Internal tandem duplication mutations in the tyrosine kinase domain of FLT3 display a higher oncogenic potential than the activation loop D835Y mutation

Abstract Background: Activating FLT3 internal tandem duplication mutations (FLT3-ITDs) occur in approximately 30% of acute myeloid leukemia (AML) patients. Expression of the FLT3-ITD receptor results in autophosphorylation of FLT3 and subsequent activation of downstream signaling. Clinically, FLT3-ITDs are associated with a dismal clinical outcome; previous explorative analyses suggest that not only FLT3-ITD per se but also the mutant/wild-type allelic ratio and/or the length of the FLT3-ITD provide prognostic information. Aims: To determine ITD insertion sites and length in FLT3-ITD mutated AML and to correlate these findings with clinical outcome. Methods: In 241 patients, DNA-based amplification of the FLT3-ITD mutation was followed by DHPLC-based separation of FLT3 mutant and wild-type fragments. Single mutated fragments were collected by a fragment collector, reamplified and sequenced. Patients [16 to 60 years of age] were entered on 3 consecutive AMLSG treatment trials [AML HD93, AML HD98A, AMLSG 07-04] all including intensive therapy. Results: Thirty-four (14.1) of the 241 patients had more than one ITD (two ITDs n=29, three ITDs n=3, four ITDs n=2). In total, 282 ITDs were analyzed. The median length was 52 nucleotides (range 15–180). For further correlations we grouped ITD integration sites according to the functional regions of FLT3: JM-domain (JMD) [amino acid (AA) 572–603, patients n =141, ITDs n=148], hinge region of JMD [AA 604–609, patients n=45, ITDs n=48], beta-1-sheet of the tyrosine kinase domain-1 (TKD1) [AA 610–615, patients n=69, ITDs n=73], and the remaining region of TKD1 [AA >615, patients n=13, ITDs n=13]. Interestingly, ITD length was strongly correlated to functional regions with shortest ITDs being present in the JMD and longest in the TKD1 (p<0.001). Clinical data were available in 239 patients showing no differences in patient characteristics (age, WBC etc.); frequencies of cooperating class II mutations (NPM1-mut n=137, CEBPA-mut n=12, MLL-PTD n=21) were equally distributed among the functionally categorized groups. The logistic regression model on induction success (IS) revealed ITD integration sites in the beta-1-sheet (odds ratio (OR) 0.25, p=0.01) and in the remaining region of TKD1 (OR 0.14, p=0.007) as well as logarithm of WBC count (OR 0.36, p=0.002) and NPM1 mutations (OR 2.04, p=0.04) as significant variables. ITD insertion in the beta-1-sheet was also significant in the Cox regression analysis on overall survival (OS) (Hazard Ratio (HR) 2.7, p=0.01). In univariable analyses, event free survival (EFS) and OS were significantly inferior in patients with ITD in the beta-1-sheet (p=0.005 and p=0.001). Of note, the proportions of patients receiving an allogeneic transplantation were comparable in both groups (61% and 67%, respectively). In multivariable analyses, neither length of ITD nor mutant/wild-type allelic ratio had an impact on OS. Conclusions: FLT3-ITD insertion sites seems to be an important prognostic marker for induction failure, EFS and OS. Therefore, not only FLT3-ITD mutation status but also ITD integration site should be prospectively analyzed in future clinical trials, in particular in the context of treatment with FLT3-tyrosine kinase inhibitors.

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FLT3 INTERNAL TANDEM DUPLICATION AND D835 MUTATIONS IN PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA AND ITS CLINICAL SIGNIFICANCE

Mediterranean Journal of Hematology and Infectious Diseases ◽

10.4084/mjhid.2014.038 ◽

2014 ◽

Vol 6 (1) ◽

pp. e2014038 ◽

Cited By ~ 5

Author(s):

Ghaleb Elyamany ◽

Mohamed Awad ◽

Omar Alsuhaibani ◽

Kamal Fadalla ◽

Omer Al Sharif ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Tyrosine Kinase ◽

Tandem Duplication ◽

Lymphoblastic Leukemia ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Class Iii ◽

Internal Tandem Duplication ◽

Flt3 Mutations ◽

Flt3 Internal Tandem Duplication

The fms-like tyrosine kinase 3 (FLT3) gene is a member of the class III receptor tyrosine kinase family, mutations of FLT3 were first described in 1997 and account for the most frequent molecular mutations in acute myeloid leukemia. No data currently exist regarding FLT3 mutations in Saudi acute lymphoblastic leukemia (ALL) patients as no study has reported for FLT3 mutations in Saudi ALL patients. In this retrospective study, we have examined a cohort of 77 ALL patients, to determine the prevalence of FLT3 mutations and the possible prognostic relevance of these mutations in ALL patients and did correlations to other biologic factors, such as karyotype, molecular mutations, and leukocyte count. FLT3 internal tandem duplication (ITD) mutations and point mutation in tyrosine kinase domain (D835) mutations were analyzed in ALL patients at diagnosis by polymerase chain reaction (PCR). 2 cases (2.6%, 2/77) were positive for FLT3 mutations, one was found to have FLT3/ITD and other was found to have FLT3/D835. Our findings suggest that FLT3 mutations were not common in Saudi ALL and did not affect clinical outcome.

