OBJECTIVE: To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation, and establish a systematic evaluation system to verify the success of model construction. METHODS: The bone marrow c-kit⁺ cells of the mice were obtained by the following steps: The mice were killed by cervical dislocation, the femur, tibia and ilium were separated, and the bone marrow cells were collected. The c-kit⁺ cells were sorted after incubation with CD117 magnetic beads. The method of constructing mouse primary mutant cells is as follows: A gene mutation vector with a GFP tag was constructed by the retroviral system, and the retroviral vector was packaged into the Platinum-E cells to obtain the virus supernatant, and then used it to infect the c-kit⁺ cells of mice. The MPN mouse model was constructed as follows: the mouse primary c-kit⁺ cells containing the mutant genes were collected after infection, and then transplanted them via the tail vein into the female recipient mice of the same species which were irradiated with a lethal dose of gamma rays (8.0 Gy). The MPN mouse model was evaluated as follows: After transplantation, the peripheral blood of the mice was regularly collected from the tail vein to perform the complete blood count test, and the size of spleen and the degree of bone marrow fibrosis were estimated. RESULTS: The mouse c-kit⁺ cells with the mutant genes were successfully obtained from the bone marrow. MPN mouse model was successfully constructed: The peripheral blood cells of the MPN-transplanted mice carried exogenous implanted GFP-positive cells, and the white blood cells (WBC), platelet (PLT) and hematocrit (HCT) were all increased; the body weight loss, and the water and food intake were reduced in the transplanted mice; further pathological analysis showed that the transplanted mice displayed splenomegaly and bone marrow fibrosis. These results suggested that the MPN mouse model was successfully constructed. According to the common and different characteristics of the three MPN mouse model, a preliminary evaluation system for judging the success of MPN mouse model construction was summarized, which mainly included the following indicators, for example, the proportion of GFP-positive cells in the peripheral blood of mice; WBC, PLT and HCT; the degree of spleen enlargement and the bone marrow fibrosis. CONCLUSION The MPN mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation is successfully established by retroviral system, which can provide an important experimental animal model for the research of MPN pathogenesis and drug-targeted therapy.
Female ; Mice ; Animals ; Primary Myelofibrosis ; Myeloproliferative Disorders/genetics* ; Bone Marrow/pathology* ; Mutation ; Disease Models, Animal ; Neoplasms ; Janus Kinase 2/genetics*

Humans ; Lymphoma ; Lymphoma, T-Cell ; Myeloproliferative Disorders/genetics* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology* ; T-Lymphocytes/pathology*

BACKGROUND: Calreticulin (CALR) mutations were recently discovered in patients with myeloproliferative neoplasms (MPNs). We studied the frequency and type of CALR mutations and their hematological characteristics. METHODS: A total of 168 MPN patients (36 polycythemia vera [PV], 114 essential thrombocythemia [ET], and 18 primary myelofibrosis [PMF] cases) were included in the study. CALR mutation was analyzed by the direct sequencing method. RESULTS: CALR mutations were detected in 21.9% of ET and 16.7% of PMF patients, which accounted for 58.5% and 33.3% of ET and PMF patients without Janus kinase 2 (JAK2) or myeloproliferative leukemia virus oncogenes (MPL) mutations, respectively. A total of five types of mutation were detected, among which, L367fs*46 (53.6%) and K385fs*47 (35.7%) were found to be the most common. ET patients with CALR mutation had lower leukocyte counts and ages compared with JAK2-mutated ET patients. CONCLUSION: Genotyping for CALR could be a useful diagnostic tool for JAK2-or MPL-negative ET or PMF patients. CALR mutation may be a distinct disease group, with different hematological characteristics than that of JAK2-positive patients.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Amino Acid Sequence ; Base Sequence ; Calreticulin/*genetics ; DNA Mutational Analysis ; Exons ; Female ; Humans ; Janus Kinase 2/genetics ; Leukocyte Count ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Myeloproliferative Disorders/diagnosis/*genetics/pathology ; Receptors, Thrombopoietin/genetics ; Young Adult

