Detection of FLT3/ITD gene mutations in patients with hematologic malignancy and their clinical significance

Objective： To analyze Fms-like tyrosine kinase 3 （FLT3）/internal-tandem duplications （ITD） mutations in various kinds of hematologic malignancy patients. Methods： FLT3/ITD gene mutations were detected by polymerase chain reaction （PCR） in 103 acute myeloid leukemia （AML） cases, 63 acute lymphocytic leukemia （ALL） cases, 53 chronic myelogenous leukemia （CML） cases in chronic phase （CML-CP）, 34 CML cases in blast crisis （CML-BC）, 11 chronic lymphatic leukemia （CLL） cases, 36 myelodysplastic syndrome （MDS） cases, 9 multiple myeloma （MM） cases and 13 non-hodgkin＇s lymphoma （NHL） cases with marrow infiltration. Results： The expressions of FLT3/ITD gene mutations were detected in 22.3% AML cases, in 6.5% CML-BC cases, in 5.6% MDS cases and in 2.6% ALL cases. The two ALL cases with FLT3/ITD mutation were diagnosed as ALL-L2 with morphology and both with myeloid antigen expression, but finally were diagnosed as acute mixed-lineage leukemia after immunology examination. FLT3/ITD gene mutations were not detected in CML-CP, MM, NHL and CLL cases. In the 23 AML patients with FLT3/ITD gene mutation, including 2 of 8 M1 （2.5%）, 8 of 33 M2 （24.2%）, 7 of 24 M3 （29.3%）, 2 of 11 M4 （18.2%）, 3 of 21 M5 （14.3%）, 1 of 5 M6 （20%）, and 0 of 1 M7 cases, and there were no significant differences in the positive rates of FLT3/ITD mutations between the FAB subtypes （P 〉 0.05）. Statistical analyses showed that in AML patients, FLT3/ITD was associated with a higher peripheral blood white cell （WBC） counts [（41.23 ± 32.56） x 109/L vs （11.36 ± 9.89） × 10^9/L （P 〈 0.01 ）], higher percentage of bone marrow blast cells [（72.78 ± 21.79）% vs （51.26 ± 20.78）% （P 〈 0.05）], and higher cumulative relapse rates （63.6% vs 27.7%, P 〈 0.025） than those negative. Conclusion： FLT3/ITD gene mutation mainly occurred in AML patients, and might be a strong prognostic factor which was associated with high peripheral WBC counts, bone marrow blast cell proportion and a increased relapse risk in AML. Detection of FLT3/ITD gene mutation might provide insights to explore a more accurate genotyping of leukemia, differential diagnosis between AML and ALL, subdivide risk level in AML and estimate prognosis of leukemia.

4-Hydroxy phenyl Retinamide Preferentially Targets FLT3 Mutated Acute Myeloid Leukemia via ROS Induction and NF-κB Inhibition

FMS-like tyrosine kinase 3(FLT3)mutation is strongly associated with poor prognosis in acute myeloid leukemia(AML).Though many FLT3 inhibitors have been developed for clinical application with 34%-56%complete remission rate,patients would develop resistance sooner or later after initial response to tyrosine kinase inhibitors(TKIs),such as gilteritinib.And increasing studies have shown that several resistance related mutations of FLT3 emerged during the AML progression.Thus,further investigation is warranted for these FLT3mu,AML patients to achieve a better treatment outcome.4-Hydroxyphenyl retinamide(4-HPR)has been investigated extensively in animal models and clinical trials as an anticancer/chemopreventive agent and is currently used for protection against cancer development/recurrence,with minimal side effects.In this study,we performed gene-set enrichment analysis and found that down-regulated genes induced by 4-HPR were associated with FLT3-ITD gene sets.CD34+ AML stem/progenitor cells separated from 32 AML samples were treated with 4-HPR.Correlation analysis showed that AML cells with FLT3-ITD genetic alteration were more sensitive to 4-HPR treatment than those without FLT3-ITD.Next,we treated 22 primary AML cells with 4-HPR and found that 4-HPR was more toxic to AML cells with FLT3-ITD.These results indicated that 4-HPR was preferentially cytotoxic to all FLT3-ITD AML cells irrespective of stem/progenitor cells or blast cells.4-HPR-induced reactive oxygen species(ROS)production and NF-kB inhibition might be the reason of 4-HPR selectivity on FLT3 mutated AML cells.

Establishment of Xenotransplantation Model of Human CN-AML with FLT3-ITD^(mut)/NPM1 in NOD/SCID Mice

Summary： Patients with FLT3-ITD^mmutt/NPM1- cytogenetically normal acute myeloid leukemia （CN-AML）, as high-risk molecular group in CN-AML, are associated with a worse prognosis than other CN-AML patients. It is beneficial to generate xenotransplantation model of FLT3-ITD^mut/NPM1- CN-AML to better understand the pathogenesis and therapeutic strategies of such AML subtype. The purpose of present study was to establish the xenotransplantation model in NOD/SCID mice with FLT3-ITD^mut/NPM1- CN-AML primary cells. The FLT3-ITD^mut/NPM1- CN-AML primary cells from 3 of 7 cases were successfully transplanted into NOD/SCID mice, and human CD45 positive cells were detected in the peripheral blood, spleen and bone marrow of mice by using flow cytometry. Infiltration of human leukemia cells in various organs of mice was observed by using immunohistochemistry. Gene analysis confirmed sustained FLT3/ITD mutation without NPM1 mutation in mice. By performing serial transplantation, it was found that characteristics of the leukemia cells in secondary and tertiary genera- tion models remained unchanged. Moreover, in vivo cytarabine administration could extend survival of NOD/SCID mice, which was consistent with clinical observation. In conclusion, we successfully estab- lished xenotransplantation model of human FLT3-ITD^mut/NPM1- CN-AML in NOD/SCID mice. The model was able to present primary disease and suitable to evaluate the curative effects of new drugs or therapy strategies.