| Expression and Clinical Significance of IKAROS6isoform in acute lymphoblastic leukemia patientsObjective:The purpose of the study was to investigate the expression of Ikaros6in adult acute lymphoblastic leukemia(ALL). Moreover, this study focused on the relationship between Ikaros6and other molecular markers, as well as prognostic value of Ikaros6.Methods:We examined the expression of Ikaros6in bone marrow from179de novo patients with ALL (B-ALL, n=105and T-ALL, n=74) from Sep2006to Jul2013. RNA was extracted from mononuclear cells of bone marrow, and then the expression of Ikaros6was detected by reverse transcription-PCR, and confirmed the results by gene scanning and sequencing. The expression of BCR-ABL1and Ikaros6were dynamically monitored by gene scanning and real-time PCR from initial diagnosis to relapse. Furthermore, we analysed the clinical feature of patients positive for Ikaros6, the relationship between Ikaros6and other molecular markers and prognostic significance of Ikaros6in ALL.Results:In the total of179adult patients with ALL (B-ALL, n=105and T-ALL, n=74), Ikaros6was observed in53patients (29.61%). Among them,37of105cases with B-ALL (35.24%) and16of74cases with T-ALL (21.62%) were detected Ikaros6. In B-ALL, Ikaros6was predominantly found in the common B-ALL subtype (24of37,64.86%). Ikaros6was principally found in high-risk group (23of37,62.16%), especially in cases of Ph+ALL (17of37,45.95%). Ikaros6was found to be associated with BCR-ABL1(P=0.011) significantly. Moreover, the expression patterns of BCR-ABL1and Ikaros6were similar from initial diagnosis to relapse. Patients positive for Ikaros6,26of37(70.27%) expressed myeloid-associated antigens and there was a significant association between Ikaros6and myeloid-associated antigens (P=0.032). Thus, there was close correlation between BCR-ABL1, myeloid-associated antigens and Ikaros6. However, Ikaros6was associated with increased risk of relapse, which was independent of BCR-ABL1and myeloid-associated antigens. In multivariable Cox analysis, Ikaros6was an independent prognostic marker for overall survival (P=0.043, hazard ratio=1.905), event-free survival (P=0.044, hazard ratio=1.822) and relapse-free survival (P=0.002, hazard ratio=4.992). In74T-cell patients, Ikaros6was principally found in medullary T-ALL subtype (8of16,50.0%), moreover, it was found negatively correlated with pro-T-ALL (P=0.036). Extramedullary infiltration occurred in10of16(62.50%) patients positive for Ikaros6, which was more frequently than cases negative for Ikaros6(34.48%), and the difference had statistical significance (P=0.043). Meanwhile these had inferior survival (OS, P=0.032; EFS, P=0.014) and increased risk of relapse (RFS, P=0.011) as compared with the Ikaros6-negative patients.Conclusion:The incidence of Ikaros6was high in adult ALL patients. Patients positive for Ikaros6were more likely to have some clinical features. Ikaros6was closely related to BCR-ABL1and myeloid-associated antigens, but Ikaros6predicted worse RFS was independent on other prognostic factors. Thus Ikaros6may serve as a valuable factor for risk stratification and prognosis evaluation in adult ALL. Investigation of IKAROS6isoform in Acute Myeloid LeukemiaObjective:The purpose of the study was to investigate the expression and prognostic value of Ikaros6in acute myeloid leukemia (AML). Moreover, we explored the relationship between Ikaros6and other molecular markers. Then, we constructed a lentiviral vector carrying IK6gene and observed the expression of IK6as well as related biologic feature in THP1and ML-2cells.Methods:We examined the expression of Ikaros6in bone marrow and peripheral blood from329de novo patients with AML from Jul2009to Jul2013. RNA and DNA were extracted from mononuclear cells of bone marrow and peripheral blood. The IKZF1△4-7deletion was detected by genomic gene PCR, and confirmed the results by genomic gene scanning and sequencing. The expression of Ikaros6was detected by nest-PCR, and confirmed the results by gene scanning and sequencing too. Moreover, IK6protein was detected by Western blot. Furthermore, we analysed the clinical feature of patients positive for Ikaros6, the relationship between Ikaros6and other molecular markers, and prognostic significance of Ikaros6in adult AML. IK6gene was recombined with the linearizing vector to construct a recombinant lentiviral vector named pGC-FU-IK6-GFP.293T cells were transfected with pGC-FU-IK6-GFP by using Lipofectamine2000. After examining the titer of the virus, it was used to transfect myeloid cell lines. The transfection efficiency was detected by flow cytometry, the expression level of mRNA was confirmed by RT-PCR and IK6-GFP fusion protein was detected by Western blotting. Then we detected the impact of IK6on apoptosis and cell cycle.Results:In the total of329adult patients with AML, Ikaros6was observed in32patients (9.73%). Among them, Ikaros6was predominantly found in AML-M5. Ikaros6was principally found in11q23non t (9;11) subgroup (42.86%,6/14) and there were significant differences in the distribution of the frequency karyotypes (P=0.001). Of note, Ikaros6was associated with the presence of MLL (P<0.001). Ikaros6was associated with adverse outcomes (OS, P=0.002; EFS, P<0.001) and increased risk of relapse (RFS, P<0.001) in adult AML. IK6gene was connected into the linearized lentiviral vector to successfully constructed target plasmid named pGC-FU-IK6-GFP with Amp resistant. The target plasmid was transfected into293T cells and the virus titer was2.0×109TU/ml. Next, THP1and ML-2cells were transfected with pGC-FU-IK6-GFP and the efficiency was up to90%. IK6mRNA and IK6-GFP fusion protein were detected in target cells. IK6could promote target cell clones formation and inhibit apoptosis, but had no significant effect on the cell cycle.Conclusion:The incidence of Ikaros6was low in adult AML. Ikaros6was predominantly found in AML-M5and was associated with the presence of MLL. Ikaros6was associated with adverse outcomes and increased risk of relapse.Thus Ikaros6may serve as a valuable factor for risk stratification and prognosis evaluation in adult AML. pGC-FU-IK6-GFP had been successfully constructed and IK6expressed in target cells stably. Biology studies showed IK6was likely to interfere with the function of normal Ikaros protein as tumor suppressor, and it exertes a potential anti-apoptotic effect. Thus, IK6could promote leukemia cells growth. |
