Effects of Leukapheresis on Hemostatic Function in Patients with Hyperleukocytic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To analyze and compare the effects of leukapheresis on hemostatic function in patients with hyperleukocytic leukemia.@*METHODS@#A total of 139 patients with AML, ALL and CML who underwent leukapheresis from June 2009 to February 2020 and did coagulation test before and after operation were included in this study. The clearance efficiency of each group and the difference among three groups were evaluated, as well as hemostatic function including platelet counts, coagulation indicators, CDSS score and incidence of adverse events. The difference of hemostatic function caused by leukapheresis in different leukemia patients were compared.@*RESULTS@#After leukapheresis, the WBC counts were decreased significantly in the three groups of patients (P<0.001), and the clearance efficiency was highest in ALL patients. However, the platelet counts also were decreased significantly (AML:P＜0.001, ALL: P＜0.001, CML: P＜0.01) in the three groups of patients, particularly for acute leukemia patients with a positive correlation with WBC clearance efficiency(r=0.284). After leukapheresis, fibrinogen decreased, PT and APTT prolonged. For acute leukemia patients, higher CDSS score was related to an elevated incidence of bleeding events (P<0.05).@*CONCLUSION@#Leukapheresis is an effective method to decrease the leukemic burden, but it is necessary to monitor the impact on hemostatic function. It is recommended to assess the CDSS socre for acute leukemia patients, in order to identify the predictive value for bleedings.

Comparison of the efficacy of decitabine combined with micro-transplantation or priming regimen as consolidation treatment for older patients with acute myeloid leukemia / 中华血液学杂志

Objective: To probe the feasibility of decitabine (DAC) combined with micro-transplantation as consolidation treatment for older patients with acute myeloid leukemia (AML). Methods: Between November 2012 and September 2015, 37 consecutive patients with AML ≥60 years of age were analyzed. Of them, 19 patients received consolidation therapy with DAC followed by micro-transplantation (microtransplant group). Another 18 ones (chemo group) were treated with DAC plus priming regimen as consolidation chemotherapy in the same period. Results: There were no significant differences in terms of age, WBC count, and disease status of onset between the microtransplant and chemo groups (P>0.05). The two regimens were well tolerated. There was no difference of CTC grade 3-4 nonhematologic toxicities between the microtransplant and chemo groups (36.8% vs 27.8%, χ(2)=0.347, P=0.728). The median recovery durations for neutrophil and platelet in the microtransplant group were similar to those in the chemo group (12 vs 13 days, z=1.599, P=0.110; 14 vs 12 days, z=-1.314, P=0.189, respectively). No graft-versus-host disease was observed in the microtransplant group. The 2-year leukemia-free survival and overall survival were better in microtransplant group (50.7% and 54.9%, respectively) than in chemo group (24.3% and 30.0%, respectively) (P=0.047 and P=0.071, respectively). Conclusion: DAC combined with micro-transplantation as a consolidation regimen may be a safe and promising option for older patients with AML.

Ex vivo inducing cultured Epstein-Barr virus specific cytotoxic T lymphocytes and evaluation of their killing effect / 中国实验血液学杂志

This study was aimed to explore the method for induction and expansion of EB virus specific cytotoxic T lymphocytes (EBV-CTL) in vitro, and to detect their killing effect. Peripheral blood mononuclear cells (PBMNC) were collected from 6 EBV seropositive healthy donors, and EBV-transformed B lymphoblastoid cells (BLCL)were used as the antigen-presenting cells and antigen stimulant which was irradiated by 40 Gy (60)Co irradiator. The autologous PBMNC and irradiated BLCL were cultured to induce and expand the EBV-CTL, and the immunophenotype was identified by the flow cytometry. The killing effect of the EBV-CTL against the autologous BLCL (autoBLCL), the autologous PHA cultured B lymphoblastoid cells( PHA-BLCL), the allogeneic BLCL (alloBLCL) and the K562 cells were measured with LDH release assay under different effector-to-target ratio. The results showed that the 6 cell lines of EBV-CTL were induced and expanded from the EBV seropositive healthy donors, the overall increase in cell numbers varied from 18.6 to 55.0 times. After 10 stimulations, the specific killing efficiency of the EBV-CTL for the autoBLCL were 59.4%, 43.2% and 29.0% under the effector-to-target ratio of 20: 1, 10: 1 and 5: 1. The nonspecific killing efficiency for the PHA-blast, alloBLCL and K562 cells were 7.1%, 9.4% and 10.3% (P < 0.05) under the 20: 1 ratio; 6.6%, 8.3% and 8.1% (P < 0.05) under 10: 1; 5.4%, 7.3% and 6.3% (P < 0.05) under 5: 1, respectively. It is concluded that the EBV-CTL can be successfully induced and expanded ex vivo for specific killing of HLA matched BLCL and may become a potential treatment for EBV related post-transplant lymphoproliferative disorders.

