|
 
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| ORIGINAL ARTICLE |
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| Year : 2017 | Volume
: 8
| Issue : 2 | Page : 68-74 |
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Cytogenetics and molecular markers of acute myeloid leukemia from a tertiary care center in Saudi Arabia
Abdulaziz I Alrajeh1, Halah Abalkhail2, Salem H Khalil2
1 Hematopathology Section, Regional Lab, Ministry of Health, Riyadh, Saudi Arabia
2 Molecular Genetics Section, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
| Date of Web Publication | 17-Jul-2017 |
Correspondence Address:
Abdulaziz I Alrajeh
MD Senior Registrar Hematopathology and Blood Bank, Regional Lab, Ministry of Health, Riyadh
Saudi Arabia
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/joah.joah_57_16
Background/Purpose: Acute myeloid leukemia (AML) is a phenotypically and genetically heterogeneous disease. This heterogeneity is attributed to alterations in genetic bases. AML classification based on these abnormalities is essential for accurate diagnosis, risk stratification, prognostic value, monitoring of minimal residual disease, and developing targeted therapies. This study evaluates frequency of each karyotype and molecular abnormality at our institution with comparison to other international studies.
Materials and Methods: We reviewed 100 bone marrow samples, which represent all AML diagnosed cases at our hospital from 2012 to 2014 by conventional karyotyping, specific AML–FISH panel, and variety of AML-specific mutations using Sanger sequencing.
Results: Out of 100 AML patients investigated with median age of 29 years, 98 were successfully karyotyped, and 64% of cases had an abnormality. In addition, all 100 AML–FISH panel and molecular studies were informative with an abnormality reaching 50 and 45%, respectively. Conventional and molecular cytogenetic studies revealed trisomy 8 (15%), t(8;21) in 12%, trisomy 21(8%), inv(16) in 7%, t(15;17) in 6%, 11q rearrangements (6%), and inv(3) in 2%. The mutational analysis showed nucleophosmin 1 (12%), FMS-like tyrosine kinase-3–internal tandem duplication (9%), IDH2 (7%), IDH1 (6%), WT1 (5%), DNMT3A (4%), CEBPA (4%), and c-KIT (3%).
Conclusion: The incidence of most mutational analysis is lower, whereas abnormal karyotype showed almost similar frequency when compared to different international centers. This is the first cytogenetic data from Saudi Arabia for AML, including all these genetic mutations. Therefore, a multicenter collaboration and comprehensive study is recommended to confirm these findings.
Keywords: AML, FISH, mutation
How to cite this article:
Alrajeh AI, Abalkhail H, Khalil SH. Cytogenetics and molecular markers of acute myeloid leukemia from a tertiary care center in Saudi Arabia. J Appl Hematol 2017;8:68-74 |
How to cite this URL:
Alrajeh AI, Abalkhail H, Khalil SH. Cytogenetics and molecular markers of acute myeloid leukemia from a tertiary care center in Saudi Arabia. J Appl Hematol [serial online] 2017 [cited 2023 Sep 30];8:68-74. Available from: https://www.jahjournal.org/text.asp?2017/8/2/68/210832 |
| Introduction | |  |
Acute myeloid leukemia (AML) is the most common acute leukemia affecting adults and results from clonal expansion of myeloid blasts in the peripheral blood and bone marrow. AML is a clinically, morphologically, and genetically heterogeneous disease, involving one or all myeloid lineages. The heterogeneity of AML is reflected by differences in morphology and immunophenotyping as well as cytogenetic and molecular abnormalities.
The diagnosis of AML is made when the blast percentage is >20% in the peripheral blood and/or bone marrow.[1] In addition, the diagnosis can be made regardless of blast count in the peripheral blood or bone marrow if there is an associated chromosomal abnormality, including t(8;21)(q22;q22), inv(16)(p13.1q22), t(16;16)(p13.1;q22), or t(15;17)(q22;q12). These karyotype abnormalities lead to rearrangements of the RUNX1, CBFB, or RARA genes, respectively,[2] and the formation of fusion genes encoding chimeric proteins that impair myeloid differentiation and proliferation.
