Interferon-alpha-2b induces molecular responses of patients with polycythemia vera and its post-polycythemic myelofibrosis / 中国实验血液学杂志

To evaluate the efficacy and safety of interferon-alpha-2b (IFN-α-2b) in polycythemia vera patients(PV patient) with or without post-polycythemic myelofibrosis (post-PV MF), 30 patients with mutated JAK2V617F were enrolled in this study, from which 29 patients were evaluable. The percentage of mutated JAK2V617F allele (V617F%) was evaluated by real-time polymerase chain reaction (RT-PCR) before and after treatment with IFN-α-2b. The correlation of V617F allele burden with the major clinical outcomes was studied. Adverse effects appeared in patients was observed. The results showed that the median follow-up was 24 (12 - 42) months for 29 evaluable patients. Complete hematologic response was achieved in 10%, 48%, 72% and 78% of patients after treatment for 6, 12, 24 and 36 months respectively. The detection of V617F allele burden revealed that the molecular remission of patients (V617F%) was achieved in 41%, 76%, 89% and 89% after treatment for 6, 12, 24 and 36 months respectively. Molecular complete remission (JAK2V617F undetectable) was achieved in 4 patients, lasted from 6 to 12 months after IFN-α-2b discontinuation. The decrease of V617F% in patients with post-PV MF was significantly higher than that in patients without post-PV MF (53 ± 18% vs 32 ± 22%, respectively; p = 0.031) after treatment for 12 months. PV patients had a good tolerance to IFN-α-2b. It is concluded that IFN-α-2b can decrease the mutated V617F allele burden. Patients with PV, especially with post-PV MF, can achieve molecular remission after treatment with IFN-α-2b.

Relation of HLA-DRB1*15 with pathogenesis in 162 childhood cases of acute lymphoblastic leukemia / 中国实验血液学杂志

To unravel the relation of HLA-DRB1*15 with childhood acute lymphoblastic leukemia (ALL), 162 childhood patients with ALL were selected for this investigation. 1 000 normal umbilical cord blood samples were used as control.HLA-DRB1*15 and HLA-DRB5* were typed by polymerase chain reaction (PCR) analysis. The relation of HLA-DRB1*15 with childhood ALL was studied by calculating the chi-square test and relative risk. The results showed that the antigen frequencies and allele frequencies of HLA-DRB1*15 in childhood patients with ALL were 40.12% and 22.62% respectively, while the antigen frequencies and allele frequencies of HLA-DRB1*15 in control were 30.80% and 16.81% respectively, there were significant difference between them (chi(2) = 5.560, p = 0.018, RR = 1.506). In conclusion, the antigen frequencies and allele frequencies of HLA-DRB1*15 in childhood patients with ALL were higher than those in control, so the HLA-DRB1*15 gene is one of the genetic risk factors for childhood ALL. These preliminary data may be useful for further study on the pathogenesis of childhood ALL.

Studying of clinical and laboratory features of chronic eosinophilic leukemias /hypereosinophilic syndrome / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical and laboratory features of chronic eosinophilic leukemias (CEL) and hypereosinophilic syndrome (HES).</p><p><b>METHODS</b>The clinical manifestations, laboratory parameters were retrospectively analyzed in 20 patients with HES/CEL. Detection of the FIP1L1-PDGFRA fusion gene was performed by nested RT-PCR. JAK2 V617F mutation screening was processed through allele-specific PCR combined with sequence analysis. PCR-RFLP was used to discriminate homozygous from heterozygous mutation patterns. TCR gamma rearrangement was detected by PCR.</p><p><b>RESULTS</b>Of the 20 patients, 19 were males and one female, with a median age of 33 (20 to 57) years. The FIP1L1-PDGFRA fusion gene positivity in bone marrow mononuclear cells in 12 cases was identified. All the breakpoints were identified by direct sequencing of cloned RT-PCR products in FIP1L1 intron 10 - 12 and in PDGFRA exon 12. In CEL the most common involved organs were lungs, heart and nervous system. Splenomegaly was significantly more frequent in CEL than in HES (92.5% vs 42.5%, P = 0.031). Anemia and myelofibrosis were common in CEL. There was no significant difference in circulating absolute eosinophil, leukocyte, platelet counts, hemoglobin level and percentages of eosinophil and blast cell in bone marrow between CEL and HES. The morphological abnormalities of eosinophils on bone marrow smear were easily found in CEL, including hypogranularity, and cytoplasmic vacuolization, increased basophilic granule. One patient with HES was found to have heterozygous JAK2 V617F mutation. Six patients had TCR gamma rearrangement, including 4 CEL and 2 HES.</p><p><b>CONCLUSIONS</b>(1) There is a male predominance in HES/CEL, and the median age was in the thirties. (2) The most common involved organs in CEL were lung, heart and nervous system. Bone marrow morphology might be of a little help in diagnosis of CEL. (3) JAK2 V617F may be involved in the pathogenesis of HES. (4) Patients with CEL carried the FIP1L1-PDGFRA fusion gene and TCR gamma rearrangement concurrently, their relationship warrants further study.</p>

