Survival Outcomes in Patients with FLT3-Itd Positive Acute Myeloid Leukaemia Relative to Biological Stratification: Smaller FLT3-Itd Clone Size Predict Better Overall Survival

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1460-1460 ◽  
Author(s):  
Justin Ching Ting Loke ◽  
Susanna Akiki ◽  
Joanne Ewing ◽  
Syed W Bokhari ◽  
Deepak Chandra ◽  
...  
Keyword(s):  
Overall Survival ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Conflicts Of Interest ◽  
Wild Type Allele ◽  
Wild Type ◽  
Relative Expression ◽  
Type Allele ◽  
The Impact ◽  
Acute Myeloid

Abstract Abstract 1460 Background: FLT3 internal tandem duplication (itd) mutations are found in 25% of adult patients with acute myeloid leukaemia (AML) and are associated with an adverse prognosis. This mutation results in constitutive activation of downstream pathways. Distinct biological subgroups can be identified based on FLT3-itd mutation type: clones with heterozygous FLT3-itd mutated/wildtype; homozygous mutated FLT3 allele; FLT3 heterozygous biallelic mutant and clones with evidence of overexpression of FLT3-itd. There is evidence to suggest that these mechanisms are important in the clonal evolution of AML cells. We sought to investigate their clinical impact. Method: Itd mutation analysis within exon 14 and/or exon 15 of the FLT3 gene was carried out at diagnosis by PCR of genomic DNA (gDNA) and cDNA for all new AML referrals in the region over 8 years. PCR products were identified and sized using fluorescent based fragment analysis. Allelic ratio (AR) and expression ratio (ER) (mutation: wild type ratio in gDNA and cDNA respectively) was determined from the relative peak heights. High relative expression was defined as 10 fold difference of ER/AR. Copy neutral loss of heterozygosity for the wild type allele resulting in homozygosity of the FLT3-itd mutation (acquired isodisomy (AID)) was determined by analysis of microsatellite markers along chromosome 13. Overall survival (OS) (time of diagnosis to death), event free survival (EFS) (time from diagnosis to induction failure, relapse or death) was calculated. Survival rates were estimated by the Kaplan-Meier method. Differences between the survival distributions were compared with the log-rank test. Results: 177 patients positive for the FLT3-itd mutation were identified. Median follow-up for patients alive was 3.4 years. A separate group of 49 patients tested negative for this mutation during this period had better OS and EFS (p=0.02) compared to the patients who were FLT3-itd positive (median survival 1715 and 307 days respectively). Patients who were FLT3-itd positive had statistically significant (p<0.05) differences in outcomes based on age, presenting white cell count, treatment intensity and cytogenetic risk. The characteristics of this group of patients are described below. Patients with lower AR (less than/equal to 0.3) as compared to higher AR (greater than 0.3) had an improved OS (p=0.018) and EFS (p=0.02). The impact of AR on OS had borderline significance (p=0.05) when only patients treated with intensive chemotherapy were considered. AID provides true evidence of FLT3 mutant homozygosity and was detected in 15 (142 tested) patients. In 38 patients who relapsed and had samples at these stages, 5 had developed AID, but were heterozygous (mutant/wildtype) at diagnosis. 6 patients with multiple FLT3-itd products may comprise patients who have multiple different mutant/wildtype clones but may include patients with a second, independent FLT3-itd mutation resulting in biallelic heterozygous mutations, although this cannot be confirmed. A high relative expression level of FLT3-itd was seen in 12 patients. The clinical significance of these findings is uncertain due to the small numbers. Conclusion: The impact of FLT3-itd mutation and other known prognostic factors has been confirmed in a heterogeneous, real life cohort of patients. An AR over 1 provides firm evidence of loss of the wild type allele (9 patients). AID also occurs at an AR of less than 1 because of the presence of normal cells or due to preferential amplification of the wildtype allele. Direct testing for AID is a more sensitive measure of FLT3 mutant homozygosity (detected in 14% of tested patients). The development of AID in patients who relapse may be an important mechanism by which an AML clone gains a further advantage. Lower AR was associated with improved survival. In the context of a high blast count (median bone marrow blast 80%), this implies having a small sub-clone of FLT3-itd positive cells is advantageous to having a larger FLT3-itd clone in their population of AML cells. Disclosures: No relevant conflicts of interest to declare.