Roughly 25% of AML patients will have a “favorable” cytogenetic profile, 10 to 20% of patients will have an “adverse” cytogenetic profile, and the remaining 45 to 60% of patients will be defined as “intermediate”.[3] The favorable cytogenetic group involves chromosomal aberrations such as t(8;21) (RUNX1/RUNX1T1), chromosome 16 inversion (CBFB/MYH11), and t(15;17) (PML/RARA). Monosomy 5 or 7 as well as other monosomies that is, −5q and −7q, and complex abnormalities, define the adverse cytogenetic group. Patients without either the favorable or adverse cytogenetic profiles are considered intermediate.[3]
FMS-like tyrosine kinase 3 (FLT3) maps to chromosome 13 and encodes a tyrosine kinase receptor involved in hematopoietic cell differentiation and proliferation. The most common form of the FLT3 mutation is an internal tandem duplication (ITD) in exons 14 and 15 that maps to the juxta-membrane domain and occurs in 25 to 35% of cytogenetically normal (CN)-AML patients.
The presence of this mutation confers a higher risk of relapse and death. However, in cases in which the allelic ratio (ratio of mutant to wild-type FLT3) is low, patients appear to have an outcome similar to those patients without FLT3–ITD mutation, whereas an increased allelic ratio predicts poor survival.[4]
FLT3 mutations may occur in association with t(15;17) in Acute promyelocytic leukemia (APL) and t(6;9) and with normal karyotype or other mutations such as those in nucleophosmin (NPM1), Isocitrate dehydrogenase 1 (IDH1), andisocitrate dehydrogenase (NADP(+)) 2, mitochondrial (IDH2).[14] Accordingly, 26% of our AML cases developed combined mutations at the time of diagnosis, mainly in FLT3 (ITD) in addition to either NPM1 or IDH1 mutations.[5],[6]
Mutation of the NPM1 gene has recently been described as the most frequent mutation in AML, accounting for 50 to 60% of all adult CN AML cases[7] and 2 to 8% of childhood AML cases. De novo mutations are typically heterozygous and occur in exon 12. The most common mutation in NPM1 is tetra nucleotide TCTG duplication, the mutation precedes other mutations such as FLT3–ITD and is consistently retained at relapse.[8]
As the genetic abnormalities, leading to AML, involves multistep, heterogeneous, and complex processes, molecular analysis could be particularly important for accurate diagnosis, prognostic stratification, and monitoring of minimal residual disease to develop targeted therapies.
In this retrospective study, the frequencies of a variety of AML-associated karyotypes, cytogenetic abnormalities, and genetic mutations were evaluated among Saudi patients with AML that is, mutation screening in FLT3, NPM1, IDH1, IDH2, CCAAT/enhancer-binding protein alpha (CEBPA), DNA (cytosine-5)-methyltransferase 3A (DNMT3A), KIT proto-oncogene receptor tyrosine kinase (c-KIT), Wlm’s Tumor1 (WT1), RUNX−, and PML/RARA genes were investigated in addition to karyotype and fluorescence in situ hybridization (FISH) panel for RUNX1T1 translocation (8;21), PML/RARA translocation (15;17), CBFB (16q22) rearrangement, mixed-lineage leukemia (MLL) (11q23) rearrangement, RPN1/MECOM translocation (3;3), or inv(3).
| Purpose | | |
This retrospective study evaluates frequency of each karyotype and molecular abnormality in AML patients at our institution (King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia) between 2012 and 2014 with comparison to other international studies and reports.
| Materials and Methods | | |
A total of 100 bone marrow samples were collected from adult and pediatric AML cases referred by our hematology/oncology clinics, which represent all new cases of AML to KFSH between 2012 and 2014 (the study included the cases relapsed or pre-Bone Marrow transplant (BMT) which are new patients to our hospital). The cases were diagnosed by morphology and flow cytometry and were sent for cytogenetic karyotyping, FISH–AML panel, and diagnostic molecular profiling. This retrospective study was conducted with ethics clearance from Office of Research Affairs at our hospital.
The presence of eight different genes mutation and two translocations were assessed according to Laboratory Protocols.
Cytogenetic analysis of cells obtained by marrow aspiration was performed in 98% of patients using standard cytogenetic techniques. Marrow cells were collected in heparin (10/15 UI/mL) and placed in RPMI 1640 with 40% fetal calf serum at a concentration of 2′ 106-nucleated cells/mL. The cultures were incubated overnight at 37°C (approximately 24 h). No direct preparations were used. No mitotic stimulants were used. Standard harvesting procedure was utilized.
Colcemid (0.01 mg/mL) was added to the cultures for 15 min. For the hypotonic treatment, 0.075 M KCl was utilized for 10 min at 37°C. The fixation procedure consisted of three changes of 3:1 absolute methanol: glacial acetic acid solution with 10-min waiting period between each change.
