Research Progress of FLT3 Mutation in Acute Myeloid Leukemia --Review / 中国实验血液学杂志

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic tumor originated from hematopoietic stem cells. FLT3 is an important receptor tyrosine kinase in cell signal transduction pathway and one of the common mutated genes in AML. AML patients with FLT3-ITD mutation have a poor prognosis and tendency to relapse. Therefore, early identification of FLT3 gene mutation and selection of appropriate treatment are particularly important. Currently, the small moleculetargeted drugs have been new treatment methods for AML patients with FLT3-ITD mutation, but accompanied drug resistance need to be solved. This paper reviews the mechanism of FLT3 mutation, the clinical significance of FLT3 mutation in AML, FLT3 inhibitors and drug resistance mechanism.

Clinical Value of Translocator Protein Gene in Evaluating the Efficacy of FLT3-ITD/DNMT3A R882 Double-Mutated Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To observe the clinical significance of translocator proteins (TSPO) gene in the treatment of FLT3-ITD/DNMT3A R882 double-mutated acute myeloid leukemia (AML).@*METHODS@#Seventy-six patients with AML hospitalized in the Department of Hematology of the Affiliated People's Hospital of Ningbo University from June 2018 to June 2020 were selected, including 34 patients with FLT3-ITD mutation, 27 patients with DNMT3A R882 mutation, 15 patients with FLT3-ITD/DNMT3A R882 double mutation, as well as 19 patients with immune thrombocytopenia (ITP) hospitalized during the same period as control group. RNA was routinely extracted from 3 ml bone marrow retained during bone puncture, and TSPO gene expression was detected by transcriptome sequencing (using 2-deltadeltaCt calculation).@*RESULTS@#The expression of TSPO gene in FLT3-ITD group and DNMT3A R882 group at first diagnosis was 2.02±1.04 and 1.85±0.76, respectively, which were both higher than 1.00±0.06 in control group, but the differences were not statistically significant (P=0.671, P=0.821). The expression of TSPO gene in the FLT3-ITD/DNMT3A R882 group was 3.98±1.07, wich was significantly higher than that in the FLT3-ITD group and DNMT3A R882 group, the differences were statistically significant (P=0.032, P=0.021). The expression of TSPO gene in patients who achieved complete response after chemotherapy in the FLT3-ITD/DNMT3A R882 group was 1.19±0.87, which was significantly lower than that at first diagnosis, and the difference was statistically significant (P=0.011).@*CONCLUSION@#TSPO gene may be used as an indicator of efficacy in FLT3-ITD /DNMT3A R882 double-mutated AML.

Research Advance of Gene Mutation and Targeted Drug Therapy in Childhood Acute Myeloid Leukemia --Review / 中国实验血液学杂志

The clinical therapeutic regimen for acute myeloid leukemia (AML) is not significantly different between adults and children, which is mostly based on IA (idarubicin and cytosine arabinoside) induction chemotherapy. With the rapid development of sequencing technique, people's understandings towards the molecular and biological abnormalities of AML are increasing, diverse AML gene mutation-based targeted drugs have been rapidly developed and applied. In this review, several commonly gene mutations in AML (such as FLT3, NPM1 and C/EBPA) was described, and the therapeutic effects and differences of targeted drugs that used in clinical treatment or had been reported (like tyrosine kinase inhibitor, IDH1 mutation inhibitor and epigenetic modification inhibitor) in child and adult AML patients were summrized.

Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML / 中国实验血液学杂志

OBJECTIVE@#Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes.@*METHODS@#The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib.@*RESULTS@#In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate.@*CONCLUSION@#The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

Association of Next Generation Sequencing Based Genotypic Profiling with MICM Characteristics in NPM1 Mutated Acute Myeloid Leukemia / 中国实验血液学杂志

