scholarly journals Mutation topography and risk stratification for de novo acute myeloid leukaemia with normal cytogenetics and no nucleophosmin 1 ( NPM1 ) mutation or Fms‐like tyrosine kinase 3 internal tandem duplication ( FLT3‐ ITD)

2020 ◽  
Vol 190 (2) ◽  
pp. 274-283 ◽  
Author(s):  
Ya‐Lan Zhou ◽  
Li‐Xin Wu ◽  
Robert Peter Gale ◽  
Zi‐Long Wang ◽  
Jin‐Lan Li ◽  
...  
Keyword(s):  
Risk Stratification ◽  
Acute Myeloid Leukaemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukaemia ◽  
Tandem Duplication ◽  
De Novo ◽  
Internal Tandem Duplication ◽  
Npm1 Mutation ◽  
Normal Cytogenetics ◽  
Acute Myeloid

Zinc Finger Nuclease (ZFN) Targeted Homologous Recombination Assay Detects Elevated Inter Homologous Recombination Activity and Copy Neutral Loss of Heterozygosity (CN-LOH) in Internal Tandem Duplication Mutated FLT3 (FLT3-ITD) Acute Myeloid Leukaemia (AML)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1219-1219
Author(s):  
Terry J Gaymes ◽  
Ghulam J Mufti
Keyword(s):  
Homologous Recombination ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Zinc Finger ◽  
Tandem Duplication ◽  
Neutral Loss ◽  
Internal Tandem Duplication ◽  
Transfected Cells ◽  
Aggressive Disease ◽  
Acute Myeloid

Abstract Mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor whether it is internal tandem duplication (ITD) of its juxtamembrane domain or point mutations in its kinase domain are one of the most common mutations in normal karyotype acute myeloid leukaemia (NK-AML). The presence of FLT3-ITD mutation in NK-AML results in a more aggressive disease, resistance to therapy and poor survival. Acquired copy neutral loss of heterozygosity (CN-LOH) also referred to as uniparental disomy (UPD) is a common phenomenon of myeloid malignancies where an oncogenic allele is duplicated on the other chromosome. The use of single nucleotide phenotype analysis (SNP-A) karyotyping detects CN-LOH at the FLT3 locus, 13q associated with a FLT3-ITD mutation in NK-AML that results in an even more aggressive disease compared to NK-AML + FLT3-ITD without CN-LOH. It has been proposed that CN-LOH is the result of a homologous recombination (HR) DNA repair event. However, the underlying mechanisms that confer CN-LOH have yet to be determined. To elucidate the mechanisms that produce CN-LOH in NK-AML we developed a Zinc Finger Nuclease (ZFN) inter homologous recombination (iHR) targeting assay. The assay, based on the DR-GFP reporter developed by Pierce et al (1999), (Genes Dev. 13(20):2633-8) relies on the sequential targeting of two overlapping, but disrupted GFP repeat sequences to the adeno associated virus integration site 1 (AAVS1) on separate 19q alleles. Productive recombination between heterologous chromosomes in the region of 19q would result in restoration of GFP expression. The AML cell lines NB4 and THP-1 were transfected with a ZFN specific for AAVS1 integration (CompZr, Sigma) and the PZDonor AAVS1 puromycin derived construct DR1-Puro (Figure 1A,C) that possesses a 5' GFP sequence interrupted by an 18bp recognition sequence for the rare restriction enzyme, I-SCE I. Puromycin selected cells were then transfected with pZDonor derived DR2-Blast (Figure 1B,D) possessing the 3' GFP sequence and a blastocidin resistance gene. Puromycin and blastocidin selected cells were verified for correct integration of the plasmids to separate AAVS1 loci using location specific PCR and FISH, Figure 1F-H. Clones with correct integration were then expanded and transfected with FLT3-ITD, WT FLT3 expressing or empty vectors. Transfected cells were treated with an I-SCE I expression viral supernatant to induce a double strand break in DR1-Puro. Cells were cultured for a further 5 days before FACS analysis. FLT3-ITD transfected cells demonstrated GFP positive cells (inter HR events, 0.15% compared to WT-FLT3 and empty vector transfected cells (0.15% vs 0%, p<0.05, n=3) (Figure 1I). Furthermore, we were able to abolish inter HR events with co-culture with the FLT3 inhibitor, ACC220. To verify that GFP positive cells in this assay were the result of HR and not false positives, hot start PCR using primers designed at the DR1 locus was carried out on FACS sorted GFP positive cells. PCR products from GFP positive cells could only be digested by Bcg 1 and not I-SCE I in primary AML confirming the presence of iHR dependent GFP positive cells. Evaluation of HR events in primary AML was also investigated using ZFN targeted integration. Using the PZDonor AAVS1 puromycin derived construct HR substrate, PZD-DR1/DR2-Puro (Figure 1C), primary FLT3-ITD demonstrated significantly increased single locus targeted HR activity compared to primary WT FLT3, (0.62 vs 0.17%, p<0.01, n=22). Spontaneous HR activity (without I-SCE I cleavage) was observed in more than half the primary FLT3-ITD and was not seen in primary WT FLT3. Moreover, HR activity was significantly inhibited in primary AML with ACC220 (0.65% vs 0.1%, p<0.01, n=3) correlating with the reduction in RAD51 expression. The findings that FLT3-ITD mutation directly confers iHR activity provide a strong proof of principle that constitutive FLT3-ITD kinase activity augments HR activity, shifting the cellular milieu in favour of illegitimate recombination events such as CN-LOH that are selected for leukaemic progression. As both JAK2 and p53 mutations demonstrate enhanced HR repair and acquire CN-LOH at 9p and 17p respectively, we have identified a common mechanism of mutagenesis that furthers significantly our understanding of leukaemic progression and relapse in NK-AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Prevalence of FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD+) in de novo acute myeloid leukemia patients categorized according to cytogenetic risk