Loss of the Wild-Type Allele Contributes to Myeloid Expansion and Disease Aggressiveness in FLT3/ITD Knock-in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 866-866
Author(s):  
Li Li ◽  
Emily Bailey ◽  
Sarah M Greenblatt ◽  
David Huso ◽  
Donald Small
Keyword(s):  
Median Survival ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Point Mutations ◽  
Kinase Domain ◽  
Wild Type Allele ◽  
Wild Type ◽  
Type Allele ◽  
Lower Ratio ◽  
The Impact

Abstract Abstract 866 Activating mutations of FLT3, either in the form of internal tandem duplication (ITD) mutations in the juxtamembrane domain or point mutations in the kinase domain, are one of the most frequent mutations in acute myeloid leukemia (AML). AML patients with FLT3/ITD mutations have poor prognosis. Loss of the wild-type FLT3 allele is associated with even worse prognosis when compared to those FLT3/ITD AML patients with the wild-type FLT3 allele still present. Also, FLT3/ITD patients with a high mutant-to-wild-type ratio have a significantly worse outcome than FLT3/ITD patients with a lower ratio. We have previously reported that heterozygous FLT3wt/ITD “knock-in” mice develop a slowly fatal MPN. In order to study the roles wild-type FLT3 play in the development of leukemia associated with FLT3/ITD mutations, we crossed FLT3wt/ITD mice with themselves or with FLT3 “knockout” (FLT3−/−) mice to obtain hemizygous (FLT3−/ITD) or homozygous (FLT3ITD/ITD) FLT3/ITD mice. Investigating phenotypic differences among them reveals the impact of wild-type FLT3 on the development of MPN resulting from FLT3/ITD mutations, and by extension, the effect on acute leukemia. FLT3−/ITD mice, with the loss of the wild-type allele, displayed a more severe MPN, as evidenced by even larger spleen, higher white blood counts and shorter survival, compared to FLT3wt/ITD mice. FLT3ITD/ITD mice had an even severe MPN compared to the FLT3−/ITD and FLT3wt/ITD mice. Fully transformed leukemia developed in some of the FLT3ITD/ITD (7%, 9/129), but not FLT3wt/ITD or FLT3−/ITD mice, with latency ranging from 139 to 304 days. Compared to FLT3wt/ITD mice, FLT3−/ITD and FLT3ITD/ITD mice displayed a further increase in the fraction of primitive hematopoietic cells, with notable increases in ST-HSCs and MPPs. Phosphorylation of STAT5, one of the key downstream targets for constitutively activated FLT3, was increased in FLT3wt/ITD, FLT3−/ITD and FLT3ITD/ITD mice compared to the wild-type control. FLT3wt/ITD, FLT3−/ITD and FLT3ITD/ITD BM also showed increased PU.1 expression and decreased GATA-1 expression, resulting in the subsequent expansion of granulocytic/monocytic/lymphocytic progenitors and a decrease in megakaryocytic/erythrocytic progenitors. It appears that the extent of myeloproliferation in FLT3/ITD mice correlates with loss of the wild-type allele (FLT3wt/ITD vs. FLT3−/ITD) and with the dose of mutant allele (FLT3−/ITD vs. FLT3ITD/ITD). In order to further explore the potential moderating effect of wild-type FLT3 expression on FLT3/ITD-associated MPN, we transduced wild-type FLT3 (wtFLT3, with the lentiviral vector co-expressing GFP) into lineage-depleted FLT3−/ITD CD45.2 BM cells and injected them into lethally irradiated CD45.1 recipients. When injected with sorted (GFP+) BM, vector alone-transduced GFP+FLT3−/ITD BM recipients died of MPN, with a median survival of 62 days. 100% of the recipients in the other three groups, i.e., those injected with vector alone-transduced GFP+ wild-type BM, wtFLT3-transduced GFP+ wild-type BM or wtFLT3-transduced GFP+ FLT3−/ITD BM, remained viable even after the point in time at which all of the recipients in the vector alone-transduced GFP+FLT3−/ITD group died. Similarly, recipients transplanted with unselected (including GFP+ and GFP− populations) vector alone-transduced FLT3−/ITD BM also died early, with a median survival of 73 days and overt signs of MPN. The percentages of GFP+ and GFP− cells in the BM of the dying recipients were comparable to those shortly after transplantation, indicative of the similar expansion ability of the GFP+ and GFP− populations in the BM. In contrast to the wtFLT3-transduced GFP+FLT3−/ITD BM recipients, which have a very prolonged survival, recipients injected with unselected wtFLT3-transduced FLT3−/ITD BM died of MPN, with a median survival of 91 days. Interestingly, 99% of the BM cells in the BM of the dying recipients were GFP−, demonstrating a proliferative/survival advantage for the FLT3−/ITD cells that had not been successfully transduced with wild-type FLT3. These results suggest that the presence of wild-type FLT3 delays and moderates the development of MPN caused by FLT3/ITD mutations. These results suggest that loss of the wild-type allele contributes to the development of a more severe phenotype. Thus, the wild-type FLT3 allele seemingly functions as a “tumor suppressor” in leukemia harboring FLT3/ITD mutations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The impact of TNF superfamily molecules on overall survival in acute myeloid leukaemia: correlation with biological and clinical features