OBJECTIVE@#To explain the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) by analyzing the association between next-generation sequencing (NGS) profiles and MICM characteristics in patients with this AML subtype.@*METHODS@#Data of 238 NPM1mut patients with available NGS information on 112 genes related to blood disease was collected, and χ2 test and nonparametric test were used to analyze the distribution association between NGS-detecting mutations and conventional MICM parameters.@*RESULTS@#In entire NPM1mut cohort, totaling 240 NPM1 mutation events were identified, of whom 10 (10/240, 4.2%) were missense mutations, which did not involve any W288 or W290 locus and were found exclusively in NPM1mut/FLT3-ITD- group. All but one of these missense mutations (9/10, 90%) were accompanied by AML subtype-defining recurrent cytogenetic or molecular abnormalities, of which 7 cases were in the low risk and 2 in the high risk. NPM1mut occurred solely as an insertion/deletion (indel) type in the NPM1mut/FLT3-ITD+ group. The incidence of favorable plus unfavorable karyotypes in NPM1mut/FLT3-ITD- group was higher than in NPM1mut/FLT3-ITD+ group (6.4% vs. 0, P=0.031). The positive rates of CD34 and CD7 in NPM1mut/FLT3-ITD+ group were significantly higher than in NPM1mut/FLT3-ITD- group (CD34: 47.9% vs. 20.6%, P<0.001; CD7: 61.5% vs. 29.9%, P<0.001). Logistic analysis showed that FLT3-ITD independently predicted for CD34+ and CD7+ [odds ratio (OR)=5.29, 95%CI: 2.64-10.60, P<0.001; OR=3.47, 95%CI: 1.79-6.73, P<0.001; respectively]. Ras-pathway mutations independently predicted for HLA-DR+ (OR=4.05, 95%CI: 1.70-9.63, P=0.002), and KRAS mutation for MPO- (OR=0.18, 95%CI: 0.05-0.62, P=0.007). TET2/IDH1 mutations independently predicted for CD34- and CD7- (OR=0.26, 95%CI: 0.11-0.62, P=0.002; OR=0.30, 95%CI: 0.14-0.62, P=0.001; respectively), and MPO+ (OR=3.52, 95%CI: 1.48-8.38, P=0.004). DNMT3A-R882 independently predicted for CD7+ and HLA-DR+ (OR=3.59, 95%CI: 1.80-7.16, P<0.001; OR=13.41, 95%CI: 4.56-39.45, P<0.001; respectively), and DNMT3A mutation for MPO-(OR=0.35, 95%CI: 1.48-8.38, P=0.004).@*CONCLUSION@#Co-existing FLT3-ITD in NPM1mut AML independently predicts for CD34+ and CD7+, co-existing Ras-pathway mutation for HLA-DR+ and MPO-, co-existing TET2/IDH1 mutation for CD34-, CD7-, and MPO+, and co-existing DNMT3A mutation for HLA-DR+, CD7+, and MPO-, thereby providing a new mechanism explanation for the immunophenotypic heterogeneity of these AML patients.

Effect of FLT3-ITD Length on 32D Cell Proliferation, Apoptosis and Sensitivity to FLT3 Inhibitor / 中国实验血液学杂志

OBJECTIVE@#To study the effects of FLT3-ITD length on 32D cell proliferation, apoptosis and sensitivity to FLT3 inhibitor, so as to provide references for stepwise therapy of FLT3-ITD mutated acute myeloid leukemia patients.@*METHODS@#Three different FLT3-ITD mutants with same or adjacent insert sites were selected and constructed in an eukaryotic expression vector. FLT3-ITD mutants stably expressed 32D cell strains were selected with the help of lentivirus system and IL3 free cell culture medium. The proliferation and apoptosis of 32D cell strains after AC220 treatment were detected.@*RESULTS@#FLT3-ITD mutants (ITD1, ITD2 and ITD3) stably expressed 32D cell strains were constructed successfully. In the absence of IL3 factor, the proliferation number of ITD1, ITD2 and ITD3 cell strains were mounted up to 2.3 folds, 3.7 folds, and 4.3 folds after 48 hours, respectively. Under the exposure of FLT3 inhibitor AC220, the IC@*CONCLUSION@#FLT3-ITD mutant expressed cell strains with longer ITD show higher capacity of proliferation and higher tolerance to AC220 treatment.

