|Year : 2012 | Volume
| Issue : 4 | Page : 200-206
Nucleophosmin (NPM1-A) and FMS-like tyrosine kinase 3 (FLT3/ITD) mutations in acute myeloid leukaemia with a normal karyotype ( frequency and prognostic significance)
Hosneia K.H. Akl1, Heba H Gawish1, Fouad M Abou Taleb2
1 Department of Clinical Pathology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Medical Oncology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
|Date of Submission||22-Apr-2012|
|Date of Acceptance||01-Jun-2012|
|Date of Web Publication||21-Jun-2014|
Hosneia K.H. Akl
Department of Clinical Pathology, Faculty of Medicine, P.O. Box 61519, Zagazig University, Zagazig
Source of Support: None, Conflict of Interest: None
Treatment of acute myeloid leukaemia (AML) with a normal karyotype (NK) has always been a challenge for clinicians. This is because it affects the majority of AML patients and falls into the intermediate-risk group that needs further risk stratifications.
Aim of the study
The current study aimed to verify the importance and significance of analysis of NPM1-A and FLT3/ITD mutations, alone and combined, and their relation to prognostic criteria.
Patients and methods
Thirty-five patients with de-novo AML with a median age of 33 years (19–60) and ten healthy volunteers (the control group) were included. All participants were subjected to clinical examination, routine laboratory investigations and (for patients only) bone marrow examination and cytogenetic analysis by the G-banding technique. Using the genomic PCR (RT and conventional) technique, NPM1 type A and FLT3/ITD mutations were studied in the NK-AML patients and controls. Their impacts were evaluated alone and combined.
After cytogenetic analysis 20/35 (57.1) patients proved to be of NK; their median age was 37.5 (19–60) years. This NK-AML group and control group were examined for NPM1 and FLT3/ITD mutations. None of the controls had either mutation. The NK group was categorized into NPM1+ or NPM1− and FLT3+ or FLT3−. Their frequencies were 40% (8), 60% (12) and 40% (8), 60 (12), respectively. These subgroups were assessed after induction chemotherapy for achievement of complete remission (CR), which was recorded in 62.5% of NPM1-positive patients and 0% of NPM1-negative patients, showing a significant difference with a P value of 0.004, whereas nonsignificant difference between FLT3-positive and FLT3-negative patients was recorded. The NK patients were further subdivided into four genetic subgroups: NPM+/FLT3−, NPM−/FLT3−, NPM+/FLT3+ and NPM−/FLT3+, with frequencies of 15, 45, 25 and 15%, respectively. Sixty per cent of patients who achieved CR were NPM1+/FLT3−. Furthermore, 100% of this subgroup achieved CR, 40% of NPM+/FLT3+ also achieved CR, but none of the NPM−/FLT3− and NPM1−/FLT3+ patients did.
Genotypes defined by the mutational status of NPM1, FLT3 are associated with the outcome of treatment in NK-AML patients.
Keywords: AML, FLT3/ITD, NPM1-A, PCR, prognosis
|How to cite this article:|
Akl HK, Gawish HH, Abou Taleb FM. Nucleophosmin (NPM1-A) and FMS-like tyrosine kinase 3 (FLT3/ITD) mutations in acute myeloid leukaemia with a normal karyotype ( frequency and prognostic significance). Egypt J Haematol 2012;37:200-6
|How to cite this URL:|
Akl HK, Gawish HH, Abou Taleb FM. Nucleophosmin (NPM1-A) and FMS-like tyrosine kinase 3 (FLT3/ITD) mutations in acute myeloid leukaemia with a normal karyotype ( frequency and prognostic significance). Egypt J Haematol [serial online] 2012 [cited 2019 Dec 9];37:200-6. Available from: http://www.ehj.eg.net/text.asp?2012/37/4/200/134965
| Introduction|| |
Acute myeloid leukaemia (AML) is a heterogeneous clonal disorder of haemopoietic progenitor cell ‘blasts’, which lose the ability to differentiate normally and respond to normal regulators of proliferation. This loss leads to fatal infection, bleeding or organ infiltration, typically in the absence of treatment, within 1 year of diagnosis 1.
