Molecular Diagnosis, Clinical Features, And Treatment Outcome Of Adult Patients With De Novo Leukemia With11Q23 Chromosome Translocations And Establishment Of Primary Mll Cells Xenotransplantation Mice Model

Objective:Acute leukemia (AL) with Ilq23/MLL gene arrangements is a recurrent cytogenetic abnormality. Numerous partners of the translocations and the various types of arrangements involving 11q23/MLL, it is difficult for conventional cytogenetic assay (CCA) to disclose all MLL arrangements. To improve and validate the detection of 11q23/MLL chromosomal aberrations in leukemias, we designed a combined application of karyolysis with multiplex RT-PCR. Nμmerous molecular markers have been recently discovered as potential prognostic factors in AL. It has become of critical importance to thoroughly evaluate their interrelationships and relative prognostic importance. We determined molecular mutations and other specific immunophenotype of AL Patients with 11q23/MLL gene arrangements, which may be helpful for risk-based treatment of AL.Methods:Karyotype studies and Multiplex RT-PCR assays were performed in 347 consecutive adult patients diagnosed with leukemia between 2009 to 2011, while determined the presence of molecular mutations (NPM1, FLT3, CEBPa, C-KIT, DNMT3A, IDH1 and IDH2). Patients received standard chemotherapeutic protocols and survival outcome was determined. Clinical analysis was mainly carried out in the AL with 11q23/MLL and the normal karyotype AL without molecular mutations. The Cox proportional hazards model was implemented to identify independent prognostic factors for EFS and OS.Results:Among the AL, only 2 patients with 11q23 chromosome translocations were identified by karyotype studies, while 28 patients harboring various MLL rearrangements were identified by multiplex RT-PCR. Within the 28 11q23/MLL patients, no NPM1, CEBPa, C-KIT and IDH1 mutations were detected. Furthermore, FLT3, DNMT3A and IDH2 mutations were found in 4 (14.28%),1 (3.5%) and 1 (3.5%) of these28 patients, respectively. Multivariable Cox regression analysis shows that these molecular mutations were not associated with an adverse outcome for 11q23/MLL gene arrangements patients. Conclusions:Conventional cytogenetics and molecular-based methods are complementary tests for the detection of clonal genetic aberrations in 11q23/MLL gene arrangements patients. Cytogenetic and molecular findings are used in determining the best therapy strategy for 11q23/MLL gene arrangements patients, especially the determination of allogeneic stem cell transplantation is needed. Purpose:To establish xenotransplantation model in NOD/SCID mice by using MLL cells.Methods:Peripheral mononuclear cells from leukemia patients with MLL/AF4 rearrangement into NOD/SCID mice were injected via the tail vein. The mice were weighed weekly, and the percent of CD45+ cells in their peripheral blood were detected by flow cytometry at the 14th day after injection. The mouse was euthanized when they become droopy, tired, pale, pilo-erected, or lose weight up to 20 percent. Dissected the mice, examined spleens and livers by HE, and detected the percent of CD45+ cells in the peripheral blood, marrow, spleen and lymph nodes suspension.Results:CD45+ hμman cells were detected in the peripheral blood of mice 35±4 days after injection by flow cytometry, utilizing peripheral mononuclear cells from control group as negative gating. Those cells were found to increase gradually, and reach 61.0%±3.5% in the end. Spleens, livers and lymph nodes of mice were observed to be enlarged, and typical malignant tμmor cells were found under microscope. A group of CD45+hμman cells was detected by flow cytometry in peripheral blood, marrow, lymph nodes and spleens of the mice.Conclusion:Xenotransplantation model in NOD/SCID mice was established successfully using primary hμman MLL/AF4 cells in this research.