49% were females. The mean weight of the delivered babies was 3178 gm (range 220-4160), and mean placenta weight was 526 gm (range 400-655). Seven percent of the donating mothers reported that they were current smokers.


Table 1 Table 1. Maternal and neonatal characteristics

Analysis of umbilical cord blood samples. Cord blood cell counts were analyzed within 24 hours of collection. The mean volume of UCB collected (not including the 28 ml of anticoagulant) was 68.9 ml (range 40-115 ml). The mean viability was 94.9% (range 80-99%), the mean total nucleated cell (TNC) count was 6.5 x 108 (range 1-32), with 10.6% have TNC of more than 1 x 109 and 4% of more than 1.2 x 109. The mean total mononuclear cell count (MNC) was 3.4 x 108 (range 0.5-14.9), the mean total CD34+ cell count was 3.8 x 106 (range 0.2-11.8), and the mean total CFU-GM was 9.9 x105 (range 2-25). The UCB unit’s data are summarized in Table 2.
The results of the univariate analysis correlation are presented in Table 3. The volume of UCB units collected was positively correlated with TNC (p=0.008), cell viability (p=0.001), MNC (p=0.018), CD34+ cell count (p=0.034) and with the umbilical cord length (p=0.011). There was also a trend towards obtaining higher UCB volume from mothers with increasing number of prior live births (p=0.086). Our results showed that higher TNC is correlated with MNC (p=0.001), CD34+ cell count (p=0.009), and increased viability (p=0.001). Finally, our study demonstrated an inverse correlation between CFU-GM concentration and the gestation duration (P = 0.038). There was no significant effect of gestational age on TNC or CD34+ cell count of the collected UCB.


Table 2 Table 2. Analysis of umbilical cord blood units (No 75)
Table 3 Table 3. Univariate analysis for correlation


Discussion

This is the first study to show that collection of cord blood is feasible and can result in adequate TNC collection and viability in a developing country in the Middle East. Of the 124 women enrolled in the study, the umbilical cord blood of only 17 (13.7%) did not contain adequate volume of blood despite the fact that untrained technicians were present at the delivery to collect the UCB. An additional 25.8% of the UCB units had to be excluded either because some data were missing, or because the UCB did not reach the lab in the required time for processing. Among the 75 units that met the predefined eligibility criteria, the volume, nucleated cell dose and CD34 count was similar to what has been previously published.[5,7,8,10,14]
Previous studies concluded that UCB yield of TNC, CD34+ cells, and CFU-GM is influenced not only by neonatal and maternal factors but also by ethnicity of the parents.[7,15] In this study, a total of 75 cord blood samples from Jordanian neonates were analyzed in order to investigate any neonatal and maternal factors that might influence UCB unit in terms of TNC and CD34+ cell content, and CFU-GM yields. In the current study, the average age of the donor mothers included was 28 years and both the TNC and CD34+5,8-10] We found no significant correlation between birth order and UCB volume, TNC, and CD34+ levels. Our findings are different from prior studies which showed that women with few previous live births produced UCB units with higher TNC yields.[9,10] Cell yield was not influenced by maternal age. While the majority of published data showed similar observation, the study by Nakagawa et al. which analyzed 572 UCB units, showed that increasing mother’s age was associated with lower TNC cell yield. 
The volume of UCB collected was not influenced by any of the maternal or neonatal factors except the umbilical cord length, as longer cords were associated with higher volume collected. This correlation has only been observed in one prior study from Japan.[8]
We also found an inverse correlation between the CFU-GM concentration and gestational age, which indicates that there is a loss of hematopoietic potential with a longer gestational age. In a study by Ballen et al. of 1269 UCB units, there was an 11% decrease in CFU-GM with each additional week of gestation.[10] Since TNC and CD34+ cell counts are the most important predictors of the outcome following cord blood transplant, collecting units with a larger volume is desired. In our current study, the only positive predictor of improved cell count is the volume of UCB collected.
The chance of finding a matched related donor in Jordan is significantly higher than what has been observed in other countries (65% versus 25%) due to more homogeneous ethnic group in the region.[16]
Approximately 10-16% of UBC units collected in the international cord blood banks have TNC of more than 1.2 x 109. In Our study, 10.6% of UCB units collected have TNC of more than 1 x 109 and 4% of more than 1.2 x 109. Although our cell dose was slightly lower than what has been reported by established cord blood banks, this is dependent on the experience of the collection staff which always improves with time.
We believe that establishing a cord blood bank in Jordan will further increase the possibility of identifying donors for patients who lack related donor options. Taking into consideration the geographical and cultural similarities between Jordan and its neighboring Arabic countries, a cord blood bank in Jordan will help patients throughout the region. Additional training and better logistical support are needed to collect UCB units in order to decrease the percentage of unacceptable units collected. We need also more efforts towards education of the parents about the benefits and safety of UCB collection, as only 70% of the approached mothers agreed to participate in this study. A proper cost-effective analysis should be carried out before establishing national cord blood banks in countries with limited resources.
In conclusion, we have found that collection of cord blood units in Jordan is feasible and can result in similar cell content compared to other developed countries. Efforts toward establishing public cord blood banks in our area are warranted.