The genetic variant rs16754 of Wilms tumor gene 1 (WT1) has recently been described as an independent prognostic factor in AML patients. It is of great interest to test whether WT1 single nucleotide polymorphism can be used as a molecular marker in other types of cancer, to improve risk and treatment stratification. We performed sequencing analysis of exons 7 and 9 of WT1, which are known mutational hotspots, in a total of 73 patients with BCR-ABL1-negative myeloproliferative neoplasm (MPN) and 93 healthy controls. No previously reported WT1 mutations were identified in the present study. In Korean patients with BCR-ABL1-negative MPN, WT1 genetic variant rs16754 had no significant impact on clinical outcomes. We observed a significant difference in the allelic frequencies of WT1 rs16754 in Koreans between BCR-ABL1-negative MPN cases and healthy controls. Individuals carrying variant G alleles of WT1 rs16754 showed a relatively low prevalence of BCR-ABL1-negative MPN, compared with those carrying wild A alleles of WT1 rs16754 (Hazard ratio 0.10-0.65, P<0.05). Therefore, possession of the variant G allele of WT1 rs16754 may reduce the risk of developing BCR-ABL1-negative MPN.
Adult ; Aged ; Aged, 80 and over ; Alleles ; Asian Continental Ancestry Group/*genetics ; Case-Control Studies ; Exons ; Female ; Fusion Proteins, bcr-abl/genetics ; Gene Frequency ; Genotype ; Humans ; Leukemia, Myeloid, Acute/pathology ; Male ; Middle Aged ; Myeloproliferative Disorders/*genetics/pathology ; Polymorphism, Single Nucleotide ; Prognosis ; Proportional Hazards Models ; Republic of Korea ; Risk ; Sequence Analysis, DNA ; WT1 Proteins/*genetics ; Young Adult

No abstract available.
Adult ; Biomarkers, Tumor/genetics ; Bone Marrow/pathology ; Calreticulin/genetics ; Erythropoietin/blood ; Female ; Fusion Proteins, bcr-abl/*genetics ; Hematocrit ; Hemoglobins/analysis ; Humans ; Janus Kinase 2/genetics ; Male ; Middle Aged ; Mutation ; Myeloproliferative Disorders/*diagnosis/genetics ; Polycythemia Vera/*diagnosis/genetics ; Receptors, Thrombopoietin/genetics ; Thrombocythemia, Essential/diagnosis ; World Health Organization

The 8p11 myeloproliferative syndrome (EMS) is named as stem cell leukemia/lymphoma syndrome, and is an aggressive neoplasm associated with chromosomal translocations involving the fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase gene on chromosome 8p11-12. EMS is a syndrome characterized by peripheral blood leucocytosis with eosinophilia, myeloid hyperplasia of bone marrow, and T-cell lymphoblastic leukemia/lymphoma. Clinically, EMS is an aggressive disease with a short chronic phase before rapid transformation into acute leukemia. Its prognosis is poor. The only curative option for patients with EMS at this time appears to be bone marrow or stem cell transplantation. At the molecular level, all cases carry a chromosomal abnormality involving the FGFR1 gene at chromosome 8p11. The novel chimeric proteins foster dimerization and ligand-independent activation of FGFR1 tyrosine kinase, subsequently promoting activation of downstream pathways involved in proliferation and malignant transformation of cells. Currently, 13 translocations and 1 insertion have been identified. Here, the current review mainly focuses on molecular genetic features, pathogenic mechanisms and therapy of EMS.
Chromosomes, Human, Pair 8 ; Humans ; Myeloproliferative Disorders ; classification ; genetics ; pathology ; Receptor, Fibroblast Growth Factor, Type 1 ; genetics

