Expressions of BAP1 and TET2 proteins in bone marrow of patients with chronic myelomonocytic leukemia and their significances / 白血病·淋巴瘤

Objective To investigate the expressions of BAP1 and TET2 proteins in bone marrow of patients with chronic myelomonocytic leukemia (CMML) and their relationship with the prognosis of CMML. Methods The bone marrow paraffin specimens of 41 cases from 41 adult CMML patients diagnosed by Shanghai Sino-US Joint Leukemia Coordination Group from September 2003 to May 2007 were collected. The immunohistochemistry was used to detect the expressions of BAP1 and TET2 proteins in 41 CMML patients. The expressions of TET2 and BAP1 proteins were also detected by the same method in 40 adult patients with acute myelomonocytic leukemia (AMML) and 20 patients with iron deficiency anemia (IDA) diagnosed at the same time as the comparison. The clinical data of 41 CMML patients were analyzed by using retrospective cohort study. The count data were compared by using chi-square test. The correlation between expressions of BAP1 and TET2 proteins was analyzed by using Pearson correlation analysis. Kaplan-Meier method was used to calculate the survival time, and Log-rank test was used for single factor analysis. Results In 41 CMML patients, the positive expression rate of BAP1 was 31.7％ (13/41), including 28.6％ (8/28) in CMML-1 patients and 38.5％ (5/13) in CMML-2 patients; the positive expression rate of TET2 was 41.5％ (17/41), including 39.3％ (11/28) in CMML-1 patients and 46.2％ (6/13) in CMML-2 patients. In 40 AMML patients, the positive expression rate of BAP1 was 32.5％ (13/40), and the positive expression rate of TET2 was 35.0％ (14/40). In 20 IDA patients, the positive expression rate of BAP1 was 5.0％ (1/20), and TET2 had no positive expression. There was no significant difference in the expressions of BAP1 and TET2 proteins between CMML-1 and CMML-2 patients (χ 2 = 0.40, P = 0.53; χ 2 = 0.17, P = 0.68). There was no significant difference in the expressions of BAP1 and TET2 proteins between CMML and AMML patients (χ 2 = 0.01, P = 0.94; χ 2 = 0.36, P = 0.64). There were significant differences in the positive expression rate of BAP1 and TET2 proteins between hematological neoplastic disease (CMML+AMML) and hematological non-neoplastic disease (IDA) (χ 2 = 6.01, P < 0.05; χ 2 = 11.04, P < 0.01). Pearson correlation analysis showed that there was no correlation between expressions of BAP1 and TET2 proteins (r = 0.35, P = 0.27). Univariate analysis showed that anemia (Hb < 60 g/L), mature monocyte count ≥ 2.0×109/L, neutrophil count (1.5×109/L), abnormal karyotype were associated with poor prognosis for CMML. Protein expressions of BAP1 and TET2 were not related with the prognosis of CMML (χ 2 = 0.28, P = 0.600; χ 2 = 0.53, P = 0.460). Conclusion Both BAP1 and TET2 proteins have high positive expression rates in CMML patients, but the expressions of them are not related to the prognosis.

Déficit adquirido de factor X en leucemia mielomonocítica crónica: reporte de un caso / Isolated factor X deficiency in chronic myelomonocytic leukemia: Report of one case

Bleeding disorders are commonly associated with hemato-oncologic diseases. We report a 68 years old male with a chronic myelomonocytic leukemia derived from a long lasting mielodysplastic syndrome that did not respond to treatment with Azacitidine. The patient was hospitalized due to tonic clonic seizures. A CAT scan showed a hematoma in the frontal lobe. A new assessment of hemostasis revealed an isolated deficiency of Factor X. We speculate that this deficit could be secondary to consumption due to the chronic Myelomonocytic Leukemia.

Construction of eukaryotic expression vectors of FGFR3 gene and their expressions in human leukemia K562 cell line / 吉林大学学报(医学版)

Objective To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3) MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN, and to detect their expressions in human chronic myeloid leukemia(CML)K562 cell line.Methods The full-length FGFR3 (fgfr3-WT)and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR). The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry. Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method, and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared with control (K562 MSCV)group,the expression level of FGFR3-WT in MSCV/puro-fgfr3-WT transfection (K562-WT)group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN transfection (K562-DN)group,but there was no expressions in control(K562 MSCV)group and K562-WT group.The flow cytometry results showed that the high expressions of FGFR3-WT were in 57.5% cells in K562-DN group and the high expressions of FGFR3-DN were in 41.5% cells in K562-DN group. Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.