The Expression of WTAP Gene in Acute Myeloid Leukemia and Its Clinical Significance / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of WTAP gene in acute myeloid leukemia (AML) and its clinical significance.@*METHODS@#74 acute myeloid leukemia patients with non-M3 type and 19 normal donors were selected, and real-time quantitative polymerase chain reaction was used to detect the mRNA expression level of WTAP gene in their bone marrow cells. The relationship between the mRNA expression level of WTAP gene and the clinical characteristics was analyzed.@*RESULTS@#The relative mRNA expression of WTAP gene in the non-M3 AML group was significantly higher than that in the healthy control group, and the difference showed statistically significant (P0.05) according to the classification of FAB. The mRNA expression level of WTAP gene in FLT3-ITD mutated AML patients was higher than that in FLT3-ITD unmutated group (P=0.016), and the mRNA expression level of WTAP gene in AML patients with CEBPα mutation was lower than that in CEBPα unmutated group (P=0.016). The expression level of WTAP mRNA was positively correlated with WT1 expression (r=0.6866, P0.05). The expression level of WTAP mRNA showed no obvious effect on the complete remission of patients after first treatment. The different expression level of WTAP gene at initial diagnosis showed also no effect on the overall survival time of patients.@*CONCLUSION@#The expression level of WTAP gene is increasing in new diagnosed non-M3 acute myeloid leukemia. There is a positive correlation between the expression level of WTAP gene and the expression level of WT1 fusion gene. WTAP mRNA always shows higher expression in patients with FLT3-ITD mutation than that in patients without FLT3-ITD mutation, and shows lower expression in patients with CEBPα mutation than that in unmutated group.

Successful treatment of post-transplant relapsed acute myeloid leukemia with FLT3 internal tandem duplication using the combination of induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Report of three cases / Tratamento bem-sucedido de leucemia mieloide aguda recorrente após transplante com duplicação interna em tandem FLT3 usando combinação de indução por quimioterapia, infusão de linfócitos de doador, soferanib e azacitidina. Relato de três casos

ABSTRACT Acute myeloid leukemia is a hematopoietic stem cell neoplastic disease associated with high morbidity and mortality. The presence of FLT3 internal tandem duplication mutations leads to high rates of relapse and decreased overall survival. Patients with FLT3 internal tandem duplication are normally treated with hematopoietic stem cell transplantation in first complete remission. Nevertheless, the incidence of post-transplant relapse is considerable in this group of patients, and the management of this clinical condition is challenging. The report describes the outcomes of patients with FLT3 internal tandem duplication positive acute myeloid leukemia who relapsed after allogeneic hematopoietic stem cell transplantation and were treated with the combination of re-induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Three cases are described and all patients achieved prolonged complete remission with the combined therapy. The combination of induction chemotherapy followed by donor lymphocyte infusion, and the maintenance with azacitidine and sorafenib can be effective approaches in the treatment of post-hematopoietic stem cell transplant and relapsed FLT3 internal tandem duplication positive acute myeloid leukemia patients. This strategy should be further explored in the context of clinical trials.

RESUMO A leucemia mieloide aguda é uma doença neoplásica de células-tronco hematopoiéticas com alta morbimortalidade. A presença de mutações de duplicação em tandem de FLT3 leva a altas taxas de recorrência e a menor sobrevida global. Os pacientes com duplicação em tandem de FLT3 são normalmente tratados com transplante de células-tronco hematopoiéticas na primeira remissão completa. No entanto, a incidência de recidiva pós-transplante é considerável neste grupo de pacientes, e a conduta, nestes casos, é um desafio. O relato descreve os resultados do tratamento de pacientes com leucemia mieloide aguda positiva e duplicação em tandem de FLT3 que recidivaram depois do transplante alogênico de células-tronco hematopoiéticas e que foram tratados com combinação de quimioterapia de reindução, infusão de linfócitos de doador, sorafenib e azacitidina. São descritos três casos, e todos os pacientes apresentaram remissão completa prolongada com a terapia combinada. A combinação de quimioterapia de indução, seguida de infusão de linfócitos do doador, e a manutenção com azacitidina e sorafenib podem ser abordagens eficazes no tratamento da recorrência pós-transplante em pacientes com leucemia mieloide aguda e duplicação em tandem de FLT3. Essa estratégia deve ser mais explorada no contexto de ensaios clínicos.