The prognosis of patients with AML depends on several well-defined factors including age of the patient, intensity of postremission therapy (in younger adults), biological characteristics of the disease and, the most important, the karyotype at the time of diagnosis. Patients with cytogenetically normal AML comprise the largest subgroup of AML patients 2. These patients pose considerable challenges in pathogenesis, risk stratification and prognosis because of their heterogeneous nature at the molecular level 3.
To date, karyotype at diagnosis provides the most important prognostic information for adult patients with AML, and cytogenetic data are used to assign patients to distinct prognostic groups within risk-adapted treatment protocols to improve treatment outcome and minimize toxicity of therapies 4–6.
Three distinct groups of patients, favourable, intermediate and unfavourable risk groups, can be categorized on the basis of their leukaemic cell karyotype 5. This cytogenetic testing at diagnosis yields critical prognostic information, but more refinement in prognosis is required. Within the cytogenetic subgroups, further important subgroup definitions are possible based on the mutation status and expression analysis of genes such as C-KIT, feline McDonough sarcoma (FMS)-like tyrosine kinase 3 (FLT3), nucleophosmin (NPM)1 and CCAAT/enhancer-binding protein α (CEBPA), although defining therapies on the basis of such mutations remains controversial 7.
The FMS-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinase (RTK), with a structure that resembles KIT, FMS and the platelet-derived growth factor (PDGF) receptor 8.
The most common mutation in the FLT3 gene is the FLT3 internal tandem duplication (FLT3/ITD) of the region coding for the juxtamembrane domain of the FLT3 receptor 9.
AML with FLT3/ITD mutation is an aggressive haematologic malignancy with a generally poor prognosis. It can be successfully treated into remission with intensive chemotherapy, but the patient routinely relapses 10.
Moreover, Rockova et al. 11 found that patients with FLT3/ITD mutations have lower overall survival and event-free survival.
Nucleophosmin (NPM1) is a nucleocytoplasmic shuttling protein that plays a key role in a variety of cellular processes including promotion of ribosome biogenesis, maintenance of genomic stability, regulation of transcription and modulation of tumor-suppressor transcription factors 12.
To date, NPM1 gene mutations represent the most commonly known genetic abnormality in AML. Moreover, these alterations have been shown to carry prognostic significance because they seem to identify patients with better response to chemotherapy 13. Mutations of the NPM1 gene in AML were investigated and six main types were found (A, B, C, D, E and F), but the most common was type A. Cytoplasmic NPM1 is a characteristic feature of a large subgroup of patients with AML who have a normal karyotype (NK), NPM gene mutations and responsiveness to induction chemotherapy 14.
There was a significant correlation between worse overall survival, relapse risk and increased white blood cell count with increased mutant level in FLT3 in patients with AML, and patients with the FLT3 duplication had a worse risk of relapse compared with patients without the FLT3 duplication. However, the presence of an NPM1 mutation had a beneficial effect on the remission rate in patients with and without FLT3 duplications, most likely because of a lower rate of RD. AML was classified into three prognostic groups: good in those with only an NPM1 mutation; intermediate in those with either no FLT3 or NPM1 mutation or mutations in both genes; and poor in those with only FLT3 mutations 15.
Falini et al. 14 found a higher frequency of FLT3 mutation in cases with mutant NPM1, indicating a possible pathogenic link between these two gene mutations.
This study aimed to evaluate the frequency and prognostic significance of FLT3/ITD and NPM1 type A mutations and their relationship with different parameters in AML patients with NKs.
| Patients and methods|| |
The present study was carried out on randomly selected adult patients with newly diagnosed de-novo AML before receiving induction chemotherapy during the period from March 2009 to April 2011. Informed consents were obtained from all of them to use their samples for research. There were 20 male and 15 female patients; their ages ranged from 19 to 60 years with a median of 33 years. All patients were diagnosed according to FAB cooperative group criteria using standard methods including morphology, cytochemistry and immunophenotyping.
After cytogenetic analysis, AML patients with a NK, 20 patients (12 male and 8 female; ages ranged from 19 to 60 years with a median of 37.5 years) were evaluated for the presence of NPM1-A and FLT3/ITD mutations.