Acknowledgments

We are grateful for the effort of the attending physicians, residents and nursing staff of Obstetrics and Gynecology department at Al-Isra'a Hospital. We gratefully acknowledge the Deanship of Research and Graduate Studies, The Hashemite University, Jordan for financial support.
Author contribution: Study design (AAH, RMB, LHT, AZE), study analysis and interpretation of the data (AAH, RMB, LHT, HF, AZE). All authors contributed to the writing of the manuscript and approval of the final version.

References

  1. Markovic A, MacKenzie KL, Lock RB: FLT-3: a new focus in the understanding of acute leukemia.Int J Biochem Cell Biol 2005, 37:1168-1172 http://dx.doi.org/10.1016/j.biocel.2004.12.005   PMid:15778081    
  2. Takahashi S. Downstream molecular pathways of FLT3 in the pathogenesis of acute myeloid leukemia: biology and therapeutic implications. J Hematol Oncol. 2011 Apr 1;4: 13. http://dx.doi.org/10.1186/1756-8722-4-13 PMid:21453545 PMCid:PMC3076284
  3. Gilliland DG, Griffin JD. The roles of FLT3 in hematopoiesis and leukemia. Blood 2002 Sep 1; 100(5): 1532-42. http://dx.doi.org/10.1182/blood-2002-02-0492 PMid:12176867     
  4. Yamamoto Y, Kiyoi H, et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 2001; 97: 2434-2439. http://dx.doi.org/10.1182/blood.V97.8.2434  PMid:11290608     
  5. Kiyoi H, Towatari M, Yokota S, Hamaguchi M, Ohno R, Saito H and Naoe T. Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product. Leukemia 1998; 12: 1333-1337. http://dx.doi.org/10.1038/sj.leu.2401130  PMid:9737679     
  6. Levis M and Small D. FLT3: ITD does matter in leukemia. Leukemia 2003; 17: 1738-1752. http://dx.doi.org/10.1038/sj.leu.2403099 PMid:12970773     
  7. Stirewalt DL, Radich JP: The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer 2003; 3(9):650-665. http://dx.doi.org/10.1038/nrc1169 PMid:12951584     
  8. Leung AY, Man CH, Kwong YL. FLT3 inhibition: a moving and evolving target in acute myeloid leukaemia. Leukemia. 2013 Feb;27(2):260-8. http://dx.doi.org/10.1038/leu.2012.195 PMid:22797419     
  9. Xu F, Taki T, Eguchi M, et al. Tandem duplication of the FLT3 gene is infrequent in infant acute leukemia. Leukemia. 2000;14:945-947. http://dx.doi.org/10.1038/sj.leu.2401760 PMid:10803532    
  10. Nakao M, Janssen JWG, Erz D, Seriu T, Bartram CR. Tandem duplication of the FLT3 gene in acute lymphoblastic leukemia: a marker for the monitoring of minimal residual disease. Leukemia. 2000;14:522-524. http://dx.doi.org/10.1038/sj.leu.2401695 PMid:10720156     
  11. Armstrong SA, Kung AL, Mabon ME, et al. Inhibition of FLT3 in MLL: validation of a therapeutic target identified by gene expression based classification. Cancer Cell. 2003;3:173-183. http://dx.doi.org/10.1016/S1535-6108(03)00003-5 
  12. Armstrong SA, Mabon ME, Silverman LB, Li A, Gribben JG, Fox EA, Sallan SE, Korsmeyer SJ. FLT3 mutations in childhood acute lymphoblastic leukemia. Blood 2004, 103(9):3544-3546. http://dx.doi.org/10.1182/blood-2003-07-2441  PMid:14670924     
  13. Andersson A, Paulsson K, Lilljebjorn H, Lassen C, Strombeck B, Heldrup J, Behrendtz M, Johansson B, Fioretos T: FLT3 mutations in a 10 year consecutive series of 177 childhood acute leukemias and their impact on global gene expression patterns. Genes Chromosomes Cancer 2008, 47(1):64-70. http://dx.doi.org/10.1002/gcc.20508 PMid:17943971     