Clinical study on relationship between JAK2 V617F mutation and chronic myeloproliferative disorders / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate JAK2V617F mutation and its clinical significance in patients with chronic myeloproliferative disorders (cMPD).</p><p><b>METHODS</b>A retrospective study was performed on 523 cMPD patients diagnosed according to the current World Health Organization (WHO) criteria. Allele-specific PCR (ASP) was used to identify JAK2V617F mutation, the mutation status was analyzed by PCR-RFLP, and the results were confirmed by sequence analysis. The mutation burden was calculated by the ratio of T/G. The correlation between the allele burden and the clinical and hematologic features was analysed. For those without JAK2 V617F, MPL W515L mutation was analyzed.</p><p><b>RESULTS</b>JAK2 V617F was detected in 66% of all patients (94% in PV, 80% in ET, 78% in CIMF, 75% in CMPD-U and 14% in HES). The majority of patients carried JAK2 V617F mutation were heterozygous , homozygote was found in only 5 cases (4 in PV and 1 in ET). The mutation burden in most patients (71.5%) was low with PV>ET>CIMF (P =0.003). Hemoglobin level was significantly related to high mutation burden in PV (r = 0. 203, P =0.033). Bone marrow megakaryocyte counts were found to be marked increased in ET with high JAK2 V617F loads (P = 0.024), and hepatomegaly in CIMF was significantly associated with high JAK2 V617F mutation burden (r = 0.315, P = 0.001).</p><p><b>CONCLUSIONS</b>1) Most cMPD patients, especially those with PV, carry JAK2 V617F mutation, except for CML. 2) .98% of JAK2 V617F mutation occurs of heterozygous status. 3) The mutation burden is PV>FT>CIMF. High JAK2 V617F loads are significantly associated with higher hemoglobin level in PV and higher bone marrow megakaryocyte counts in ET. 4) The positive correlation between hepatomegaly and JAK2 V617F mutation burden is found in CIMF.</p>

Long-term therapeutic outcome of patients with acute promyelocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the long-term therapeutic outcome of patients with acute promyelocytic leukemia(APL).</p><p><b>METHODS</b>Newly diagnosed APL patients were treated with ATRA as induction therapy followed by 3-4 courses of combined consolidation chemotherapy and 2 year maintenance therapy with ATRA and 6-MP + methrotrexate, alternatively. Patients were regularly monitored with nested RT-PCR for PML-RARalpha fusion transcript at the end of consolidation chemotherapy and in the following 4 to 5 years.</p><p><b>RESULTS</b>A total of 81 patients with APL were entered the trial, 75 (92.6%) patients achieved CR. Early death (ED) rate was 6.6%. ED patients had significantly higher WBC count and higher percentage of peripheral promyelocyte than those achieved CR. Of 65 patients received consolidation, 60 (92.3%) were proved PML-RARalpha fusion gene negative at the end of the 3rd courses and 3 (4.6%) the end of the 4th courses of consolidation. The mean follow-up was 21.2 (8-64) months, 6 patients relapsed (relapse rate 9.2%). The 5-year Kaplan-Meier estimates of overall survival (OS) rate was (86.6 +/- 4.6)%. For 65 patients received consolidation therapy, the 5-year relapse-free survival (RFS) rate was 82.7%. COX-regression analyses showed only high WBC count (>10 x 10(9)/L) had an adverse prognostic influence on OS.</p><p><b>CONCLUSION</b>More than 80% of APL patients treated with systemic therapy could experience long-term relapse-free survival.</p>