Ten haematologically normal individuals (controls) of matched age and sex were also included in the study to verify the presence or absence of NPM1-A and FLT3/ITD mutations.
All studied persons were subjected to the following:
Full history taking, thorough clinical examination and radiological studies including chest radiograph, computed tomography scans and pelvi-abdominal sonar (if indicated).
Routine laboratory investigations including:
Complete blood picture with staining of peripheral blood and bone marrow (BM) films with Leishman stain and myeloperoxidase cytochemistry to confirm diagnosis.
Liver and kidney function tests.
Immunophenotyping by flow cytometry16 (patients only). The following labeled monoclonal antibodies were used: B-cell markers CD 19, CD20 and CD22; T-cell markers CD3, CD5 and CD7; early markers HLA-DR, CD34, CD10 and TdT; myeloid markers CD13, CD33 and CD14; and control markers IgGla and IgG2a (FACScan; Becton Dickinson, San Diego, California, USA).
Special Investigations such as:
Cytogenetic analysis using the G-banding technique (Cell imaging analyzer; IMSTAR, Paris, France) for patients.
Detection of FLT3-ITD and NPM1-A mutations by Genomic PCR in patients with NK and in healthy controls.
RT-PCR of NPM1 exon 12 mutation A
RNA extraction, reverse transcription by RT-PCR and detection by agarose gel electrophoresis were carried out in the sequence mentioned.
A volume of 1 ml of sterile whole-blood (or BM aspirate for patients) samples collected on sterile EDTA vacutainers (Becton Dickinson and Company, Franklin Lakes, New Jersey) were subjected to treatment with an RNA Extraction Kit (E.Z.N.A. Blood RNA Kit; Omega Bio-Tek Incorporation, Norcross, Georgia, USA).
Reverse transcription-PCR (RT-PCR) reaction was performed with each sample using ‘Illustra Ready-to-Go RT-PCR Beads’ supplied by General Electric Healthcare (Waukesha, Wisconsin, USA) to amplify fragments of NPM1 exon 12 mutation A.
For amplification of NPM1, we used the forward primer NPM-A and the reverse primer NPM-REV-6, and their sequences were as follows:
The forward primer contains an intentional mismatch at the third, specifically designed to exclude amplification of NPM1 pseudogenes 13.
As internal PCR control, we used ABL gene amplification. ABL gene is a housekeeping gene that, with successful amplification, should be detected in all samples. The same RT-PCR conditions were used, but with specific forward primer ABL-A2B- reverse primer ABL-A3E-. Their sequences were as follows:
Amplification protocol: The following temperature scheme was applied for samples and for negative control reactions 13. For amplification, the thermal cycler Gene Amp PCR System 9700 supplied by Perkin-Elmer, (California, USA), was used: preheating at 95°C for 7 min, 35 amplification cycles at 95°C for 30 s, denaturation at 67°C for 45 s, annealing at 72 C for 45 s and elongation and final extension at 72°C for 7 min.
Detection by Agarose Gel Electrophoresis: The PCR products were electrophoresed on 2% agarose gels and analysed by direct visualization after ethidium bromide staining. A DNA molecular weight marker of 100–1000 bp (Bioron, Ludwigshafen, Germany) was used. The size of any resulting band could be detected using the molecular size marker, which gave different bands ranging from 100 to 1000 bp. Detection of a 320-bp product indicated the presence of NPM1 exon 12 mutation A. ABL product detection at 258 bp was used as an internal control of successful extraction and amplification.
Detection of FLT3/ITD by genomic PCR technique
Three steps were performed: DNA extraction, PCR amplification, and the detection of amplicons using agarose gel electrophoresis.
Genomic DNA was prepared from the peripheral blood of normal individuals and from BM specimens or from the peripheral blood of patients obtained at the time of diagnosis using a GeneJET Genomic DNA Purification Kit (Fermentas; Thermo Fisher Scientific Inc., Surrey, UK) according to the standard procedure described by the manufacturer.