AAcute WT1-positive promyelocytic leukemia with hypogranular variant morphology, bcr-3 isoform of PML-RARα and Flt3-ITD mutation: a rare case report / Leucemia promielocítica aguda WT1-positivo com morfologia variante hypogranular, isoforma bcr-3 da PML-RARα e mutação Flt3-ITD: relato de caso raro

ABSTRACT CONTEXT: Acute promyelocytic leukemia (APL) accounts for 8% to 10% of cases of acute myeloid leukemia (AML). Remission in cases of high-risk APL is still difficult to achieve, and relapses occur readily. CASE REPORT: Here, we describe a case of APL with high white blood cell counts in blood tests and hypogranular variant morphology in bone marrow, together with fms-like tyrosine kinase-3 with internal tandem duplication mutations (FLT3-ITD), and bcr-3 isoform of PML-RARα. Most importantly, we detected high level of Wilms’ tumor gene (WT1) in marrow blasts, through the reverse transcription polymerase chain reaction (RT-PCR). To date, no clear conclusions about an association between WT1 expression levels and APL have been reached. This patient successively received a combined treatment regimen consisting of hydroxycarbamide, arsenic trioxide and idarubicin plus cytarabine, which ultimately enabled complete remission. Unfortunately, he subsequently died of sudden massive hemoptysis because of pulmonary infection. CONCLUSION: Based on our findings and a review of the literature, abnormal functioning of WT1 may be a high-risk factor in cases of APL. Further studies aimed towards evaluating the impact of WT1 expression on the prognosis for APL patients are of interest.

RESUMO CONTEXTO: Leucemia promielocítica aguda (LPA) compreende 8% a 10% dos casos de leucemia mieloide aguda (LMA). A remissão em casos de LPA de alto risco ainda é dificilmente conseguida, e recorrência é comum. RELATO DE CASO: Descrevemos aqui um caso de LPA com glóbulos brancos elevados no exame de sangue e a morfologia variante hipogranular na medula óssea, juntamente com fms-like tirosina-quinase-3 com mutações de duplicação em tandem interna (FLT3-ITD) e a isoforma bcr-3 de PML- RARα. Mais importante, detectamos alto nível de gene do tumor de Wilms (WT1) em blastos medulares por RT-PCR (reverse transcription polimerase chain reaction). Até agora, não há conclusões claras sobre a associação entre os níveis de expressão WT1 e APL. Este paciente recebeu sucessivamente regime de tratamento combinado, de hidroxicarbamida, trióxido de arsênico e idarrubicina e citarabina, alcançando finalmente a remissão completa. Infelizmente, em seguida, ele morreu de repente de hemoptise maciça devido a uma infecção pulmonar. CONCLUSÃO: Com base em nossos resultados e numa revisão da literatura, a função anormal de WT1 pode ser um fator de alto risco em casos de APL. Novos estudos, com o objetivo de avaliar o impacto da expressão de WT1 no prognóstico dos doentes com APL, são de interesse.

Detection of TET2, KRAS and CBL variants by Next Generation Sequencing and analysis of their correlation with JAK2 and FLT3 in childhood AML

Background: Acute myeloid leukemia [AML] is a heterogeneous clonal disorder in terms of cytogenetic and molecular aberrations. Ten-Eleven-Translocation 2 [TET2], Kirsten rat sarcoma viral oncogene homolog [KRAS], and Casitas B-cell lymphoma [CBL] have an important role pathogenesis of acute myeloid leukemia [AML] and their activated mutations confer proliferative and survival signals

Aim: In this study, we aimed to find possible genetic markers for molecular analysis in childhood AML by screening hot-spot exons of TET2, KRAS, and CBL using Next Generation Sequencing [NGS] analysis. In addition, association between found variants and mutations of Januse Kinase-2 [JAK2] and Fms-Related Tyrosine Kinase [FLT3] were analyzed which are important prognostic risk factors for AML

