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Objective: To investigate the frequency and mutational status of JAK2V617F mutation in Chinese patients with chronic myeloproliferative disorders (CMPD) and to study the relative quantification of mutated JAK2 mRNA and the clinical significance., Methods: JAK2V617F mutation and the mutational status were screened with amplification-refractory mutation system polymerase chain reaction (ARMS-PCR), the relative quantification of mutated JAK2 mRNA was studied by using capillary electrophoresis., Results: A higher prevalence of JAK2V617F in either the heterozygote or homozygote status in essential thrombocythemia (ET) was observed in elderly patients with ET (P < 0.05). The presence of JAK2V617F was found to be significantly correlated with the age at diagnosis (P < 0.05); patients with age > or = 60 years showed significantly higher JAK2 mutated RNA levels than those with age < 60 years (P < 0.05); the presence of JAK2V617F in polycythemia vera (PV) and ET was found to be significantly associated with higher hemoglobin level and higher leukocyte count (P < 0.05). In addition, higher leukocyte count was observed in homozygous ET patients than in heterozygous ET patients (P < 0.05). The frequency of JAK2V617F mutation and the prevalence of homozygote in PV patients were higher than those in ET patients (P < 0.05). The differences of JAK2V617F mRNA levels among PV, ET and chronic idiopathic myelofibrosis (IMF) were not significant., Conclusions: ARMS-PCR technique can be used to detect the frequency and mutational status of JAK2V617F mutation owing to its sensitivity and along with capillary electrophoresis, quantitative assay for mutated JAK2 mRNA, diagnosis of CMPD and judgement of prognosis become possible.

Objective: To study T-cell reconstitution by the assessment of T-cell receptor excision circle (TRECs) and T-cell receptor clonal repertoire after HLA-matched sibling bone marrow transplantation (MSD-BMT) in leukemia patients and try to reveal the rule of T-cells repopulation in MSD-BMT., Methods: Real-time quantitative PCR detection of TRECs was carried out in the DNA of peripheral blood mononuclear cells from 23 leukemia patients who underwent MSD-BMT. The content of TRECs in 70 normal donor individuals was also detected to determine the normal range of TRECs in healthy subjects. Among them, 10 patients received RT-PCR to amplify 24 subfamily genes of T-cell receptor B variable (TCRBV) and five normal donors served as control. The PCR products were further analyzed with genescan to evaluate the clonality of BV subfamily and calculate the usage rate of BV families., Results: The mean value of TRECs in normal donors was (3351.1 +/- 3711.1) copies/10(5) cells. There was an inverse correlation between the value of TRECs and the age of healthy subjects. Before transplantation, all the patients were detected for the number of TRECs, the mean TRECs number was (307.9 +/- 433.3) copies/10(5) cells and it was far lower than that of healthy subjects. From one month to five months after bone marrow transplantative (BMT), the TRECs levels were low and in some patients they even could not be detected. Six months later, TRECs levels increased obviously and maintained that high nearly one year. 24 months after BMT, the number of TRECs increased and reached the pretransplantation status. One patient was detected 2 m and 3 m after transplantation and found that there was a tendency of additional use of BV families and an increase of the expression of CDR3 polymorphism. 2-15 months after transplantation, in ten of the patients, there were 7-16 BV subfamilies expressing and in more than 48% the expression was polyclinic. Monoclones and oligoclones existed in 24 BV subfamilies., Conclusions: At an early stage after BMT, the number of TRECs was low and remained so for a long time. TCRBV CDR3 repertoire showed certain BV families expressing. 6 months after BMT, thymus produced certain number of naive T cells and TCRBV CDR3 repertoire showed additional use of BV families and increased expression of polymorphism.

Objective: To investigate the frequency and clinical implication of JAK2V617F mutation in Chinese patients with myeloproliferative disorders (MPD)., Methods: Genomic DNA from bone marrow or blood mononuclear cells of 137 cases of MPD was screened with allele-specific polymerase chain reaction (PCR) and JAK2V617F mutation was detected with gel electrophoresis. There were 57 cases with polycythemia vera (PV), 68 with essential thrombocythemia (ET), 12 with idiopathic myelofibrosis (IMF)., Result: JAK2V617F mutation was detected in 42 (73.7%) of the 57 patients with PV, 40 (58.8%) of the 68 with ET and 8 (66.7%) of the 12 with IMF. Sequence analysis of PCR products from selected patients confirmed the coexistence of both mutant and wild-type alleles. A higher prevalence was observed in elderly patients with MPD (P < 0.05). Cytogenetic analysis was performed in 115 of the 137 patients. Among the 108 patients with normal karyotype, JAK2V617F mutation was detected in 74 patients (68.5%) as compared with 5 of the 7 patients with karyotypic abnormalities (71.4%)., Conclusion: JAK2V617F mutation occurs in a significant percentage of Chinese patients with the myeloproliferative disorders. There is a higher prevalence in elderly patients.

Objective: To investigate the clinical significance of Wilms tumor gene (WT1) expression levels in the bone marrow (BM) of chronic myelogenous leukemia patients., Methods: Real-time quantitative retroversion polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1 and GAPDH expression levels in the BM of 109 CML and 23 non-leukemic patients with Light Cycler. Normalized WT1 expression level (WT1(N)) was determined as a ratio between WT1 and GAPDH expression times 10(4)., Results: The median expression levels of WT1(N) in 23 CML patients of accelerate phase (CML-AP) and 22 CML patients of blast crisis (CML-BC) were statistically higher than those of 64 CML patients of chronic phase (CML-CP) and 23 non-leukemic controls (103.71 and 129.44 vs 3.44 and 1.47). No statistic differences were found between the CML-CP group and control group, nor between the CML-AP group and CML-BC group. Furthermore, the WT1(N) levels were correlated to the BCR/ABL fusion gene expression levels. Dynamic change of WT1(N) levels in 7 CML patients before or after allogeneic bone marrow transplantation showed that WT1 expression levels could predict relapse of leukemia., Conclusion: WT1 expression levels in CML patients in accelerate phase or blast crisis were strikingly higher than those in non-leukemic patients or CML patients in chronic phase, in whom WT1 expression was undetectable or showed low expression. WT1 gene could be an useful marker for detecting MRD and evaluating therapeutic efficacy in CML.

Objective: To study the expression of gelatinase A (MMP2) in acute leukemic cells and its clinical implication., Methods: The bone marrow samples from 46 acute leukemia patients were investigated by reverse transcription polymerase chain reaction (RT-PCR) and Gelatin Zymography method. Matrigel invasion assay was studied on U937, NB4, K562 and SHI1 cells in vitro., Results: The expression of MMP2 was positive in 24 of 46 patients with AL (52.2%). In MMP2 positive and negative groups of AL, the extramedullary infiltration rates were 50.0% and 18.2% (P < 0.05), whilst the percentage of blast cell in peripheral white blood cells were (77.21 +/- 13.9)% and (62.95 +/- 17.2)% (P < 0.01), respectively. The positive expression of TIMP2 and MT1-MMP in 46 AL patients were 27 (58.7%) and 33 (71.7)%. The matrigel invasion assay suggested that MMP2 is involved in migration of leukemic cell through extracellular matrix (Matrigel)., Conclusion: The active form of MMP2 might be essential to the egress of leukemic cells from bone marrow into peripheral blood, followed by an extramedullary infiltration.