Air-dry slide preparations were made using wet slides at room temperature. Chromosomes were Q banded using quinacrine fluorescence (QFQ banding). The slides for QFQ banding were placed in quinacrine 0.05% dilution in distilled water for 30 min and then washed in McIlvaine buffer pH 7.0. Whenever possible, at least 20 metaphases were analyzed, both for structural and numerical chromosomal abnormalities. Karyotypes were assigned according to the recommendations of the International System for Human Cytogenetic Nomenclature.[14]
The observation of a minimum of two mitoses with an identical structural rearrangement or extrachromosomes or three cells with the same missing chromosome was considered evidence for the existence of an abnormal clone.
The FISH panel included five translocations according to Laboratory Protocols which are RUNX1/RUNX1T1 t(8;21), PML/RARA t(15;17), CBFB inv(16), MLL t(9;11), and PRN1/MECOM in v3.
Mutation analysis of eight genes and two translocations was performed, including the following: FLT3, NPM1, IDH1, IDH2, CEPBA, DNMT3A, c-KIT, WT1, RUNX1/RUNX1T1, and PML–RARA, on bone marrow samples. Genetic screening was performed for PML/RAR alpha fusion gene and RUNX1/RUNX1T1 fusion gene using reverse transcription-polymerase chain reaction (RT-PCR) on an ABI 7900HT system (Applied Biosystems, Foster City, California, USA).
Briefly, extracted ribonucleic acid (RNA) was subjected to reverse transcription and amplification using TaqMan® One-Step RT-PCR kit (Applied Biosystems, Foster City, California, USA), using BIOMED group PCR protocol.[9],[10],[11]
Fragment analysis
Genomic deoxyribonucleic acid (DNA) was extracted from bone marrow samples in EDTA using MagNa Pure 96automated nucleic acid extraction system (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Extracted DNA was subjected to Polymerase Chain reaction (PCR) using fluorescently labeled primers (description of the primers used in Supplementary [Table 1]) targeting the region on the FLT3 gene that is, susceptible to ITD and the region susceptible to insertion on the NPM1 gene separately. | Table 1: Frequency of molecular abnormalities in acute myeloid leukemia (our study comparison to multiple international studies)
Click here to view |
Briefly, 1 μL of the diluted labeled PCR product (1:20) was mixed with 8.5 μL Hi-Di formamide and 0.5 μL of Genescan Liz 500 size standard. The samples were then denatured at 95°C for 3 min and then chilled for 3 min in −20°C freezer. The size of the PCR product was then determined by capillary electrophoresis using the ABI 3500xL Genetic Analyzer (Applied Biosystems, Foster City, California, USA) using POP4™ polymer (Applied Biosystems, Foster City, California, USA) as per manufacturer specifications.
Sequence analysis
Genomic DNA was extracted from bone marrow samples in EDTA using MagNa Pure 96 automated nucleic acid extraction system (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. PCR was performed with 100 ng DNA using FastStart Taq DNA Polymerase, 5 U/μL using specific in house primers designed to amplify exon 4 of IDH1, exon 4 of IDH2, exon 26 of DNMTA3, the entire coding region of CEBPA, exon 7 and exon 9 of WT1, and exons 8, 9, 11, 13, and 17 of c-KIT separately (Supplement [Table 1]).[5] The PCR products were purified using magnetic beads with the Agencourt® AMPure® XP kit (Beckman Coulter, Brea, California, USA), followed by direct sequencing with a BigDye Terminator v3.1 cycle sequencing ready reaction kit (Applied Biosystems, Foster City, California, USA) and purification using Agencourt® CleanSEQ® kit (Beckman Coulter) according to manufacturer’s instructions.
The amplified sequences were analyzed on an ABI 3730XL automated sequencer from both strands. Analysis and mutation nomenclature is based on GenBank accessions: NM_005896 (IDH1), NM_002168 (IDH2), NM_022552 (DNMTA3), NM_004364 (CEBPA), NM_024426.4 (WT1), and NM_000222.2 (c-KIT).
| Results | | |
A total of 100 cases diagnosed as AML were assessed with median age of 29 years and average of 31 years, the study had 58 male patients and 42 female patients, 76 adult patients and 24 pediatric patients. Of the 100 cases, 74 had de novo AML, nine had secondary AML [to Myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN), or therapy related], 12 relapsed AML, and five AML patients prehematopoietic stem cell transplantation (the relapsed and pre-BMT cases included only if they were diagnosed first time outside KIng Faisal specialist Hospital & Research centre) [Figure 1].
The secondary AML cases were mainly progressed from myelodysplastic syndrome (5/9), two cases from chronic myeloid leukemia, one case from primary myelofibrosis, and one case from therapy-related leukemia.