Methods: Eight patients who were diagnosed with pediatric AML at Losante Pediatric Hematology- Oncology Hospital were included to the study. Hot-spot exons of TET2, KRAS and CBL genes were screened using the NGS method. Furthermore, FLT3-Internal Tandem Duplicate [FLT3-ITD] and JAK2-V617F were analyzed by Real Time Polymerase chain Reaction [Real Time-PCR]

Results: In total, we identified 20 variants in studied genes by NGS. In our patient group, 16 variants in the TET2 [seven novel, seven missense and two silent], two variants in the KRAS [one missense and one intronic] and two variants in the CBL [two novel] were found. All of AML patients were found negative for JAK V617 F. Three of the eight patients [37.5%] showed mutations of both FLT3-ITD and TET2, KRAS, CBL

Conclusion: We found novel mutations forTET2, KRAS, and CBL. The detected variants in this article seem to be the first screening results of genes studied by NGS in childhood AML patients. Our results also showed some degree of association between FLT3-ITD and TET2, KRAS, CBL mutations

Correlation of NPM1 Type A Mutation Burden With Clinical Status and Outcomes in Acute Myeloid Leukemia Patients With Mutated NPM1 Type A

BACKGROUND: Nucleophosmin gene (NPM1) mutation may be a good molecular marker for assessing the clinical status and predicting the outcomes in AML patients. We evaluated the applicability of NPM1 type A mutation (NPM1-mutA) quantitation for this purpose. METHODS: Twenty-seven AML patients with normal karyotype but bearing the mutated NPM1 were enrolled in the study, and real-time quantitative PCR of NPM1-mutA was performed on 93 bone marrow (BM) samples (27 samples at diagnosis and 56 at follow-up). The NPM1-mutA allele burdens (represented as the NPM1-mutA/Abelson gene (ABL) ratio) at diagnosis and at follow-up were compared. RESULTS: The median NPM1-mutA/ABL ratio was 1.3287 at diagnosis and 0.092 at 28 days after chemotherapy, corresponding to a median log10 reduction of 1.7061. Significant correlations were observed between BM blast counts and NPM1-mutA quantitation results measured at diagnosis (γ=0.5885, P=0.0012) and after chemotherapy (γ=0.5106, P=0.0065). Total 16 patients achieved morphologic complete remission at 28 days after chemotherapy, and 14 (87.5%) patients showed a >3 log10 reduction of the NPM1-mutA/ABL ratio. The NPM1-mutA allele was detected in each of five patients who had relapsed, giving a median increase of 0.91-fold of the NPM1-mutA/ABL ratio at relapse over that at diagnosis. CONCLUSIONS: The NPM1-mutA quantitation results corresponded to BM assessment results with high stability at relapse, and could predict patient outcomes. Quantitation of the NPM1-mutA burden at follow-up would be useful in the management of AML patients harboring this gene mutation.

Molecular Features of Three Children Diagnosed With Early T-Cell Precursor Acute Lymphoblastic Leukemia

No abstract available.

Minor BCR-ABL1-Positive Acute Myeloid Leukemia Associated With the NPM1 Mutation and FLT3 Internal Tandem Duplication

No abstract available.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

BACKGROUND: N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. METHODS: We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. RESULTS: The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. CONCLUSIONS: There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

BACKGROUND: N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. METHODS: We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. RESULTS: The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. CONCLUSIONS: There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

Impact of FLT-3 mutations on clinical features and response to the therapy in acute promyelocytic leukemia patients