Recent advances on diagnosis and therapy of chronic myelomonocytic leukemia / 白血病·淋巴瘤

Chronic myelomonocytic leukemia (CMML) is a rare malignant disorder of hematopoietic system.It is classified by WHO as MDS/MPN and many gene mutations are invovled such as RAS,TET2 and CBL.Hypomethylating drugs are effective in the management of CMML and may alter the prognosis.Allogeneic stem cell transplantation is still the only way to cure the disorder.

A clinical and laboratory investigation of myelodysplastic/myeloproliferative neoplasms / 中华检验医学杂志

ObjectiveTo investigate distingwished clinical and experimental characteristics of the four main subtypes in myelodysplastic/myeloproliferative neoplasms (MDS/MPNs).MethodsMDS/MPNs 53 cases from Provincial Hospital Affiliated to Shandong University,including 24 cases CMML,13 cases aCML,12 cases JMML,4 cases MDS/MPN-U,were analyzed regarding to 2001 WHO classification.Morphology (M) of peripheral and bone marrow blood cells were observed under microscope.FCM was used in immunological(Ⅰ) analyse on blasts and myelomonocytes in peripheral blood and bone marrow.G-banding technique was used in cytogenetic (C)examination.PCR was used in molecular genetic (M) mutation detection.Numeric data,such as mean Hb,WBC,PLT et al,among several groups,were compared using Single-factor analysisof variance.Student-Newman-Keulstestwasuseincomparingmeansof two groups.Proportions,such as percentage of clinical features,immunological and cytogenetic abnormal cases among different groups,were compared using Chi-square test or Fisher exact test.Results( 1 ) In the course of MDS/MPNs,there were 46 cases (86.8％ ) had paleness and fatigue 33 cases (62.5％ ) had palpable spleen.JMML had most fever and enlargement of lymph node (75.0％,75.0％ ),statistically distinguished from CMML ( 12.5％,12.5％ ) (x2 =14.89,17.98,P ＜ 0.05 ).(2) The hemoglobin was ( 83.1 ± 24.6 ) g/L.WBC counts were ( 19.8 ± 8.1 ) × 109/L.PLT counts were ( 158.7 ± 108.2) x 109/L.Immature neutrophils and blasts were found in peripheral blood.(3)JMML and CMML had most monocytes absolute counts among the subtypes (4.25 ±0.76) (3.62 ±0.76).(4) Almost 100％ JMML had monocytes abnormalities.(5)For 15 cases were detected immunological characteres by FCM,13 cases showed abnormalities.(6)For 29 cases of MDS/MPNs had been analyzed chromosome karyotypes and 12 out of them (41.4％) were abnormal,Ph chromosomes and those AML-defining translocations were all negative,+ 8 and 7-involved- karyotypes were more frequent.(7)23 cases were detected molecular genetic features,in which were all negative.BCR/ABL1 and JAK2 V617F mutation were all negative in the 13 cases of aCML.JAK2 V617F mutation was positive in 1 case of MDS/MPN-U.ConclusionsMost MDS/MPNs had paleness and fatigue,light to mild anemia,cytosis,monocytes low grade of blast and immature neutrophils in peripheral blood with dysplasia in bone marrow.JMML seems has more severe clinical features and more distinguishing laboratory characters.Immunological abnormalities and abnormalkaryotypes are found frequently in MDS/MPNs with no statistical differences among the four subtypes.There is no specific molecular abnormals in MDS/MPNs.( Chin J Lab Med,2012,35:832-837)

Laboratory diagnosis of chronic myelomonocytic leukemia and progression to acute leukemia in association with chronic lymphocytic leukemia: morphological features and immunophenotypic profile