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Internal tandem duplication and tyrosine kinase domain mutations in FLT3 alter the response to daunorubicin in Ba/F3 cells

Biomedical Reports ◽

10.3892/br.2015.541 ◽

2015 ◽

Vol 4 (1) ◽

pp. 83-86 ◽

Cited By ~ 1

Author(s):

SHINICHIRO TAKAHASHI ◽

KUMI SHIRAHAMA

Keyword(s):

Tyrosine Kinase ◽

Tandem Duplication ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Internal Tandem Duplication ◽

Kinase Domain Mutations

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Insertion of FLT3 internal tandem duplication in the tyrosine kinase domain-1 is associated with resistance to chemotherapy and inferior outcome

Blood ◽

10.1182/blood-2009-03-209999 ◽

2009 ◽

Vol 114 (12) ◽

pp. 2386-2392 ◽

Cited By ~ 152

Author(s):

Sabine Kayser ◽

Richard F. Schlenk ◽

Martina Correa Londono ◽

Frank Breitenbuecher ◽

Kerstin Wittke ◽

...

Keyword(s):

Prognostic Factor ◽

Tyrosine Kinase ◽

Tandem Duplication ◽

Kinase Domain ◽

Hazard Ratio ◽

Adult Patients ◽

Tyrosine Kinase Domain ◽

Internal Tandem Duplication ◽

Unfavorable Prognostic Factor ◽

Functional Regions

AbstractTo evaluate internal tandem duplication (ITD) insertion sites and length as well as their clinical impact in younger adult patients with FLT3-ITD–positive acute myeloid leukemia (AML), sequencing after DNA-based amplification was performed in diagnostic samples from 241 FLT3-ITD–mutated patients. All patients were treated on 3 German-Austrian AML Study Group protocols. Thirty-four of the 241 patients had more than 1 ITD, leading to a total of 282 ITDs; the median ITD length was 48 nucleotides (range, 15-180 nucleotides). ITD integration sites were categorized according to functional regions of the FLT3 receptor: juxtamembrane domain (JMD), n = 148; JMD hinge region, n = 48; beta1-sheet of the tyrosine kinase domain-1 (TKD1), n = 73; remaining TKD1 region, n = 13. ITD length was strongly correlated with functional regions (P < .001). In multivariable analyses, ITD integration site in the beta1-sheet was identified as an unfavorable prognostic factor for achievement of a complete remission (odds ratio, 0.22; P = .01), relapse-free survival (hazard ratio, 1.86; P < .001), and overall survival (hazard ratio, 1.59; P = .008). ITD insertion site in the beta1-sheet appears to be an important unfavorable prognostic factor in young adult patients with FLT3-ITD–positive AML. The clinical trials described herein have been registered as follows: AML HD93 (already published in 2003), AML HD98A (NCT00146120; http://www.ClinicalTrials.gov), and AMLSG 07-04 (NCT00151242; http://www.ClinicalTrials.gov).

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Internal Tandem Duplication (ITD) in the Tyrosine Kinase Domain of FLT3 Displays Higher Oncogenic Potential in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.5118.5118 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5118-5118 ◽

Cited By ~ 1

Author(s):

Alissa Marhäll ◽

Thomas Fischer ◽

Florian H. Heidel ◽

Julhash U. Kazi ◽

Lars Rönnstrand

Keyword(s):

Acute Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Tandem Duplication ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Internal Tandem Duplication ◽

Transfected Cells ◽

Flt3 Mutations ◽

Acute Myeloid

Abstract Up to 30% of patients with acute myeloid leukemia (AML) harbor a mutation in FMS like tyrosine kinase 3 (FLT3). This mutation is not only the most frequent, but also the most clinically challenging, because it is associated with increased risk of relapse and poor overall survival. The most common oncogenic FLT3 mutation is the internal tandem duplication (ITD) in the juxtamembrane domain (ITD-JM). Other less frequently occurring mutations include ITD mutations in the tyrosine kinase domain (ITD-TDK) and point mutations in the TKD (e. g. D835Y-TKD). Use of FLT3 inhibitors has shown initial promise, though an acquired resistance remains a problem. A need therefore remains for additional therapeutic targets, and in order to identify these we need to have a better understanding of the mechanisms by which the FLT3 mutations drive leukemogenesis. Although ITD-JM and D835Y-TKD mutations have been studied extensively, the role of the recently identified ITD-TKD remains poorly understood. In this study we compared the ITD-TKD domain mutations with other well-studied FLT3 mutations. We observed that transfection of cytokine-dependent Ba/F3 cells with ITD-TKD is sufficient to induce formation of colonies in semisolid medium in the absence of cytokines. Number and size of colonies were comparable to that of ITD-JM, while D835Y-TKD transfected cells failed to form colonies suggesting that the ITD-TKD mutations have stronger transforming potential than other TKD mutations. Similar to colony formation assays, proliferation and cell survival was significantly higher in ITD-TKD transfected cells compared to cells transfected with D835Y-TKD. ITD-TKD selectively enhanced STAT5 and AKT phosphorylation while ERK1/2 and p38 phosphorylation remained unchanged. Collectively our data suggest that ITD-TKD is a more potent oncogenic mutant compared to the TKD mutants. Disclosures Fischer: Novartis: Consultancy, Honoraria.