This study was purposed to explore the application value of fluorescence in situ hybridization (FISH) detection in differential diagnosis of chronic myeloproliferative disorders (CMPD) and Ph(+) acute lymphoblastic leukemia (Ph(+) ALL), as well as in dynamic monitoring of minimal residual disease (MRD) after treatment. The BCR/ABL fusion gene of newly diagnosed and treated cases was detected by using BCR/ABL (ES) probe and BCR/ABL (DF) probe respectively. The results showed that among 49 newly diagnosed cases considered as CMPD, 28 cases met the criterion of CML morphologically, out of them 23 cases were eventually diagnosed to be CML and with morphological consistent rate 82.1% (23/28), the sensitivity and specificity all were 100% (23/23). The BCR/ABL positive rate of eventually diagnosed cases was 81.3% ± 17.7%. Among 13 cases received allogeneic haemopoietic stem cell transplantation (allo-HSCT), 9 cases achieved long-term disease-free survival and 4 cases relapsed, the several monitoring for whom after donor lymphocyte infusion (DLI) and imatinib treatment or allo-HSCT showed BCR/ABL negative. Among 16 cases treated with imatinib, 11 cases remained BCR/ABL negative after 1 year; 5 cases showed BCR/ABL positive during 6, 7 and 10 years after treatment, respectively, but out of them BCR/ABL positive in 1 case turned negative after allo-HSCT. It is concluded that the FISH is sensitive and specific diagnostic technique, the detection of BCR/ABL fusion gene in newly diagnosed and treated cases by using 2 different probes can help to fast and accurately determine the differential diagnosis for CML and Ph(+) ALL, and dynamically monitor the MRD after treatment with imatinib and allo-HSCT.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Diagnosis, Differential ; Female ; Fusion Proteins, bcr-abl ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Middle Aged ; Myeloproliferative Disorders ; diagnosis ; pathology ; Neoplasm, Residual ; pathology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; diagnosis ; pathology ; Retrospective Studies ; Sensitivity and Specificity ; Young Adult

No abstract available.
Adult ; Alleles ; Fusion Proteins, bcr-abl/*genetics ; Heterozygote ; Humans ; Hydroxyurea/*therapeutic use ; Janus Kinase 2/*genetics ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis ; Leukocytes, Mononuclear/pathology ; Leukocytosis/diagnosis ; Male ; Middle Aged ; Mutation ; Myeloproliferative Disorders/drug therapy/*genetics ; Phenotype ; Splenomegaly/diagnosis ; Thrombocytosis/diagnosis ; Translocation, Genetic

Animals ; Eosinophilia ; genetics ; pathology ; Gene Rearrangement ; Humans ; Lymphoma ; genetics ; pathology ; Myeloproliferative Disorders ; genetics ; pathology ; Receptor, Fibroblast Growth Factor, Type 1 ; genetics ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; Receptor, Platelet-Derived Growth Factor beta ; genetics

This study was aimed to investigate the therapeutic effect of two molecular targeted therapeutic drugs, tyrosine kinase inhibitors gefitinib and lapatinib, on JAK2 V617F positive myeloproliferative disorders (MPD). The human leukemia cell line (HEL cell line) carrying JAK2 V617F mutation was treated with gefitinib (0.5, 1, 5, 10, 25 µmol/L) and lapatinib (0.5, 1, 2, 4, 8, 16 µmol/L) respectively. MTT method was used to detect HEL cell proliferation. The apoptotic rate and cell cycle were measured by flow cytometry. The results showed that gefitinib could significantly inhibit the proliferation of HEL cells in a dose-dependent manner, it's correlation coefficients for 24 and 48 h were 0.991 and 0.895 respectively. IC(50) at 48 h was 5.4 µmol/L. Gefitinib could effectively induce apoptosis of HEL cells in a dose-dependent manner (r = 0.896). Otherwise, gefitinib could arrest HEL cells at G(0)/G(1) phase. The inhibitory effect of lapatinib was less than gefitinib, it's IC(50) of inhibiting proliferation of HEL cells was 19.6 µmol/L. It is concluded that both gefitinib and lapatinib can inhibit the proliferation of HEL cells. These two tyrosine kinase inhibitors can be used for researching of targeted therapy of JAK2 V617 positive MPD.
Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Janus Kinase 2 ; genetics ; Mutation ; Myeloproliferative Disorders ; metabolism ; pathology ; Protein Kinase Inhibitors ; pharmacology ; Quinazolines ; pharmacology