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Morphological, cytogenetic and molecular characterisation of FLT3 mutations in Pakistani patients with de novo acute myeloid leukaemia: A single centre experience

Faiz M, Rashid F

Malays J Pathol, 2023 Aug;45(2):205-214.
PMID: 37658530

INTRODUCTION: Mutations in FLT3 are the most commonly reported genetic changes in AML patients. These mutations are normally identified in approximately one third of newly diagnosed patients and are reported to have prognostic significance.
MATERIALS AND METHODS: Peripheral blood samples was collected from 63 AML patients to study their morphological, cytogenetic and molecular features. PCR was used to determine the prevalence of FLT3 mutations; internal tandem duplication (ITD) and tyrosine kinase domain (TKD) in AML patients.
RESULTS: Among 63 AML patients, 42 were males and 21 were females with male to female ratio 2:1 with median age of 32 years. AML-M2 was the predominant French-American-British (FAB) subtype (42%) followed by M4 (27%), M3 (8%), M1 (8%), M0 (8%) and M5 (7%) respectively. Cytogenetic analysis of 60 patients showed 58% as cytogenetically normal (CN) whereas 42% had aberrant karyotype.The most frequent aberrations were trisomy8, t(8;21), t(15;17) (8.3%) each, inversion16 (5%), and different deletions (12%) respectively. FAB-M4 subtype showed most of the chromosomal anomalies. Among 63 AML patients, 22% showed FLT3/ITD while 6.4% had D835 mutation after molecular analysis. FLT3 mutations were found in most of the FAB subtypes and cytogenetic groups. FLT3/ITD mutations were more common in patients with normal karyotype (26%) and usually present with hyperleukocytosis but association between two was not significant.
CONCLUSION: The cytogenetic data of adult AML from Pakistan showed presence of favourable prognostic karyotype with comparable prevalence as reported in international data. Moreover, FLT3/ITD mutations are commonly found in our patients as determined by molecular analysis. Therefore, inclusion of this unfavourable prognostic marker should be routine in molecular diagnostic testing of AML.

Matched MeSH terms: fms-Like Tyrosine Kinase 3/genetics
RUNX1/ETO regulates reactive oxygen species (ROS) levels in t(8,21) acute myeloid leukaemia via FLT3 and RAC1

Azlan A, Khor KZ, Rajasegaran Y, Rosli AA, Said MSM, Yusoff NM, et al.

Med Oncol, 2023 Jun 21;40(7):208.
PMID: 37341821 DOI: 10.1007/s12032-023-02075-w

Reactive oxygen species (ROS) homeostasis is crucial for leukaemogenesisand deregulation would hamper leukaemic progression. Although the regulatory effects of RUNX1/ETO has been extensively studied, its underlying molecular mechanims in ROS production in t(8,21) AML is yet to be fully elucidated. Here, we report that RUNX1/ETO could directly control FLT3 by occupying several DNA elements on FLT3 locus. The possible hijacking mechanism by RUNX1/ETO over FLT3 mediated ROS modulation in AML t(8;21) was made apparent when suppression of RUNX1/ETO led to decrement in ROS levels and the direct oxidative marker FOXO3 but not in FLT3 and RAC1 suppressed t(8,21) AML cell line Furthermore, nuclear import of RUNX1/ETO was aberrated following RUNX1/ETO and RAC1 suppression suggesting association in ROS control. A different picture was depicted in non t(8;21) cells where suppression of RAC1 and FLT3 led to decreased levels of FOXO3a and ROS. Results alltogether indicate a possible dysregulation of ROS levels by RUNX1/ETO in t(8,21) AML.

Matched MeSH terms: fms-Like Tyrosine Kinase 3/genetics; fms-Like Tyrosine Kinase 3/metabolism
Fulltext Misfolded N-CoR is Linked to the Ectopic Reactivation of CD34/Flt3-Based Stem-Cell Phenotype in Promyelocytic and Monocytic Acute Myeloid Leukemia