Primers for the PCR assay (Sigma, St. Louis, USA) were selected for amplification of exons 14 and 15 of the FLT3 gene. The primers were:
14 Forward: 5′ CAATTTAGGTATGAAAGCCAGC-3′
15 Reverse: 5′-CTTTCAGCATTTTGACGGCAACC-3′
The reaction was performed with a Thermal Cycler Gene Amp PCR System 9700 (Perkin-Elmer) using the following PCR cycling protocol: an initial denaturation step at 94°C for 4 min, followed by 40 cycles of PCR, with each cycle consisting of denaturation at 94°C for 1 min, annealing at 52°C for 30 s and extension at 72°C for 30 s. Then a final extension step was carried out at 72°C for 5 min.
Each run of PCR amplification included negative controls that had no template DNA to avoid false-positive results caused by suspected contamination.
Detection of PCR products was performed by the same procedure as used in NPM1 mutations. A fragment of 328 bp, which is the normal FLT3 gene product, is produced from wild-type alleles. Any sample displaying an additional PCR product (longer than 328 bp) was considered as FLT3/ITD positive. Hence, FLT3/ITD-positive cases showed an extra PCR fragment that was larger than the wild-type fragment. The sizes of the mutant bands were variable, as indicated by different electrophoretic mobility of the various bands 17.
All patients received induction chemotherapy in the form of —one to two courses of the standard 3+7 induction regimen 18 (adriamycin 25 mg/m2 intravenously daily from days 1–3, cytarabine 100 mg/m2 continuous intravenous infusion daily from days 1–7). Complete blood counts and BM aspirates were obtained 28 days after receiving induction therapy to evaluate the remission status. Complete remission (CR) was defined as normocellular BM containing less than 5% blasts. However, most of the cases defined as CR achieved a neutrophil count of l×109/l and platelet count of 100×l09/l 19. Remission failures were classified as either partial remission (PR, defined as 5–15% blasts or <5% blasts but a hypocellular BM), resistant disease (RD, >15 blasts in the BM), or induction death (ID, related to treatment or hypoplasia) 19.
Data were analysed using SPSS win statistical package version 17 (SPSS Inc., Chicago, Illinois, USA). The χ2-test or Fisher’s exact test was used to examine the relationship between qualitative variables. For not normally distributed quantitative data, comparison between two groups was made using the Mann–Whitney test. A P value less than 0.05 was considered significant.
| Results|| |
This study was conducted on 35 de-novo AML patients. After cytogenetic analysis only 20 patients proved to be of NK. The demographic data and some laboratory data of these NK patients are shown in [Table 1].
|Table 1: Some demographic and laboratory data of AML patients with a normal karyotype|
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The NK group and 10 healthy volunteers were examined for NPM1 and FLT3/ITD mutations. They were classified into NPM1+ or NPM1− and FLT3+ and FLT3−.
[Table 2] shows demographic data and laboratory data according to the presence or absence of either mutation, together with a comparative Mann–Whitney test (nonparametric t-test) for some parameters and the χ2 t-test for the rest. BM blast showed a nonsignificant difference between NPM1-positive and NPM1-negative groups, being higher in the NPM1-negative group (P=0.181). The percentage of CD34 showed a nonsignificant difference between NPM1 groups (P=0.157). A nonsignificant difference was found with respect to all parameters between FLT3-positive and FLT3-negative groups. It showed a nonsignificant difference with regard to the monocytic leukaemia group (M4 and M5) as it is more frequent in the NPM-positive group (P=0.170). Moreover CD14 showed a nonignificant difference between both groups as it was more frequent in the NPM1-positive group (P=0.161) (data not shown).
|Table 2: Demographic and laboratory data for the presence or absence of NPM1 or FLT3 mutations|
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[Table 3] shows a comparison between both CR and nonremission (NR) groups, a highly significant difference concerning BM blast % as it was higher in the NR group (P<0.001) and a significant difference concerning CD34 percentage as it was lower in the CR group (P<0.05).
|Table 3: Comparison between CR and NR groups regarding demographic and laboratory data|
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[Table 4] shows a comparison between NPM1-positive and NPM1-negative groups concerning FLT3 positivity. It showed a nonsignificant difference as it was higher in NPM1-negative cases (P=0.167).