FLT3 mutations are associated with poor outcome in acute myeloblastic leukemia [AML] patients. Only limited information is available about effects of FLT3 mutation on Acute Promyelocytic Leukemia [APL]. We investigated the prevalence and impact of FLT3 mutations on the clinical characteristics and the response to treatment in APL patients treated with arsenic trioxide [As[2]O[3]]. Blood samples were collected from 115 untreated APL patients and genomic DNA was extracted by the salting-out method. FLT3-ITD and FLT3-D835 mutations were investigated by PCR-RFLP. Mann-Whitney U test and Chi-square were used for data analysis. FLT3-ITD and FLT3-D835 mutations were detected in 16 [14%] and 13 [11%] of the patients, respectively. Both mutations were identified in two patients, so overall frequency of FLT3 mutations was estimated to be 23.5%. Patients positive for FLT3-ITD mutation had a higher rate of white cell counts [p= 0.005] and more frequent bcr3 type of PML/RARA fusion [p=0.04]. We have not found any significant association between FLT3-D835 mutation and the clinical characteristics of patients. Between the group with FLT3 Mutations and the group without, there was no significant difference in response to therapy. Complete remission induction with As[2]O[3] may be independent of FLT3 mutation status, so As[2]O[3] may be the first choice of APL especially in patients with FLT3 mutations. However, further studies on a large group of patients are necessary to confirm our findings

Detection of nucleophosmin and FMS-like tyrosine kinase-3 gene mutations in acute myeloid leukemia

Nucleophosmin gene mutations are frequently reported in acute myeloid leukemia [AML] patients with normal karyotype, which is also frequently associated with internal tandem duplication mutations in the FMS-like tyrosine kinase-3 gene. We sought to detect the nucleophosmin and FMS-like tyrosine kinase-3 [FLT3] internal tandem duplication [ITD] mutations among Iranian patients with AML and to assess the relationship between these mutations and the subtypes of the disease. Cross-sectional study of patients referred during 2007 through 2009. Bone marrow and peripheral blood samples of 131 AML patients were randomly collected at the time of diagnosis and prior to treatment and the DNA extracted. After amplifying the nucleophosmin and FLT3 gene regions, positive cases were screened by conformation-sensitive gel electrophoresis and agarose gel electrophoresis techniques. Of 131 patients, 23 [17.5%] [0.95% Cl=0.107-0.244] had nucleophosmin gene mutations. The highest frequency of such mutations was found among the subtypes of M4 [30.4%], M3 [21.7%] and M5 [17.4%]. There was a high frequency of these mutations in the M3 subtype as well as a high frequency of allele D in all subtypes. Also, 21 [16.0%] samples [0.95% CI=0.092-0.229] had FLT3/ITD mutation, of which 8 samples had mutant nucleophosmin [8 of 23, 35%], and another 13 samples had wild-type nucleophosmin gene [13 of 108, 12%]. There was a high degree of association between the occurrence of nucleophosmin and FLT3/ITD mutations [P=.012]. Our data showed a high frequency of NPM1 mutations in the monocytic subtypes of AML, as well as a high degree of association between the occurrence of NPM1 and FLT3/ITD mutations

Haematological & molecular profile of acute myelogenous leukaemia in India.

Background & objectives: Recurrent balanced translocations are generally recognized to be a major parameter for prognostication in acute myeloid leukaemia (AML). The chromosomal translocation t(15;17) results in PML/RARα fusion gene, t(8;21) results in AML1/ETO fusion gene and Inv 16 generates CBFβ/MYH11 fusion gene. Patients with these mutations have a good prognosis unlike abnormalities in chromosome 5 or 7 or FLT3 genes. Therefore, we screened the AmL patients for known specific genetic abnormalities that could lead to more definitive prognoses. Methods: A total of 113 AML patients were evaluated at diagnosis based on routine morphology and cytochemistry and classified according to the WHO criteria. The distribution of AML subtypes was M1(1), M2(32), M3(57), M4(14), M5(1), M6(1) and seven cases where morphological subtype could not be classified. RT-PCR was performed to identify PML/RARα, AML1/ETO, CBFβ/MYH11 and FLT3 internal tandem duplication (ITD). Results: Of the 57 patients with M3 subtype, 55 had the PML-RARα fusion transcript. The prevalence of bcr3 (short isoform) was higher (62%) than that of bcr1 (long isoform) (38%) and no correlation was found with age, sex or white blood cell count. FLT3 internal tandem duplication (ITD) mutations were more frequent in patients with APL than in other AML subtypes (17.5 vs. 8.9%), the frequency greater in patients with bcr3 isoform (70%) than in those with in bcr1 isoform (30%). Patients with FLT3/ITD mutations had a significantly higher median white cell count than those without these mutations (55 x 109/l vs. 6.3 x 109/l; P<0.001). More patients with FLT3/ITD mutations died early (53%) than those without these mutations (16%) (P<0.01). AML1-ETO fusion transcript was detected in 16 of 56 patients with no correlation with clinical or haematological parameters. Interpretation & conclusion: The results of the present study showed presence of bcr3 (short isoform) higher than bcr1 (long isoform). FLT3 internal tandem duplication (ITD) mutation was predominant in acute promyelocytic leukaemia patients with bcr3 isoform. Thus, patients with APL who have FLT3 mutation appear to have a poorer prognosis. Therefore, rapid identification of specific translocations at diagnosis is important for prognostic purposes and their detection should be incorporated into routine assessment.