Chronic myelomonocytic leukemia is a clonal stem cell disorder that is characterized mainly by absolute peripheral monocytosis. This disease can present myeloproliferative and myelodysplastic characteristics. According to the classification established by the World Health Organization, chronic myelomonocytic leukemia is inserted in a group of myeloproliferative/myelodysplastic disorders; its diagnosis requires the presence of persistent monocytosis and dysplasia involving one or more myeloid cell lineages. Furthermore, there should be an absence of the Philadelphia chromosome and the BCR/ABL fusion gene and less than 20% blasts in the blood or bone marrow. Phenotypically, the cells in chronic myelomonocytic leukemia can present myelomonocytic antigens, such as CD33 and CD13, overexpressions of CD56 and CD2 and variable expressions of HLA-DR, CD36, CD14, CD15, CD68 and CD64. The increase in the CD34 expression may be associated with a transformation into acute leukemia. Cytogenetic alterations are frequent in chronic myelomonocytic leukemia, and molecular mutations such as NRAS have been identified. The present article reports on a case of chronic myelomonocytic leukemia, diagnosed by morphologic and phenotypical findings that, despite having been suggestive of acute monocytic leukemia, were differentiated through a detailed analysis of cell morphology. Furthermore, typical cells of chronic lymphocytic leukemia were found, making this a rare finding.

Chronic myelomonocytic leukemia complicated with Sweet 's syndrome developed into acute myelomonocytic leukemia: a case report and review of the literature / 白血病·淋巴瘤

ObjectiveTo improve the understanding of chronic myelomonocytic leukemia associated with Sweet' s syndrome.MethodsRetrospective analysis of a case of chronic myelomonocytic leukemia associated with skin herpes was reported. Skin biopsy was performed. DA and CAG regiment were administrated.ResultsSweet's syndrome was diagnosed by skin biopsy.Corticosteroids therapy alone was not effective. Complete remission was achieved by CAG regiment and skin rash has been effectively controlled.Three months later Sweet' s syndrome relapsed and chronic myelomonocytic leukemia developed into acute myelomonocytic leukemia. ConclusionChronic myelomonocytic leukemia associated with Sweet's syndrome is rare but implies a quick progression to acute myelomonocytic leukemia.

Genetic diagnosis of a patient with non-syndromic variants of congenital neutropenia / 中华内科杂志

ObjectiveTo explore the procedures and methods for genetic diagnosis in one nonsyndromic variants of congenital neutropenia (NSVCN) patient and its pathogenic mutation.Methods Genomic DNA was prepared from one NSVCN patient who had progressed to chronic myelomonocytic leukemia and ELA2,HAX1,WASp and GFI1 genes were amplified and sequenced.Results A novel compound heterogeneous mutation consisting of two frame-shift mutations (c.430-1insG and c.655- 9del5bp) was found in HAX1 gene.ConclusionA practically genetic diagnosis procedure for NSVCN has been established,and the novel HAX1 gene mutation may contribute to the etiology of NSVCN.

Effects of imatinib on the expression of SHIP gene and apoptosis of K562 cells / 肿瘤

Objective: To observe the changes of SHIP, caspase-1, caspase-3, caspase-9 and bcl-2 gene expression levels in K562 cells after mesylate imatinib (MI) treatment and explore the possible mechanism for apoptosis-inducing effects of imatinib. Methods: K562 cells were cultured with MI at different concentrations. The cells were collected at different time points. Real-time quantitative PCR was used to detect the expression level of SHIP gene. Semi-quantitative reverse transcriptase PCR was used to detect the mRNA transcription of anti-apoptotic gene bcl-2 and pro-apoptotic genes caspase-1, caspase-3 and caspase-9. The cell proliferation was measured by MTT assay. The cell apoptosis was analyzed by Annexin V/PI double staining flow cytometry. Results: MI significantly increased the expression of SHIP gene in a time-and dose-dependent manner. Caspase-9 gene was also up-regulated after MI treatment and had linear correlation with SHIP gene expression. The expression levels of caspase-1, caspase-3 and bcl-2 had no significant changes. MI down-regulated the proliferation and induced the apoptosis of K562 cells. The morphological changes of typical of apoptosis were observed. Conclusion: MI significantly increases the expressions of SHIP gene and caspase-9 gene and induces apoptosis of K562 cells. The mechanism for the apoptosis-inducing effects of imatinib may be associated with the up-regulation of SHIP and caspase-9 gene.