  14. Case M, Matheson E, Minto L, Hassan R, Harrison CJ, Bown N, Bailey S, Vormoor J, Hall AG, Irving JA: Mutation of genes affecting the RAS pathway is common in childhood acute lymphoblastic leukemia. Cancer Res 2008, 68(16):6803-6809. http://dx.doi.org/10.1158/0008-5472.CAN-08-0101 PMid:18701506     
  15. Paulsson K, Horvat A, Strombeck B, Nilsson F, Heldrup J, Behrendtz M, Forestier E, Andersson A, Fioretos T, Johansson B: Mutations of FLT3, NRAS, KRAS, and PTPN11 are frequent and possibly mutually exclusive in high hyperdiploid childhood acute lymphoblastic leukemia. Genes Chromosomes Cancer 2008, 47(1):26-33. http://dx.doi.org/10.1002/gcc.20502  PMid:17910045    
  16. Yamamoto T, Isomura M, Xu Y, Liang J, Yagasaki H, Kamachi Y, Kudo K, Kiyoi H, Naoe T, Kojma S: PTPN11, RAS and FLT3 mutations in childhood acute lymphoblastic leukemia. Leuk Res 2006, 30(9):1085-1089 http://dx.doi.org/10.1016/j.leukres.2006.02.004 PMid:16533526     
  17. Braoudaki M, Karpusas M, Katsibardi K, Papathanassiou C, Karamolegou K, Tzortzatou-Stathopoulou F: Frequency of FLT3 mutations in childhood acute lymphoblastic leukemia. Med Oncol 2009, 26(4):460-462. http://dx.doi.org/10.1007/s12032-008-9146-z  PMid:19085113     
  18. Paietta E, Ferrando AA, Neuberg D, Bennett JM, Racevskis J, Lazarus H, Dewald G, Rowe JM, Wiernik PH, Tallman MS, Look AT. Activating FLT3 mutations in CD117/KIT(+) T-cell acute lymphoblastic leukemias. Blood. 2004 Jul 15;104(2):558-60. http://dx.doi.org/10.1182/blood-2004-01-0168  PMid:15044257     
  19. Yokota S, Kiyoi H, Nakao M, Iwai T, Misawa S, Okuda T et al. Internal tandem duplication of the FLT3gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignanciesA study on a large series of patients and cell lines.Leukemia 1997; 11: 1605-1609. http://dx.doi.org/10.1038/sj.leu.2400812 PMid:9324277     
  20. Meshinchi S, Woods WG, Stirewalt DL, Sweetser DA, Buckley JD, Tjoa TK et al. Prevalence and prognostic significance of Flt3internal tandem duplication in pediatric acute myeloid leukemia. Blood 2001; 97: 89-94. http://dx.doi.org/10.1182/blood.V97.1.89  PMid:11133746     
  21. Stirewalt DL, Kopecky KJ, Meshinchi S, Appelbaum FR, Slovak ML, Willman CL et al. FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia. Blood 2001; 97: 3589-3595. http://dx.doi.org/10.1182/blood.V97.11.3589  PMid:11369655     
  22. Abu-Duhier FM, Goodeve AC, Wilson GA, Gari MA, Peake IR, Rees DC et al. FLT3internal tandem duplication mutations in adult acute myeloid leukaemia define a high risk group. Br J Haematol 2000; 111: 190-195. http://dx.doi.org/10.1046/j.1365-2141.2000.02317.x  PMid:11091200     
  23. Kiyoi H, Naoe T, Nakano Y, Yokota S, Minami S, Miyawaki S et al. Prognostic implication of FLT3and NRAS gene mutations in acute myeloid leukemia. Blood 1999; 93: 3074-3080  PMid:10216104     
  24. Rombouts WJ, Blokland I, Lowenberg B, Ploemacher RE. Biological characteristics and prognosis of adult acute myeloid leukemia with internal tandem duplications in the Flt3gene. Leukemia 2000; 14: 675-683. http://dx.doi.org/10.1038/sj.leu.2401731 PMid:10764154     
  25. Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA et al. The presence of a FLT3internal tandem duplication in patients with acute myeloid leukaemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 2001; 98: 1752-1759 http://dx.doi.org/10.1182/blood.V98.6.1752 PMid:11535508     