Low Frequency and Variability of FLT3 Mutations in Korean Patients with Acute Myeloid Leukemia

FLT3 mutations are common genetic changes, and are reported to have prognostic significance in acute myeloid leukemia (AML). The FLT3 internal tandem duplication (ITD) and the D835 activating mutation in the tyrosine kinase domain (TKD) were analyzed by polymerase chain reaction (PCR) in the genomic DNA of Korean patients with AML at diagnosis and during follow-up. There were 226 patients with AML enrolled between March 1996 and August 2005. The incidence of ITD and TKD at diagnosis was 13% (29/226) and 3% (6/226). When compared to Western and other Asian patients with AML, Korean patients had a lower frequency by about two-thirds of ITD and TKD. Among the non-M3 cases (N=203), the patients with an ITD had a significantly shorter event-free survival when compared with those without an ITD (p=0.0079). Among 54 relapsed patients, 9 patients had the FLT3 ITD at diagnosis. Six patients demonstrated a reappearance of the ITD and 3 patients remained negative at relapse. One patient, among 45 patients who relapsed, had a negative baseline ITD but acquired a de novo ITD at relapse. There were 101 samples from 93 patients in remission; they were all negative for an ITD. Among 34 patients who failed to achieve a remission, five patients had a persistent ITD and one patient had a de novo ITD. These results support the concept of resistance of FLT3 ITD leukemic clones to chemotherapy. Therefore, effective therapy with FLT3 targeting agents may improve the prognosis of non-M3 AML patients with the FLT3 mutation.

Prevalence of FLT3 Internal Tandem Duplication in Adult Acute Myelogenous Leukemia / 대한진단검사의학회지

BACKGROUND: fms-like tyrosine kinase (FLT3), a member of the class III receptor tyrosine kinases, regulates the proliferation and differentiation of hematopoietic stem cells. An internal tandem duplication of the FLT3 gene (FLT3/ITD) has been reported in acute myelogenous leukemia (AML) and may be associated with a poor prognosis. In this study we determined the prevalence and prognostic significance of FLT3/ITD in adult AML patients. METHODS: This study included 52 adult de novo AML. Exon 14 and 15 of the FLT3 gene were amplified by PCR and the PCR products were analyzed by 3730XL DNA analyzer (Applied Biosystems, USA) and GeneMapper Software. RESULTS: FLT3/ITD was found in 15 (28.8%) of the 52 AML patients. The presence of FLT3/ITD was significantly associated with absolute leukocyte counts (P=0.002) and bone marrow blast counts (P=0.036). FLT3/ITD was also more frequent in patients with normal karyotype (7 of 18) than in those with cytogenetic aberrations (3 of 25). Patients with t (15;17) showed a higher prevalence of FLT3/ITD (2 of 7). FLT3/ITD was significantly associated with overall survival (P<0.042). CONCLUSIONS: Our data indicate that FLT3/ITD is a common alteration in adult AML patients. Although based on a study with a limited number of AML patients, FLT3/ITD is a prognostic marker in patients with AML.