Monitoring IgH levels in patients with B-cell malignancy by real-time quantitative PCR after hematopoietic stem cell transplantation and its significance / 中国实验血液学杂志

The study was purpose to evaluate the value of real time quantitative-PCR for monitoring IgH level in patients with B-cell malignancy after hematopoietic stem cell transplantation (HSCT). Quantification of IgH levels was performed on bone marrow mononuclear cells from 9 patients with B-cell malignancy before and after HSCT by PCR using the consensus JH TaqMan probe in combination with an allele-specific oligonucleotide (ASO) upstream primer. The IgH levels was normalized by control gene GAPDH. The results indicated that the reproducible sensitivity of RQ-PCR was 1 copy, the significant reduction of IgH copies was observed in bone marrow samples of 9 patients at one month post HSCT (6.67x10(3)/10(6) GAPDH vs 29/10(6) GAPDH, p<0.01). 3 out of 9 patients who achieved complete clinical and molecular cytogenetic remission (CCyR) contained persistently measurable low IgH level of 10(2)/10(6) GAPDH within 15 months and no detectable IgH at 18 months post HSCT. Whereas 5 out of 9 patients whose IgH copies were less than 10(2)/10(6) GAPDH within 3 months and less than 10(3)/10(6) GAPDH 3 months post HSCT achieved a sustained complete remission (CR). IgH copies in one patient were 4.5x10(3)/10(6) GAPDH at 3 months post HSCT, who relapsed at 4 months post HSCT. The median levels of tumor contamination in the stem cell harvests from 8 patients measured by RQ PCR were 3.68x10(2) (0-1720)/10(6) GAPDH. RQ PCR showed that PBPC harvests were less contaminated than BM harvests [75 (0-890)/10(6) GAPDH vs 1.1x10(3) (527-1720)/10(6) GAPDH, p<0.05]. 8 patients whose stem cell harvest were avaiable for RQ PCR were still in CR despite of the tumor contamination. The level of tumor contamination in stem cell harvest well correlated with IgH levels at diagnosis and one month after HSCT (r=0.810, r=0.708, p<0.05). It is concluded that RQ PCR can effectively monitor the IgH levels in patients with B-cell malignancy after auto-HSCT. 10(3)/10(6) GAPDH within 3 months post HSCT may be a cut-off level of IgH copies, which may be used to evaluate different prognoses of patients.

Detection of CBFbeta/MYH11 fusion transcripts and study of the mechanism of leukemogenesis of CBFbeta/SMHHC fusion protein / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore CBFbeta/MYH11 fusion transcripts and its expressing product CBFbeta/SMHHC fusion protein in mechanism of leukemogenesis.</p><p><b>METHODS</b>CBFbeta/MYH11 fusion transcripts were detected by combined RT-PCR with sequencing. Transcription assays were examined using pM-CSFR-Luc as reporting plasmid, and subcellular localization of encoding proteins were assayed by double immunofluorescent staining and Western blot.</p><p><b>RESULTS</b>Two types of CBFbeta/MYH11 fusion transcripts were found in 26 patients with acute leukemia, most being of type A (23/26 cases, 92%) and a few of type D (2/26 cases, 8%). The inhibition of CBF-mediated M-CSFR promotor transactivation by CBFbeta/SMHHC fusion protein was increasing with the increase in amount of the fusion protein. CBFalpha subunit (AML1) located in nucleus, both CBFbeta subunit (CBFbeta) and CBFbeta/SMHHC located in cytoplasm. When AML1 and CBFbeta were coexpressed, CBFbeta still located mainly in cytoplasm, but when AML1 and CBFbeta/SMHHC were coexpressed, CBFbeta/SMHHC located mainly in nucleus.</p><p><b>CONCLUSIONS</b>(1) The types of CBFbeta/MYH11 fusion transcripts of Chinese leukemia patients are almost the same as that reported in western literature. (2) CBFbeta/SMHHC inhibits CBF-mediated transactivation through competing with CBFbeta for binding to AML1.</p>