2009 ◽  
Vol 127 (1) ◽  
pp. 23-27 ◽  
Author(s):  
Everson Augusto Krum ◽  
Mihoko Yamamoto ◽  
Maria de Lourdes Lopes Ferrari Chauffaille
Keyword(s):  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
De Novo ◽  
Sao Paulo ◽  
São Paulo ◽  
Cross Sectional ◽  
Internal Tandem Duplication ◽  
Acute Myeloid

CONTEXT AND OBJECTIVE: The mechanism involved in leukemogenesis remains unclear and more information about the disruption of the cell proliferation, cell differentiation and apoptosis of neoplastic cells is required. DESIGN AND SETTING: Cross-sectional prevalence study at the Discipline of Hematology, Hospital São Paulo, Universidade Federal de São Paulo. METHODS: We investigated FMS-like tyrosine kinase 3/internal tandem duplication (FLT3/ITD+) in 40 adult patients with de novo acute myeloid leukemia (AML), categorized according to cytogenetic results, from September 2001 to May 2005. RESULTS: Thirteen patients (32.5%) were classified as presenting the favorable karyotype, 11 patients (27.5%) as an intermediate group, 7 patients (17%) as an undefined group and 9 patients (22.5%) as the unfavorable group. FLT3/ITD+ was found in 10 patients (25%): 3 with FLT3/ITD+ and favorable karyotype; 4 with FLT3/ITD+ and intermediate karyotype; 2 with FLT3/ITD+ and undefined karyotype; and only 1 with FLT3/ITD+ and unfavorable karyotype. Among the patients without FLT3/ITD+, 10 presented favorable karyotype, 8 intermediate, 4 undefined and 8 unfavorable karyotype. The cytogenetic results showed no correlations between FLT3/ITD presence and the prognostic groups (P = 0.13). We found that 2 patients were still alive more than 24 months later, FLT3/ITD+ did not influence the patients' survival rate. CONCLUSION: We found the same frequency of AML with FLT3/ITD+ in both the favorable and intermediate prognosis groups. Only one patient presented AML, FLT3/ITD+ and unfavorable karyotype (the hypothetical worst clinical situation). Therefore, the prognostic advantage of favorable cytogenetics among patients with FLT3/ITD+ remains to be elucidated, for it to be better understood.

FLT3, NPM1 and MLL Mutations Help Risk Stratification in Pediatric Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1574-1574
Author(s):  
Shuhong Shen ◽  
Yin Liu ◽  
JingYan Tang ◽  
Long-Jun Gu
Keyword(s):  
Acute Myeloid Leukemia ◽  
Risk Stratification ◽  
Myeloid Leukemia ◽  
Tandem Duplication ◽  
De Novo ◽  
Genetic Alterations ◽  
Npm1 Mutation ◽  
De Novo Aml ◽  
Pediatric Aml ◽  
Acute Myeloid