The NK group was further subdivided into four subgroups according to the presence or absence of NPM1/FLT3 mutations [Table 5]. It showed that 15% were in the NPM1+/FLT3− group; 45% were in the NPM1−/FLT3− group; 25% were NPM1+/FLT3+; and 15% were in the NPM1−/FLT3+ subgroup.
|Table 5: Frequency of different combinations of both mutations in the NK group|
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Demographic and laboratory parameters in the four genetic subgroups are shown in [Table 6].
|Table 6: Some demographic and laboratory data on the four genetic subgroups|
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[Table 7] and [Table 8] show the achievement of CR among the four subgroups and the frequency of CR in the NK group distributed among them.
In both [Table 2] and [Table 3] the χ2-test was used for analysis of FAB and sex, whereas the Mann–Whitney test was used for age and other laboratory tests.
| Discussion|| |
Molecular analyses of leukaemic blasts from patients with AML have revealed a striking heterogeneity with regard to the presence of acquired gene mutations and changes in gene and microRNA expression. Multiple submicroscopic genetic alterations with prognostic significance have been discovered 20. In the last decade, somatically acquired mutations have been identified in several genes in cytogenetically normal AML cases: the nucleophosmin 1 (NPM1) gene, the FMS-related tyrosine kinase 3 (FLT3) gene, the CCAAT/enhancer-binding protein α (CEBPA) gene, the myeloid-lymphoid or mixed-lineage leukaemia (MLL) gene, the neuroblastoma RAS viral oncogene homologue (NRAS) gene, the Wilms tumor 1 (WT1) gene, and the runt-related transcription factor 1 (RUNX1) gene 3,21.
In the current study 20 of 35 de-novo AML patients were of NK with a percentage of 57.1, which is in agreement with the study by Zidan et al. 22 who worked on a comparable number of Egyptian patients of whom 62% of all AML patients proved to be of NK. It is different from the study by Lo-Coco et al. 23 who had 42.8% of patients with NK, which may be because of diverse eligibility selection criteria and the difference in the number of cases between studies.
NPM1 and FLT3/ITD mutations and their relation to different parameters, single or combined, were evaluated among these NK patients and controls and none of the controls had either mutation.
The frequency of NPM1 mutation among the normal karyotype group was 40% (eight cases), which is nearly similar to that of Döhner et al. 24 who recorded 48% of normal karyotypic patients with NPM1 mutations. Other studies 25–28 proved that NPM1 had a frequency ranging from 24 to 60% of normal karyotype AML patients; the difference among studies could be attributed to difference in sample size, selection criteria and age limit for each study.
In addition, FLT3 positivity in patients with a NK was 40%, which was similar to the 41% seen in the study by Ottone et al. 13 but higher than those in other studies 22,29–31 and lower than that in the study by Kottoridis et al. 32, who recorded a frequency of 57%.
This significant discrepancy among different studies could be attributed to the fact that the addressed problem is a genetic mutation that may have a different prevalence in different ethnic populations; another reason could be the difference in detection methods.
Different demographic and laboratory data were compared according to the presence or absence of NPM1 or FLT3/ITD mutations. BM blasts showed near significant difference between NPM1-positive and NPM1-negative groups, being higher in the NPM1-negative group. This result is different from that of previous studies 21, 26, which reported that higher blasts were found in NPM1-positive cases, which may be because of the small sample size of this study.
The percentage of CD34 positivity showed a nonsignificant difference between the two groups as it was lower in NPM1-positive patients, agreeing with previous studies 24, 28, which found a favourable prognosis in the presence of NPM1 mutation.
Moreover, there was an association with monocytic FAB subtypes in the NPM1-positive group, as 60% of them were either M4 or M5, with a nonsignificant difference between the two groups (P=0.170). This is in agreement with other studies 24, 26, which found a significant association between monocytic subtypes and NPM1 positivity, but it was different from the study by Verhaak et al. 28, who did not report any association between FAB subtypes and NPM1 mutations. This may be attributed to the difference in sample size and the population studied.
There was no significant difference between FLT3-positive and FLT3-negative patients with regard to demographic data and laboratory parameters. This is different from several studies 33–38, which found that white blood cell count was significantly higher in FLT3/ITD-positive patients as compared with FLT3/ITD-negative patients and also reported that FLT3/ITD is associated with leucocytosis, which was explained by the fact that FLT3/ITD has been shown to cause constitutive activation of the RTK, leading to autonomous cytokine-independent cellular proliferation 39. This discrepancy in the results may be because of the difference in the number of studied patients, as a limited number does not always allow proper statistical analysis of the results.