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Quizartinib in the treatment of FLT3-internal-tandem duplication-positive acute myeloid leukemia

Future Oncology ◽

10.2217/fon-2019-0353 ◽

2019 ◽

Vol 15 (34) ◽

pp. 3885-3894 ◽

Cited By ~ 1

Author(s):

Shilpa Paul ◽

Adam J DiPippo ◽

Farhad Ravandi ◽

Tapan M Kadia

Keyword(s):

Acute Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Tandem Duplication ◽

Kinase Inhibitors ◽

Single Agent ◽

Tyrosine Kinase Domain ◽

Missense Mutations ◽

Internal Tandem Duplication ◽

Acute Myeloid

FLT3 mutations, characterized by an internal-tandem duplication or missense mutations in the tyrosine kinase domain, are observed in a third of patients with newly diagnosed acute myeloid leukemia. FLT3-ITD mutations are associated with high relapse rates and short overall survival with conventional chemotherapy. Several tyrosine kinase inhibitors targeting FLT3 have been developed in an effort to improve survival and therapeutic options. This review focuses on quizartinib, a second-generation FLT3 inhibitor that has demonstrated efficacy and safety as a single agent and in combination with chemotherapy. We discuss its clinical development as well as its place in the treatment of FLT3-mutated acute myeloid leukemia among the other FLT3 inhibtors currently available and its mechanisms of resistance.

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Clinical Course and Significance of the Novel FLT3-Y842C Mutation in a Patient with AML Treated with PKC412 Monotherapy.

Blood ◽

10.1182/blood.v104.11.2537.2537 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2537-2537

Author(s):

T. Kindler ◽

F. Breitenbuecher ◽

S. Kasper ◽

E. Estey ◽

F. Giles ◽

...

Keyword(s):

Sequence Analysis ◽

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Clinical Course ◽

Novel Mutation ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Activation Loop ◽

The Novel ◽

Constitutive Activation

Abstract We recently identified a novel mutation (Y842C) within the tyrosine kinase domain of FLT3 in a patient treated with PKC410 monotherapy (ASH 2003, # 4681). Here, we present follow up studies including the clinical course of the patient and frequency analysis in 110 patients with AML. In addition, we characterized the novel mutation using overexpression of FLT3-Y842C in 32D cells. AML M2 was diagnosed in a 63 year old, male patient in 1993. After having experienced his second relapse upon standard therapy the patient was refractory to alemtuzumab treatment. Due to reduced performance status the patient was not eligible to standard chemotherapy and was enrolled into a phase II trial investigating PKC412. On conventional FLT3 mutation analysis the patient was considered to be FLT3 wild-type. Upon 8 and 29 days of treatment complete clearance of PB blast counts and BM blast infiltration was observed, respectively. Daily substitution of G-CSF resulted in transient recovery or the patients ANC′s. Since the patient showed an excellent clinical responsiveness, we reasoned whether the patient may have a yet unidentified FLT3 mutation. Sequence analysis revealed a novel point mutation in exon 21 of FLT3 (Y842C). Protein analysis of primary AML blasts showed constitutive FLT3 tyrosine-phosphorylation, ex vivo treatment with PKC412 caused significant inhibition of FLT3 and STAT5 activation. Further, in vivo analysis of FLT3 tyrosine-phosphorylation during the course of PKC412 treatment showed complete suppression of FLT3 activation within 8 days. Overexpression of FLT3-Y842C in 32D cells resulted in constitutive activation of FLT3 and STAT5 as well as in factor independent proliferation. Treatment with PKC412 caused inhibition of FLT3 tyrosine-phosphorylation, factor independent growth and apoptotic cell death. To further investigate the clinical significance of the novel Y842C mutation, the tyrosine kinase domain of FLT3 was investigated in 110 patients with AML using sequence analysis. Altogether, the novel mutation Y842C was identified in 2 patients, FLT-ITD in 22 patients and D835 in 7 patients, respectively. It is interesting to note that the recently described crystal structure of FLT3 reveals a critical role for Y842 in regulating the switch from the closed to the open (=active) conformation of the FLT3 activation loop. Since our data is consistent with the concept that the Y842C mutation results in constitutive activation of FLT3, it is tempting to speculate that the exchange of tyrosine for cysteine at position 842 disrupts the autoinhibited state of the FLT3 activation loop. Given that the novel mutation described here could only be identified by direct sequencing, it is likely that the number of mutations in this region of FLT3 is currently underestimated. Thus, extended sequence analysis of this mutational hotspot may be helpful in further defining the spectrum of TKI-sensitive FLT3 mutations in AML.

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