2014 ◽  
Vol 94 (1) ◽  
pp. 35-43 ◽  
Author(s):  
L. Bolkun ◽  
D. Lemancewicz ◽  
E. Jablonska ◽  
A. Szumowska ◽  
U. Bolkun-Skornicka ◽  
...  
Keyword(s):  
Overall Survival ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Clinical Features ◽  
Tnf Superfamily ◽  
The Impact ◽  
Acute Myeloid

Identification and Analysis of Oncogenic Pathways in Deletion 20q Acute Myeloid Leukaemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1324-1324
Author(s):  
Matthew Ku ◽  
Nisha Narayan ◽  
Meaghan Wall ◽  
Ruth N. MacKinnon ◽  
Lynda J Campbell ◽  
...  
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Tyrosine Phosphatase ◽  
Wild Type ◽  
Src Tyrosine Kinase ◽  
Chromosome 20 ◽  
Stat3 Phosphorylation ◽  
Acute Myeloid

Abstract Abstract 1324 Deletion of the long arm of chromosome 20 [del(20q)] is a common recurrent chromosomal abnormality in acute myeloid leukaemia (AML). It is a key step in AML development and a better understanding of the associated molecular events is important. The abnormal chromosome 20 in del(20q) AML has been shown to have lost a “Common Deleted Region” (CDR) that contains Protein Tyrosine Phosphatase Receptor T (PTPRT), a tyrosine phosphatase that is mutated in many human cancers such as AML. We have previously reported (MacKinnon et al, Genes, Chromosomes and Cancer 2010) that del(20q) also harbours an amplified “Common Retained Region,” (CRR) which contains Haemopoietic Cell Kinase (HCK). HCK is anoncogenic Src tyrosine kinase and its aberrant activation has been shown to contribute to the pathogenesis of some haematological malignancies. We hypothesize that the amplification of HCK in the CRR cooperates with the loss of PTPRT in the CDR to cause AML. Our model proposes that AML occurs either through direct interaction between HCK and PTPRT, or through aberrant activation of Signal Transducer and Activator of Transcription 3 (STAT3), a cytoplasmic second messenger that is important in cellular signalling. Constitutively activated STAT3 has been shown to be oncogenic in several malignancies, including AML. STAT3 is a direct target of both HCK and PTPRT. It is phosphorylated (hence activated) by HCK, and dephosphorylated (hence inactivated) by PTPRT. This provides a downstream leukaemogenic pathway for our model. The ultimate aim of our experiments is to prove this hypothesis using mouse models. Murine haemopoietic stem cells (HSC) were isolated from the bone marrows of wild type C57BL/6 (WT) and PTPRT-null mice by Fluorescence Activated Cell Sorting for Lineage negative, C-kit and Sca-1 positive (LKS+) cells. Retroviral constructs of HCK were generated by cloning it into the retroviral vector pMSCViresEGFP(MIG), with GFP as reporter. Murine HSC were transduced with either retroviral HCK or MIG vector control and Phoenix cell system was used for retroviral packaging. Experiments using isolated LKS+ HSC were performed to examine for features of AML. Examination of bone marrow cells from del(20q) AML patients by quantitative PCR revealed an increase in HCK mRNA expression and a reduction in PTPRT expression. Wild type (WT) and PTPRT-null murine HSC transduced with either MIG or HCK were cultured in methylcellulose media. Colony forming units (CFU) were enumerated on day7 and day12. We found that both WT and PTPRT-null HSC transduced with HCK showed a significant increase in colony numbers compared to MIG transduced HSC. Furthermore, the fold increment in colony number was higher in the PTPRT-null genotype as shown in figure 1. Moreover, an intracellular anti-phosphoSTAT3 assay was performed to assess STAT3 phosphorylation levels in the transduced HSC. It demonstrated that in both WT and PTPRT-null HSC that have been transduced with HCK, STAT3 hyperphosphorylation, and hence overactivation, occured. This response was again more exaggerated in the PTPRT-null HSC, as seen in figure 2. We are currently transplanting transduced LKS+ HSC (either MIG or HCK) into lethally irradiated murine recipients to assess AML formation in a reconstitution study. The recipient mice will be assessed for evidence of engraftment and subsequent AML. The preliminary data reveals a likely new oncogenic-signalling cascade: that HCK amplification and loss of PTPRT in del(20q) AML may cooperate to cause AML directly, or by aberrant activity of hyperphosphorylated STAT3. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Preferential transcription of the mutated allele in NPM1 mutated acute myeloid leukaemia