In the current study none of the haematological data showed a significant difference between FLT3-positive and FLT3-negative patients in agreement with several previous studies 22,40–42.
Achievement of CR after induction chemotherapy was compared among NPM1-positive and NPM1-negative patients with a NK; 62.5% of NPM1-positive patients achieved CR, whereas 0% of NPM1-negative patients achieved CR, with a significant difference between the two groups (P=0.004). This result is slightly lower than that seen in the studies by Schnittger et al. 26 (86%) and Gale et al. 15, who found that favourable outcome was associated with NPM1 mutation, as 91% of NPM1-positive patients achieved CR in their study.
With regard to NPM1/FLT3 mutations there were four subgroups in the following frequencies: NPM1+/FLT3−, 15%; NPM1−/FLT3−, 45%; NPM+/FLT3+, 25%; and NPM−/FLT3+, 15%; these results go hand in hand with the finding of Döhner et al. 24 (28, 39, 19.6 and 12.6%, respectively) but are different from those of Lo-Coco et al. 23 (41.4, 33.3, 18.9 and 6.3% respectively). This can be explained by the fact that it is a genetic mutation that differs among ethnic populations.
Demographic and laboratory parameters showed no statistically significant difference among the four subgroups, but BM blast percentage was lower in the NPM1+/FLT3− subgroup with a median of 59% than in the other three subgroups (85% in each); the percentage of CD34 positivity was also the least in the same group with a median of 15% versus 27, 18 and 34%, respectively. This may indicate good prognosis for the NPM1+/FLT3− group and is comparable to the results of previous studies 15, 24, which reported that there was significant difference among the four groups with regard to BM blasts but it was the least in the NPM1+/FLT3− subgroup. Moreover, they recorded that CD34 positivity had a highly significant difference (P<0.001) among the four subgroups with the least positivity in NPM1+/FLT3− patients.
The four subgroups were evaluated for the achievement of CR after induction chemotherapy, and 60% of patients who achieved CR belonged to the NPM1+/FLT3− group, 40% to the NPM+/FLT3+ group and 0% belonged to the other two groups.
Moreover, 60% of cases who did not achieve CR were in the NPM1−/FLT3− group and 20% in the NPM−/FLT3+ group. This is in agreement with the results of Lo-Coco et al. 23, who found that NPM1+/FLT3− had higher disease-free survival; they stated that NPM1 status is the most important factor to have an impact on CR, whereas FLT3/ITD was the most influential on disease-free survival.
Moreover, in another study 24 both NPM1 and FLT3/ITD mutations did not appear as independent prognostic markers when tested as a single variable, but both mutations had significant interaction that influenced prognosis.
Gale et al. 15 stratified patients according to their mutational status for both NPM1 and FLT3/ITD as follows: NPM1+/FLT3−, good prognosis; NPM1+/FLT3+ and NPM1−/FLT3−, intermediate prognosis; and NPM1−/FLT3+, poor prognosis. Results of the present study partially follow the stratification by Gale et al. 15, as 60% of the CR group was NPM+/FLT3− and 100% of this subgroup achieved CR. In contrast, 0% of the NPM−/FLT3+ group achieved CR but the remaining two subgroups could not be stratified on the basis of this classification. This may be because of the small sample size that became smaller by further dividing patients into four subgroups, not allowing reliable comparative statistical analysis.
Döhner et al. 43 classified NPM1-positive patients without FLT3 mutation in the favourable genetic group, whereas the other three subgroups were classified in the intermediate I group. None of the other groups came in the intermediate II or the adverse genetic group.
| Conclusion|| |
FLT3-ITD and NPM1 mutation status can be defined in NK-AML cases. Prospective screening for these mutations is advocated in all NK-AML patients, as the genotype is of clinical importance when considering treatment options, including stem cell transplantation. Further studies with a larger number of patients are recommended to allow better understanding of NPM1 and FLT3/ITD interaction and study of other related genes for better risk stratification.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]