Abstract Abstract 1574 Poster Board I-600 Introduction Acute myeloid leukemia (AML) is a heterogeneous disease which harbors various genetic alterations. Among theses genetic events, Mutations of FLT3, NPM1, MLL and other genes often predict prognosis, particularly in cases cytogenetic normal (CN-AML). Could these be criteria for risk stratification in Pediatric AML ? Patients and Methods 155 cases of de novo AML were diagnosed routinely according to morphology, immunology, cytogenetics, and molecular biology examination on bone marrow (BM) aspirates between Jan. 2002 and Dec. 2008. All patients received chemotherapy according to the AML-XH-99 protocol, which consist of Daunorubicin, Cytosine arabinoside, Etoposide, Homoharringtonine. For acute promyelocytic leukemia, all-trans retinoic acid and Arsenic trioxide were also included. Meanwhile, total RNA of leukemic cells form all diagnostic BM samples were extracted, and then reverse transcribed. MLL partial tandem duplication (MLL/PTD) fusion transcripts were screened by real-time quantitative polymerase chain reaction. FLT3 internal tandem duplication (FLT3/ITD), FLT3 tyrosine kinase domain mutation (FLT3/TKD) and NPM1 mutation were examined by High resolution melting analysis. Results Of the 155 children with de novo AML, 121(78.1%) had received chemotherapy for more than one week with data available for analysis. Among them, 55(45.5%) was cytogenetically normal (CN-AML). In this total cohort of patients 49(27.09%) had FLT3/ITD (32.70% in CN-AML), 14 (9.03%) had FLT3/TKD (7.30% in CN-AML), 62 (40%) had NPM1 mutation (49% in CN-AML), and additional 8 (5.16%) had MLL/PTD (5.50% in CN-AML). In this cohort of patients 98 (63.22%) had at least one mutation. The clinical outcomes were listed in table 1. Generally, patients with FLT3 mutation (ITD or TKD mutation) usually have worse results after chemotherapy, as reported previously by other researchers. Meanwhile, NPM1 mutations usually predict better prognosis in our cohort of AML patients. MLL/PTD always predicts the worst outcome in AML as other MLL rearrangements in leukemia. Among CN-AML patients, 5-year EFS and OS were similar to whole cohort of patients according to those mutations. Cox regression analysis in a univariate model revealed that the presence of FLT3/ITD and NPM1 was significant prognostic factor of EFS, (P<0.05). We therefore proposed a molecular-risk classification of pediatric AML patients based on the data we got in this study. For the newly classified groups of low, medium and high risk groups, EFS rate was 62.03%±8.42%, 45.42%±4.52%, and 14.85%±2.99%, respectively, P=0.00. CRD for the 3 groups was 27.69±21.34 months, 22.62±19.64 months, 13.26±11.95 months, respectively, p=.022. Our results indicate that combinations of these couple of molecular events may be the useful tool for further classify AML in children. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genetic Profiles and Risk Stratification in Adult De Novo Acute Myeloid Leukaemia in Relation to Age, Gender, and Ethnicity: A Study from Malaysia

2021 ◽  
Vol 23 (1) ◽  
pp. 258
Author(s):  
Angeli Ambayya ◽  
Anthony V. Moorman ◽  
Jameela Sathar ◽  
Jeyanthy Eswaran ◽  
Sarina Sulong ◽  
...  
Keyword(s):  
Risk Stratification ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Ethnic Groups ◽  
De Novo ◽  
Age Groups ◽  
Hierarchical Classification ◽  
Southeast Asians ◽  
Genetic Profiles ◽  
Acute Myeloid

Hitherto, no data describing the heterogeneity of genetic profiles and risk stratifications of adult acute myeloid leukaemia (AML) in Southeast Asia are reported. This study assessed genetic profiles, Moorman’s hierarchical classification, and ELN 2017-based risk stratifications in relation to age, gender, and ethnicity in Malaysian adult AML patients. A total of 854 AML patients: male (52%), female (48%) were recruited comprising three main ethnic groups: Malays (59%), Chinese (32%) and Indians (8%). Of 307 patients with abnormal karyotypes: 36% exhibited translocations; 10% deletions and 5% trisomies. The commonest genotype was FLT3-ITD-NPM1wt (276/414; 66.7%). ELN 2017 risk stratification was performed on 494 patients, and 41% were classified as favourable, 39% as intermediate and 20% as adverse groups. More females (47%) were in the favourable risk group compared to males (37%), whereas adverse risk was higher in patients above 60 (24%) of age compared to below 60 (18%) patients. We observed heterogeneity in the distribution of genetic profiles and risk stratifications between the age groups and gender, but not among the ethnic groups. Our study elucidated the diversity of adult AML genetic profiles between Southeast Asians and other regions worldwide.