In vitro expansion of cord blood megakaryocyte progenitor / 中国医学科学院学报

<p><b>OBJECTIVE</b>To expand cord blood megakaryocyte progenitor cells in vitro.</p><p><b>METHODS</b>Cord blood CD34+ cells were selected by magnetic cell sorting (MACS), and thrombopoietin (TPO), interleukin-11 (IL-11), and heparin were used in the expansion system of megakaryocyte progenitor. The expansion efficiency was measured by fluorescence-activated cell sorting (FACS) using the megakaryocytic specific monoclonal antibodies (CD34+, CD41a+, CD61+, CD34+CD41a+, CD41a+CD61+) and colony-forming units-megakaryocyte (CFU-MK) analysis. The expanded megakaryocyte progenitor were determined by histochemistry staining using CD41a and the observation of the ultrastructure of megakaryocyte (MK) by electron microscopy. The megakaryocyte function were examined by the platelet activation in vitro and nonobese diabetic/severe combined immunodifficiency (NOD/SCID) mice transplantation in vivo.</p><p><b>RESULTS</b>CD34+CD41a+ cells was expanded (4.0 +/- 1.7) folds on day 7 in TPO (50 ng/ml) group and (10.5 +/- 4.8) fold in TPO combined with IL-11 group; after heparin was joined in on day 0, a more significantly elevated expansion was found in the heparin, TPO, and IL-11 group [(29.9 +/- 6.4) folds than the above two groups; P < 0.05]. Meanwhile, the large CFU-MK colony (> 50 cells/colony) was (106.8 +/- 26.9) folds on day 7 (P < 0.05). The megakaryocyte expanding with TPO, IL-11 and heparin for 7 days in vitro transplanted the NOD/SCID mice fasten the recovery of platelet and white blood cell account and improved the survival. Megakaryocyte under culture displayed certain development of territories membrane. Platelet activation test comfirmed that the expanding megakaryocyte progenitor had the normal function.</p><p><b>CONCLUSION</b>TPO, IL-11, and heparin combination system for ex vivo expansion is an effective expansion system of megakaryocyte progenitor.</p>

Activation of auto-mesenchymal stem cells of skeletal muscle by bone morphogenetic protein for rescuing bone marrow failure / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the effect of bone morphogenetic protein (BMP) to activate mesenchymal stem cells of skeletal muscle for rescuing bone marrow failure.</p><p><b>METHODS</b>The study was performed on lethal rat acute aplastic anemia model induced by combined 5-fluorouracil (5-FU) and busulfan. The rh-BMP-2 was implanted into the thigh muscle of the rats at 3 days before aplastic anemia was induced. In the control group the rats were implanted with agar into the thigh muscle. The blood picture, pathologic changes and the mortality in two groups were observed. At the same time, rh-BMP-2 were implanted into the thigh muscle of normal Kun-min mice for dynamic control observation of the implantation local morphological changes, colony forming units-spleen (CFU-S) and stem cell growth factor (SCF) expression of the stroma cells of ectopic ossicles induced by BMP.</p><p><b>RESULTS</b>At 7 days after BMP implantation in the mice the mesenchymal cells around BMP in muscle proliferated, and appeared in bone marrow to form an ectopic ossicles. The SCF expression of stroma cells in ectopic ossicles were higher than that of self-bone marrow. 56.3% of BMP-treated aplastic rats were survived over 3 months and its hematopoiesis was completely reconstituted and the histo-morphological picture of the spleen and bone marrow were recovered to normal. But in the control group only one of 23 rats was survived, the remainder died of hematopoietic failure.</p><p><b>CONCLUSIONS</b>BMP-implantation into the skeletal muscle could rescue the bone marrow hematopoietic failure. The mechanism might be related to the BMP activated auto-mesenchymal cells of skeletal muscles to direct hematopoietic cell differentiation. In our hands it might create a new pathway for utilization of auto-muscle derived mesenchymal cells to reconstitute hematopoiesis.</p>