Most of the secondary AML cases had complex karyotype above three abnormalities (five out of nine cases). The five cases that secondary to MDS: two cases with complex karyotype, one case with i(5),-12, one case with del(5q), and one case with del(7q). The two AMLs secondary to CML were one complex karyotype and one with inv(3). Lastly, the two cases secondary to PMF and therapy related had again complex karyotype.
Out of 100 AML patients investigated, 98 were successfully karyotyped, 64% of cases had an abnormality. In addition, all 100 AML–FISH panel studies were informative with an abnormality reaching 50%. Conventional and molecular cytogenetic (FISH) studies revealed trisomy 8 (15%), t(8;21) in (12%), trisomy 21 (8%), inv(16) in (7%), t(15;17) in (6%), 11q rearrangements (6%), and inv(3) (2%).
Furthermore, out of 74 de novo cases, 66% of cases had abnormal karyotype. Conventional and molecular cytogenetic (FISH) studies revealed trisomy 8 (15%), t(8;21) in (15%), inv(16) in (8%), trisomy 21 (7%), t(15;17) in 7%, 11q rearrangements (7%), and inv(3) (3%). These results showing high frequency of trisomy 8 and trisomy 21, also the t(8;21), are highest recurrent cytogenetic abnormality with balanced translocations/inversions and lower frequency of t(15;17) in our study comparing to different international studies [Table 1].
The cases of t(8;21) had median age of 20 years (range: 5–47 years), males 67 % (8/12) and females 33% (4/12), we noticed that 62% (5/8) of male patients had loss of Y chromosome, only one case (1/12) was positive c-KIT mutation.
The cases of t(15;17) or its variants had median age of 23 years (range: 15–32 years), four males, two females, one case was with FLT3–ITD mutation. There was one interesting case presented as relapsed AML (initially diagnosed outside KFSH with history of AML only) with variant RARA rearrangement t(5;17) accompanied with NPM1, FLT3–ITD, IDH2, and DNMT3A mutations.
Moreover, cases of inv(16) or t(16/16) had median age of 34 years (range: 10–51 years), five males, two females, with one case was positive for c-KIT mutation, whereas cases of 11q rearrangement had median age of 7 years (range: 2 months–15 years), four males, two females, and no cases with positive mutations.
Furthermore, 45% of AML cases were positive for mutations in the screened genes. The study of all 100 AML cases showed the frequency of NPM1 mutation (12%), FLT3–ITD (9%), IDH2 (7%), IDH1 (6%), WT1 (5%), DNMT3A (4%), CEBPA (4%), and c-KIT (3%). In 74 de novo AML cases, the mutation frequency were as follows: NPM1 mutation (9.5%), FLT3–ITD (9.5%), IDH2 (8%), IDH1 (5.4%), WT1 (5.4%), DNMT3A (4%), CEBPA (4%), and c-KIT (2.7%). Finally, the mutations frequency from cytogenetic normal cases (35 cases) are as follow: NPM1 mutation (23%), FLT3–ITD (17%), IDH2 (3%), IDH1 (9%), WT1 (9%), DNMT3A (9%), CEBPA (6%), and c-KIT (0%). Most of these results showing a less rate to those previously published in international studies especially for NPM1 and FLT3–ITD mutations [Table 2]. | Table 2: Frequency of cytogenetic abnormalities in acute myeloid leukemia (our study comparison to two large international studies)[21],[22]
Click here to view |
Cases positive for NPM1 mutation had a median age of 43 years (range: 10–69 years), 75% (9/12) had normal karyotype, 8% (1/12) the karyotyping was failed, and 17% (2/12) had abnormal karyotype [t(10;16)(q22;p13.1) and t(5;17)(p12;q11.2)].
Cases positive for FLT–ITD mutation had a median age of 22 years (range: 15–59 years), 67% (6/9) had normal cytogenetic karyotype, 11% (1/9) with t(15;17), 11% (1/9) with t(6;9) combined with trisomy 8, and 11% (1/9) with trisomy 8 only.
Cases positive for CEBPA mutation had a median age of 42 years (range: 10–53 years), 75% (3/4) had normal karyotype and 25% (1/4) had del(5q) with trisomy 8.