scholarly journals A Mutation Pentad Defined Outcome of De Novo and Cytogenetically Normal Acute Myeloid Leukaemia in Young Adults

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1400-1400
Author(s):  
Sze Pui Tsui ◽  
Ip HW Alvin ◽  
Chunxiao Zhang ◽  
Tommy W.F. Tang ◽  
CH Lin ◽  
...  
Keyword(s):  
Young Adults ◽  
Hong Kong ◽  
Clinical Outcome ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Research Funding ◽  
De Novo ◽  
Gene Mutations ◽  
Npm1 Mutation ◽  
Acute Myeloid

Background. Cytogenetically normal acute myeloid leukaemia (CN-AML) occurs in 50% adult AML and is a group of diseases with diverse mutations and distinct clinical outcome. CEBPα, NPM1 and FLT3 mutations that are commonly seen in CN-AML have been incorporated into risk stratification. However, prognostic impacts of other gene mutations and their combinations in this AML subtype have remained unclear. In this study, we examined a cohort of young adults with de novo CN-AML who have received uniform treatment protocol in Hong Kong and identified a mutation pentad that might define their clinical outcome. Methodology. Young adults (18-60 years old) with de novo CN-AML, diagnosed from 1st August 2003 to 7th August 2018 in 8 regional hospitals in Hong Kong, were included. They received standard "7+3" induction (Daunorubicin 60-90 mg/m2 and Cytarabine 100 mg/m2) followed by up to 4 courses of high dose cytarabine consolidation (Cytarabine 3 gram/m2 for 4-6 doses). Decision on allogeneic haematopoietic stem cell transplantation (HSCT) was based on clinical grounds and gene mutations according to ELN recommendations. Next generation sequencing (NGS) was performed in diagnostic bone marrow (BM) in 362 patients for 36 recurrent mutated genes and analyzed by in-house bioinformatics pipelines. Relapse-free survival (RFS) was defined by the time from first complete remission (CR) to relapse or death and overall survival (OS) by the time from diagnosis to death. Patients were censored at last follow up. Survivals were evaluated by Kaplan-Meier analysis and compared by log-rank test. Multivariate analyses of clinical and genetic parameters were analyzed by Cox-regression. P-values of &lt;0.05 were considered statistically significant. Results. A total of 436 patients (Male=189; Female=247) were recruited. Their median age of onset was 49 years old (Range 18-60); median presenting white cell counts (WCC) was 21.85x109/L (range 0.25-411x109/L), median circulating blast % was 46% (range 1-99). 416 patients received induction of whom 90.1% achieved CR or CRi (N=375) after 1 (N=268), 2 (N=78) or ≥ 3 courses of induction (N=29). One hundred and sixty three patients received allogeneic HSCT at CR1 (N=102), CR2 (N=51), ≥ CR3 (N=2) and relapsed state (N=8). Eight mutations with ≥ 10% prevalence occurred in 79.8% patients (Figure 1). Univariate analyses showed that mutations of CEBPα, TET2, IDH2-R172K and RAS were not associated with treatment outcome and survival. Five genetic subgroups based on NPM1, FLT3 and DNMT3A mutations could be identified: NPM1 mutation only; NPM1 mutation and FLT3-ITD; All wildtype; FLT3-ITD only; DNMT3A irrespective of NPM1 and FLT3-ITD status. These subgroups showed distinct RFS and OS (Figure 2). Impacts of IDH1-R132 and IDH2-R140Q mutations were evaluated in these 5 subgroups. Interestingly, adverse impacts of IDH1-R132 on RFS and OS were only significant in the all wildtype subgroup and the adverse impact of IDH2-R140Q was only significant for RFS in the NPM1 mutation only subgroup. Conclusion. A mutation pentad comprising NPM1, FLT3, DNMT3A, IDH1-R132 and IDH2-R140Q seemed to define distinct prognostic subgroups in young adults with de novo CN-AML. A limited gene panel based on this pentad using conventional PCR may provide a practical and cost-effective means to guide post-remission therapy in these patients, especially in places where NGS may not be readily available. Acknowledgements: SK Yee Medical Foundation; Li Shu Fan Medical Foundation, LKS Faculty of Medicine, University of Hong Kong, Hong Kong Blood Cancer Foundation Disclosures Leung: Curegenix: Research Funding; Servier: Research Funding; Merck: Research Funding; Pfizer: Research Funding.

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