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
G. D. Bailey ◽  
L. Doolan ◽  
A. Baskar ◽  
L. C. Smith ◽  
C. H. Seedhouse
Keyword(s):  
Acute Myeloid Leukemia ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Myeloid Leukemia ◽  
Splice Variants ◽  
Wild Type ◽  
Significant Bias ◽  
Acute Myeloid

Abstract Nucleophosmin is commonly both over-expressed and mutated in acute myeloid leukemia (AML). NPM1 mutations are always heterozygous. In addition, NPM1 has a number of different splice variants with the major variant encoded by exons 1–9 and 11–12 (NPM1.1). Further variants include NPM1.2 which lacks exons 8 and 10 and NPM1.3 which comprises exons 1–10 (and so lacks the region of sequence mutated in AML). In this study we quantified the expression of these three variants in 108 AML patient samples with and without NPM1 mutations and also assessed the level of expression from the wild-type and mutant alleles in variants NPM1.1 and NPM1.2. The results show that NPM1.1 is the most commonly expressed variant, however transcripts from wild-type and mutated alleles do not occur at equal levels, with a significant bias toward the mutated allele. Considering the involvement of mutant nucleophosmin in the progression and maintenance of AML, a bias towards mutated transcripts could have a significant impact on disease maintenance.

Knockdown of MicroRNA-10a in Acute Myeloid Leukaemia Cells Bearing the Nucleophosmin1 Mutation Causes Cell Death and Reduced Clonogenicity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1367-1367
Author(s):  
Adam J Bryant ◽  
Catalina A Palma ◽  
Mark Lutherborrow ◽  
Vivek Jayaswal ◽  
Yee Hwa Yang ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Cell Line ◽  
Caspase 3 ◽  
Luciferase Reporter ◽  
Unknown Etiology ◽  
Wild Type ◽  
Over Expression ◽  
Acute Myeloid

Abstract Abstract 1367 Acute Myeloid Leukaemia (AML) with a mutation in the Nucleophosmin1 gene (NPM1c+) accounts for one of the largest subtypes of AML, with an unknown etiology. MicroRNA dysregulation has now been implicated in the oncogenesis of many cancers including AML. We sought to investigate the role of microRNAs in the initiation and development of AML with the NPM1c+ mutation. MicroRNA profiling of bone marrow samples from 28 AML patients and confirmation by qRT-PCR demonstrated a unique microRNA signature in AML-NPM1c+ samples dominated by miR-10a over-expression of 19.6-fold compared to Nucleophosmin1 wild type (NPM1) samples. Functional assessments were performed in the human OCI-AML3 cell line, which is the only cell line to harbour NPM1c+. miR-10a repression was induced by transfection with miRCURY LNA microRNA knockdown probes (Exiqon). Cell growth (MTS) assay demonstrated a significant decrease of 19% in miR-10a knockdown cells compared to the Scrambled control. AnnexinV and Caspase 3 assays assessed the effect of miR-10a knockdown on apoptosis. miR-10a knockdown increased the proportion of AnnexinV positive events when compared to control treated cells by 34.9% and 39.3% at 24 and 48 hours respectively, but had no effect on Caspase 3 expression. Proliferation (BrdU uptake) assays did not show a change, however, clonogenic assays demonstrated a 26.1% decrease in colony number in miR-10a knockdown cells compared to the control. Potential mechanisms were elucidated by determining miR-10a mRNA targets in silico and confirmed by luciferase reporter assays. These included ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C. In this study, we have demonstrated that miR-10a was highly differentially expressed between AML-NPM1c+ cells compared to leukaemic cells bearing wild type NPM1. Knockdown of miR-10a in OCI-AML3 cells resulted in increased cell death as detected by AnnexinV binding (but not Caspase 3, indicating an effect independent of the classical apoptotic pathways) and reduced clonogenic capacity. These effects are thought to occur through miR-10a mediated modulation of ARNT, GTFH1, ID4, KLF4, MAPRE1, NR4A3, RB1CC1 and TFAP2C, all of which are associated with neoplastic transformation. Taken together, our results suggest that aberrant miR-10a over-expression in AML-NPM1c+ patients promotes cell survival. Disclosures: No relevant conflicts of interest to declare.