  26. Gari M, Abuzenadah A, Chaudhary A, Al-Qahtani M, Banni H, Ahmad W, Al-Sayes F, Lary S, Damanhouri G. Detection of FLT3 oncogene mutations in acute myeloid leukemia using conformation sensitive gel electrophoresis. Int J Mol Sci. 2008 Nov; 9(11):2194-204. http://dx.doi.org/10.3390/ijms9112194 PMid:19330068  PMCid:PMC2635624
  27. Elyamany GH, Awad M, Fadalla K, Al-Balawi M , Al- Abulaaly A. Frequency and Prognostic Relevance of FLT3 Mutations in Saudi Acute Myeloid Leukemia Patients. Adv Hematol. 2014; 2014:141360. http://dx.doi.org/10.1155/2014/141360 PMid:24696688 PMCid:PMC3950551
  28. Vrooman LM, Stevenson KE, Supko JG, O'Brien J, Dahlberg SE, Asselin BL, Athale UH, Clavell LA, Kelly KM, Kutok JL, Laverdière C, Lipshultz SE, Michon B, Schorin M, Relling MV, Cohen HJ, Neuberg DS, Sallan SE, Silverman LB. Postinduction dexamethasone and individualized dosing of Escherichia Coli L-asparaginase each improve outcome of children and adolescents with newly diagnosed acute lymphoblastic leukemia: results from a randomized study--Dana-Farber Cancer Institute ALL Consortium Protocol 00-01. J Clin Oncol. 2013 Mar 20;31(9):1202-10. Epub 2013 Jan 28. PubMed PMID: 23358966; PubMedCentral PMCID: PMC3595424. http://dx.doi.org/10.1200/JCO.2012.43.2070 
  29. Garcia-Manero G, Kantarjian HM. The hyper-CVAD regimen in adult acute lymphocytic leukemia. Hematol Oncol Clin North Am. 2000 Dec;14(6):1381-96, x-xi. Review. PubMed PMID: 11147229.
  30. Schnittger S, Schoch C, Dugas M, et al. Analysisof FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics,FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood. 2002;100:59-66. http://dx.doi.org/10.1182/blood.V100.1.59  PMid:12070009     
  31. Thiede C, Steudel C, Mohr B, et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood. 2002;99:4326- 4335
  32. Carow CE, Levenstein M, Kaufmann SH, et al. Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2)in human leukemias. Blood.1996;87:1089-109 PMid:8562934     
  33. Wang W, Wang XQ, Xu XP, Lin GW. Prevalence and prognostic significance of FLT3 gene mutations in patients with acute leukaemia: analysis of patients from the Shanghai Leukaemia Co-operative Group. J Int Med Res. 2010 Mar-Apr;38(2):432-42. http://dx.doi.org/10.1177/147323001003800206  PMid:20515557     
  34. Xu B, Li L, Tang JH, Zhou SY. Detection of FLT3 gene and FLT3/ITD mutation by polymerase chain reaction-single-strand conformation polymorphism in patients with acute lymphoblastic leukemia. Di Yi Jun Yi Da Xue Xue Bao. 2005 Oct;25(10):1207-10.
  35. Xu F, Taki T, Yang HW, Hanada R, Hongo T, Ohnishi H, Kobayashi M, Bessho F, Yanagisawa M, Hayashi Y. Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acutemyeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children. Br J Haematol. 1999 Apr;105(1):155-62. http://dx.doi.org/10.1111/j.1365-2141.1999.01284.x  PMid:10233379     
  36. Taketani T, Taki T, Sugita K, Furuichi Y, Ishii E, Hanada R, Tsuchida M, Ida K, Hayashi Y: FLT3 mutations in the activation loop of tyrosine kinase domain are frequently found in infant ALL with MLL rearrangements and pediatric ALL with hyperdiploidy. Blood 2004, 103(3):1085-1088. http://dx.doi.org/10.1182/blood-2003-02-0418  PMid:14504097     