Nin DS, Li F, Visvanathan S, Khan M

Front Oncol, 2015;5:210.
PMID: 26500885 DOI: 10.3389/fonc.2015.00210

Nuclear receptor co-repressor (N-CoR) is the key component of generic co-repressor complex essential for the transcriptional control of genes involved in cellular hemostasis. We have recently reported that N-CoR actively represses Flt3, a key factor of hematopoietic stem cells (HSC) self-renewal and growth, and that de-repression of Flt3 by the misfolded N-CoR plays an important role in the pathogenesis of promyelocytic and monocytic acute myeloid leukemia (AML). The leukemic cells derived from the promyelocytic and monocytic AML are distinctly characterized by the ectopic reactivation of stem cell phenotypes in relatively committed myeloid compartment. However, the molecular mechanism underlying this phenomenon is not known. Here, we report that N-CoR function is essential for the commitment of primitive hematopoietic cells to the cells of myeloid lineage and that loss of N-CoR function due to misfolding is linked to the ectopic reactivation of generic stem cell phenotypes in promyelocytic and monocytic AML. Analysis of N-CoR and Flt3 transcripts in mouse hematopoietic cells revealed a positive correlation between N-CoR level and the commitment of myeloid cells and an inverse correlation between N-CoR and Flt3 levels in primitive as well as committed myeloid cells. Enforced N-CoR expression in mouse HSCs inhibited their growth and self-renewal potentials and promoted maturation toward cells of myeloid lineage, suggesting a role of N-CoR in the commitment of cells of myeloid lineage. In contrast to AML cells with natively folded N-CoR, primary and secondary promyelocytic and monocytic AML cells harboring the misfolded N-CoR were highly positive for Flt3 and myeloid antigen-based HSC marker CD34. Genetic and therapeutic restoration of N-CoR conformation significantly down-regulated the CD34 levels in monocytic AML cells, suggesting an important role of N-CoR in the suppression of CD34-based HSC phenotypes. These findings collectively suggest that N-CoR is crucial for the commitment of primitive hematopoietic cells to cells of myeloid lineage and that misfolded N-CoR may contribute to transformation of committed myeloid cells through the ectopic reactivation of Flt3/CD34-based stem cell phenotypes in promyelocytic and monocytic AML. Moreover, these findings provide novel mechanistic insights into the formation of leukemic stem cells in subsets of AML and identify the misfolded N-CoR as a subtype-specific biomarker of AML.

Matched MeSH terms: fms-Like Tyrosine Kinase 3
Fulltext Identification of FLT3 and NPM1 Mutations in Patients with Acute Myeloid Leukaemia

Mat Yusoff Y, Abu Seman Z, Othman N, Kamaluddin NR, Esa E, Zulkiply NA, et al.

Asian Pac J Cancer Prev, 2019 06 01;20(6):1749-1755.
PMID: 31244296 DOI: 10.31557/APJCP.2019.20.6.1749

Objective: The most frequent acquired molecular abnormalities and important prognostic indicators in patients
with Acute Myeloid Leukaemia (AML) are fms-like tyrosine kinase-3 gene (FLT3) and nucleophosmin-1 (NPM1)
mutations. Our study aims to develop a cost effective and comprehensive in-house conventional PCR method for
detection of FLT3-ITD, FLT3-D835 and NPM1 mutations and to evaluate the frequency of these mutations in patients
with cytogenetically normal (CN) AML in our population. Methods: A total of 199 samples from AML patients (95
women, 104 men) were included in the study. Mutation analyses were performed using polymerase chain reaction
(PCR) and gene sequencing. Result: Sixty-eight patients were positive for the mutations. FLT3-ITD mutations were
detected in 32 patients (16.1%), followed by FLT3-D835 in 5 (2.5%) and NPM1 in 54 (27.1%). Double mutations of
NPM1 and FLT3-ITD were detected in 23 cases (11.6%). Assays validation were performed using Sanger sequencing
and showed 100% concordance with in house method. Conclusion: The optimized in-house PCR assays for the
detection of FLT3-ITD, FLT3-D835 and NPM1 mutations in AML patients were robust, less labour intensive and cost
effective. These assays can be used as diagnostic tools for mutation detection in AML patients since identification of
these mutations are important for prognostication and optimization of patient care.

Matched MeSH terms: fms-Like Tyrosine Kinase 3/genetics*
Characterisation and Clinical Significance of FLT3-ITD and non-ITD in Acute Myeloid Leukaemia Patients in Kelantan, Northeast Peninsular Malaysia

Yunus NM, Johan MF, Ali Nagi Al-Jamal H, Husin A, Hussein AR, Hassan R

Asian Pac J Cancer Prev, 2015;16(12):4869-72.
PMID: 26163606

BACKGROUND: Mutations of the FMS-like tyrosine kinase-3 (FLT3) receptor gene may promote proliferation via activation of multiple signaling pathways. FLT3-internal tandem duplication (FLT3-ITD) is the most common gene alteration found in patients diagnosed with acute myeloid leukaemia (AML) and has been associated with poor prognosis.
MATERIALS AND METHODS: We performed mutational analysis of exons 14-15 and 20 of the FLT3 gene in 54 AML patients using PCR-CSGE (conformational sensitive gel electrophoresis) followed by sequencing analysis to characterise FLT3 mutations in adult patients diagnosed with AML at Hospital USM, Kelantan, Northeast Peninsular Malaysia.
RESULTS: FLT3 exon 14-15 mutations were identified in 7 of 54 patients (13%) whereas no mutation was found in FLT3 exon 20. Six ITDs and one non-ITD mutation were found in exon 14 of the juxtamembrane (JM) domain of FLT3. FLT3-ITD mutations were associated with a significantly higher blast percentage (p-value=0.008) and white blood cell count (p-value=0.023) but there was no significant difference in median overall survival time for FLT3-ITD+/FLT3-ITD- within 2 years (p-value=0.374).
CONCLUSIONS: The incidence of FLT3-ITD in AML patients in this particular region of Malaysia is low compared to the Western world and has a significant association with WBC and blast percentage.

Matched MeSH terms: fms-Like Tyrosine Kinase 3/genetics*