Measuring the impact of a restrictive transfusion guideline in patients with acute myeloid leukaemia

Vox Sanguinis ◽  
2013 ◽  
Vol 105 (1) ◽  
pp. 81-84 ◽  
Author(s):  
R. T. Hoeg ◽  
E. B. Leinoe ◽  
P. Andersen ◽  
T. W. Klausen ◽  
H. S. Birgens
Keyword(s):  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Restrictive Transfusion ◽  
The Impact ◽  
Acute Myeloid

scholarly journals Methylation ofKLF5contributes to reduced expression in acute myeloid leukaemia and is associated with poor overall survival

2013 ◽  
Vol 161 (6) ◽  
pp. 884-888 ◽  
Author(s):  
Sonya M. Diakiw ◽  
Michelle Perugini ◽  
Chung H. Kok ◽  
Grant A. Engler ◽  
Nik Cummings ◽  
...  
Keyword(s):  
Overall Survival ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Poor Overall Survival ◽  
Acute Myeloid

scholarly journals Vitamin D Status in Children with Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3973-3973 ◽  
Author(s):  
Natalja Jackmann ◽  
Arja H Harila-Saari ◽  
Outi Mäkitie ◽  
Jan Gustafsson ◽  
Dzeneta Nezirevich Dernroth ◽  
...  
Keyword(s):  
Vitamin D ◽  
Overall Survival ◽  
Myeloid Leukaemia ◽  
Vitamin D Deficiency ◽  
Conflicts Of Interest ◽  
Significant Negative Correlation ◽  
Vitamin D Status ◽  
Cross Sectional ◽  
Pediatric Cancer Patients ◽  
The Impact

Abstract Children and adolescents with leukemia are potentially at a high risk of developing vitamin D deficiency, due to limited physical activity and sunlight exposure, poor nutrition, chemotherapy, and its complications. The prevalence of vitamin D inadequacy in European pediatric cancer patients has been reported to be high. It is not known how many patients already have vitamin D deficiency at the time of diagnosis and whether vitamin D status at the time of diagnosis influences clinical outcome. We aimed to investigate vitamin D status in children with leukemia at the time of diagnosis and explore possible factors (age, type of leukemia, gender, year and season of sampling) contributing to a low level of 25-hydroxyvitamin D (25-OHD). Furthermore, we aimed to investigate if vitamin D status at the time of diagnosis influences overall survival. We carried out a cross-sectional study including all 295 children (169 boys, 57.3%) aged <18 years who were diagnosed with leukemia in our institution between 1991 and 2016 and had a stored serum sample available from the time of diagnosis. All samples had been stored at -80C. We analysed serum 25-OHD and PTH with reagents from the same batch in January 2018; 25-OHD levels <25 nmol/L were considered deficient, 25-50 nmol/L insufficient, 50-75 nmol/L sufficient, and ≥75nmol/L optimal. Clinical data (sex, age, diagnosis, date of the diagnosis, overall survival) were collected from the Swedish Childhood Cancer Registry. Altogether 295 children were included: 232 of them had acute lymphoblastic leukaemia (ALL), 52 acute myeloid leukaemia (AML), and 11 other types of leukemia (8 chronic myeloid leukaemia and 3 juvenile myelomonocytic leukaemia). Mean 25-OHD concentration was 60.7 nmol/L (SD 23.3). One third of the children (33.2%) had a subnormal 25-OHD level (6.4% had deficiency and 26.8% insufficiency), 39.7% were sufficient and 27.1% had an optimal level. There was a significant negative correlation between serum 25-OHD and PTH (p<0.001). Season affected serum 25-OHD: it was lowest in the spring (55.2 nmol/L, SD 21.7) and highest in the summer (68.4 nmol/L, SD 19.6). Multiple linear regression with unadjusted and adjusted analyses to explore the impact of age, diagnosis, gender, season, and time of sampling (calendar year) on 25-OHD level indicated that significant predictors of lower 25-OHD level were older age (p<0.001), sampling in the spring (p<0.001), sampling in more recent calendar year (p=0.001) and sampling in the winter (p=0.001). When exploring the impact of 25-OHD on survival, we used Cox proportional hazard regression. In the whole cohort only the diagnosis and the age at diagnosis were significant. However, when the younger patients (≤ 6 year of age) were analysed separately, 25-OHD level <50 nmol/L at the time of diagnosis was associated with inferior overall survival independently of other factors (HR 3.05, p=0.03) as compared with those with 25-OHD ≥50 nmol/L. This patient group included 163 patients with 16 events. Conclusion: Subnormal 25-OHD levels are common in pediatric patients with leukemia already at the time of diagnosis. In younger children with leukemia 25-OHD level <50 nmol/L is associated with inferior survival. Disclosures No relevant conflicts of interest to declare.

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