  37. Karabacak BH, Erbey F, Bayram I, Yilmaz S, Acipayam C, Kilinç Y, Tanyeli A. Fms-like tyrosine kinase 3 mutations in childhood acute leukemias and their association with prognosis. Asian Pac J Cancer Prev. 2010;11(4):923-7.  PMid:21133602     
  38. Chang P, Kang M, Xiao A, Chang J, Feusner J, Buffler P, Wiemels J. FLT3 mutation incidence and timing of origin in a population case series of pediatric leukemia. BMC Cancer. 2010 27;10:513. http://dx.doi.org/10.1186/1471-2407-10-513  
  39. Grossmann V, Haferlach C, Weissmann S, Roller A, Schindela S, Poetzinger F, Stadler K, Bellos F, Kern W, Haferlach T, Schnittger S, Kohlmann A.The molecular profile of adult T-cell acute lymphoblastic leukemia: Mutations in RUNX1 and DNMT3A are associated with poor prognosis in T-ALL. Genes Chromosomes Cancer. 2013 Apr;52(4):410-22. http://dx.doi.org/10.1002/gcc.22039  PMid:23341344     
  40. Ishfaq M, Malik A, Faiz M, Sheikh I, Asif M, Khan MN, Qureshi MS, Zahid S, Manan A, Arooj M, Qazi MH, Chaudhary A, Alqahtani MH, Rasool M. Molecular characterization of flt3 mutations in acute leukemia patients in Pakistan. Asian Pac J Cancer Prev. 2012; 13(9):4581-5. http://dx.doi.org/10.7314/APJCP.2012.13.9.4581 PMid:23167384     
  41. Zhao X, Tang KJ, Tian Z, Chen LP, Mi YC, Wang JX. FLT3 mutation in patients with acute lymphoblastic leukemia and its clinical significance. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2009 Oct;31(5):522-6. PMid:19968062     
  42. Al-Tonbary Y, Mansour AK, Ghazy H, Elghannam DM, Abd-Elghaffar HA. Prognostic significance of foetal-like tyrosine kinase 3 mutation in Egyptian children with acute leukaemia. Int J Lab Hematol. 2009 Jun; 31(3):320-6. http://dx.doi.org/10.1111/j.1751-553X.2008.01039.x PMid:18336585     
  43. Wang L, Lin D, Zhang X, Chen S, Wang M, Wang J. Analysis of FLT3 internal tandem duplication and D835 mutations in Chinese acute leukemia patients. Leuk Res. 2005 Dec;29(12):1393-8. http://dx.doi.org/10.1016/j.leukres.2005.05.013 PMid:15996732     
  44. Nakao M, Yokota S, Iwai T, et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia. 1996;10:1911-1918. PMid:8946930     
  45. Weisberg E, Barrett R, Liu Q, Stone R, Gray N, Griffin JD: FLT3 inhibition and mechanisms of drug resistance in mutant FLT3-positive AML. Drug Resist Updat 2009, 12(3):81-89. http://dx.doi.org/10.1016/j.drup.2009.04.001 PMid:19467916     
  46. Hoehn D, Medeiros LJ, Chen SS, Tian T, Jorgensen JL, Ahmed Y, Lin P. CD117 expression is sensitive but nonspecific predictor of FLT3 mutation in T acute lymphoblastic leukemia and T/myeloid acute leukemia. Am J Clin Pathol. 2012 Feb;137(2):213-9 http://dx.doi.org/10.1309/AJCPR3N3JMSYLPFG  PMid:22261446     
  47. Abu-Duhier FM, Goodeve AC, Wilson GA, Care RS, Peake IR, Reilly JT. Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia. Br J Haematol. 2001;113: 983-988 http://dx.doi.org/10.1046/j.1365-2141.2001.02850.x  PMid:11442493 

[TOP]

Abstract views:
481

Views:
PDF
233
HTML
5930

Article Metrics

Metrics Loading ...

Metrics powered by PLOS ALM


Copyright (c) 2016 Mediterranean Journal of Hematology and Infectious Diseases

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
 
© PAGEPress 2008-2017     -     PAGEPress is a registered trademark property of PAGEPress srl, Italy.     -     VAT: IT02125780185