Cases positive for c-KIT mutation had median age of 20 years (range: 9–51 years), 100% (3/3) had abnormal karyotype: first one with t(8;21), second one with inv(16), and last one with t(5;17) accompanied with trisomy 8, trisomy 21, FLT3–ITD, IDH2, and DNMT3A mutations [Table 3]. | Table 3: Cytogenetics abnormalities of prognostic significant molecular mutations
Click here to view |
| Discussion | | |
Although morphological evaluation of bone marrow aspiration and biopsy remains as a cornerstone for the diagnosis of AML, it is clear that the presence or absence of specific cytogenetic abnormalities and acquired genetic mutations is not only useful for determining overall prognosis but is also useful for guiding treatment. Although cytogenetic abnormalities present at diagnosis enable the prediction of outcome and, in turn, the stratification to risk-adapted treatments, clonal chromosomal aberrations are not detected in 40 to 50% of AML patients.[12] For this CN group of a clinically heterogeneous subset of AML patients, the presence of acquired mutations allows for molecular-risk classification.[13],[14] In our study, the cytogenetic normal percentage is 36% for all 100 cases and 34% of the 74 de novo cases which are slightly low comparing with these studies, also we had high frequency of trisomy 8, acquired trisomy 21, and lower frequency of translocation (15/17)[Table 1].
Similarly, the mutations incidence for NPM1 and FLT3–ITD are less than 20 to 30% internationally reported. Our study showed that NPM1 and FLT3–ITD represent 12 and 9%, respectively, of all 100 tested cases, and 9.5 and 9.5% of the 74 de novo AML cases. Likewise when we compare the mutations incidence for NPM1 and FLT3–ITD with the cytogenetic normal cases (35 patients), NPM1 are 23% (9/35) and FLT3–ITD 17% (6/35) in comparison to 45 to 60% and ∼30%, respectively [Table 2].
The relevance of recurrent molecular abnormalities in CN-AML has been acknowledged by the inclusion of these markers within both the World Health Organization of 2008[1] and the European Leukemia Net classifications as a complement to cytogenetic analysis.[15],[16]
Patients with a cytogenetic profile associated with a favorable risk (i.e., those with PML–RARA, RUNX1–RUNX1T1, or MYH11–CBFB fusions) have relatively good outcomes with chemotherapy-based consolidation regimens, whereas patients with an adverse profile (monosomy karyotype or complex alterations) require allogeneic transplantation during the first remission to improve the prognosis.[17],[18] However, the majority of patients with AML have an intermediate cytogenetic risk (most commonly, a normal karyotype); some of these patients do well with chemotherapeutic consolidation, but others have a very poor outcome. For this reason, recent studies have focused on establishing new biomarkers for better classification of intermediate risk. The latest classification algorithms incorporated FLT3, NPM1, CEBPA, and c-KIT into the standard testing protocol. More recently, such testing has revealed that mutations in newly discovered AML genes (e.g., DNMT3A, IDH1/2, and TET2) may also provide prognostic information for some patients with an intermediate-risk profile.[19],[20],[21]
None of the current classification schemes are entirely accurate, which suggests that a more complete understanding of the genetic and epigenetic changes that are relevant to the pathogenesis of AML will be required for better classification of risk and, ultimately, better approaches to therapy.[22]
Grossmann et al.[23] recently published a novel hierarchical prognostic model of AML solely based on molecular mutations performed using next-generation sequencing (NGS) technology. Five distinct prognostic subgroups were identified: (1) very favorable: PML–RARA rearrangement or CEPBA double mutations with a 3-year overall survival (OS) rate of 82.9%; (2) favorable: RUNX1–RUNX1T1, CBFB–MYH1, or NPM1 mutation without FLT3–ITD with a 3-year OS of 62.6%; (3) intermediate: none of the mutations leading to assignment into groups 1, 2, 4, or 5 with a 3-year OS of 44.2%; (4) unfavorable: MLL–PTD, RUNX1, and/or ASXL1 mutation with a 3-year OS of 21.9%; and (5) very unfavorable: TP53 mutation with a 3-year OS of 0%. This comprehensive molecular characterization provides a more powerful model for prognostic prediction than cytogenetic and conventional sequencing platforms (especially if karyotype going with intermediate prognosis); therefore, we were encouraged to develop our own AML/MDS panel for Saudi Arabia that includes more than 50 gene mutations known in hematological malignancies. Further, the validation of NGS-based panel mutations and conventional sequencing will be reported and published separately.
| Conclusion | | |
The median and the average age of AML diagnosis are lower in our population. The incidence of most mutational analysis is lower in our population, whereas abnormal karyotype showed almost similar frequency compared to different international centers. Frequency of trisomy 8 and trisomy 21 is higher in our population, whereas t(15;17) is lower. This is the first cytogenetic data from Saudi Arabia for AML, including multiple genetic mutations. Therefore, a multicenter collaboration and comprehensive study is recommended to confirm these findings.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1]
[Table 1], [Table 2], [Table 3]
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