Quantitative monitoring of multi-donor chimerism after multi-donor allogeneic hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was aimed to establish a model for detecting the donor chimerism rate following the multi-donor hematopoietic stem cell transplantations, and simplify its calculation method. Patients with hematologic disease receiving allogeneic hematopoietic stem cell transplantation including single-donor and multi-donor were selected in this study and the donor cell chimerism rates were detected, using STR-PCR combined with capillary electrophoresis. The results indicated that the peaks of the sister alleles coming from the same individual were confirmed to have the approximate areas and can be replaced each other in the situation of mixed chimerism. In the calculation model, the value between reference chimerism and approximate chimerism have no significant difference using the hypothetical peak areas, and the result was confirmed to be accepted basing on typical measurement error between sister alleles (5% - 20%). It is concluded that the areas of share peaks can be replaced by non-share peaks and this conclusion can be used to calculate the double-donor CHM (DD-CHM)(%). Compared to the D alleles, R alleles show more strategic importance because it can lead to more accurate result and allowed simplifying the arithmetic calculations for DD-CHM(%).

Influence of HLA-E polymorphism on cytomegalovirus infection after HLA-matched hematopoietic stem cell transplantation / 中国实验血液学杂志

This study was aimed to explore the potential association of HLA-E polymorphism with the incidence of cytomegalovirus (CMV) infection after HLA-matched hematopoietic stem cell transplantation, 119 HLA-genoidentical sibling pairs for HLA-E polymorphism were analyzed, HLA-E DNA was amplified by polymerase chain reaction (PCR), and the amplified DNA products was also sequenced directly after purification to confirm the genotype. The results showed that the homozygous HLA-E*0101/0101 accounted for 20.17%, the homozygous HLA-E*0103/0103 accounted for 27.73%; heterozygous HLA-E*0101/0103 accounted for 52.10%; in homozygous HLA-E*0101/0101 group, 15 cases were infected with CMV and the CMV infection rate was 62.50%; in homozygous HLA-E*0103/0103 group, 16 cases were infected with CMV and the CMV infection rate was 48.48%; in heterozygous HLA-E*0101/0103 group 20 cases were infected with CMV and the CMV infection rate was 32.25%. As compared with the homozygous HLA-E*0101/0101 group, the CMV infection rate in HLA-E*0103 group displays statistical significance (P = 0.0295). The CMV infection rate occurred higher and its significance is statistical (P = 0.0074). It is concluded that the HLA-E gene polymorphism is associated with CMV infection after HLA-genoidentical bone marrow transplantation.

Culture and pluripotentiality of murine compact bone-derived mesenchymal stem cells / 中国实验血液学杂志

This study was purposed to culture murine compact bone-derived mesenchymal stem cell (MSC) and analyze the immunological and trilineage differentiation potential. Tibia and femur were extracted. Bone marrow cells were flushed out and compact bone fragments were digested with collagenase. The digested cells were cultured in 6-well plates. The immunophenotype, immunosuppressive function and trilineage differentiation potential were analysed by flow cytometry, mixed lympocyte reaction and Oil red O, von Kossa and alcian blue straining, respectively. The results indicated that the pure compact bone MSC could be isolated with in 3 weeks. The resulting MSC had trilineage differentiation potential and immunosuppressive effect on mixed lymphocyte reaction. The count per minute (CPM) value in control group of BALB/c T cells cocultured with irradiated C57BL/6 T cells was (2.56 ± 0.31) × 10(4), while CPM values of mixed lymphocyte cocultured with C57BL/6 compact bone MSC at ratios of 100:1 and 10:1 were (0.47 ± 0.12) × 10(4) and (0.28 ± 0.09) × 10(4). The CPM value of control group was higher than those of MSC cocultured group (P < 0.001). Compact bone-MSC had an immunosuppressive effect on mixed lymphocyte reaction in a dose dependent manner. It is concluded that murine compact bone has rich MSC and the primary MSC is contaminated with less hematopoietic cells. Murine compact bone-MSC have immunosuppressive effect on mixed lymphocyte reaction and trilineage differentiation potential. Compact bone-MSC have promising experimental study value.

Analysis of tyrosine kinases gene mutations in core binding factor related acute myeloid leukemia and its clinical significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To assess the prevalence of several tyrosine kinases (TKs) gene mutations including c-Kit, FLT3 and JAK2 V617F in core binding factor related acute myeloid leukemia (CBF-AML), and analyze their impact on clinical characteristics and prognosis.</p><p><b>METHODS</b>Mutations of c-Kit, FLT3-ITD and FLT3-TKD were detected by genomic DNA PCR and sequencing, and JAK2 V617F mutation screening by allele-specific PCR in 58 newly diagnosed CBF-AML patients [28 AML with inv(16) and 30 with t(8;21)], and analyze the patients clinical characteristics and prognoses.</p><p><b>RESULTS</b>c-Kit aberrations were detected in 32.8% cases, including 6 cases mutated in exon 8 (mutKIT8) and 13 mutated in exon 17 (mutKIT17). MutKIT8 was more prominent in inv(16) than in t(8;21) patients (21.4% vs 0, P = 0.009). Only 2 cases had FLT3-ITD and 7 (12.1%) FLT3-TKD mutations. The result of JAK2 V617F mutation screenings in these CBF-AML patients was negative. The frequency of receptor tyrosine kinases(RTK) mutations was 46.6% and only one case had two kinds of missense mutations (mutKIT8 & TKD(+)). Median age of onset was higher for mutKIT17 than for wide-type c-Kit (wtKIT) patients (55 vs 31, P = 0.003). c-Kit mutations were significantly associated with decreased overall survival (OS) and continuous complete remission (CCR) rates (P = 0.053, and 0.048 respectively), and so did more for exon17 mutated patients reduced (P = 0.005, and 0.013 respectively). FLT3-TKD mutation showed no effects on prognosis of CBF-AML patients.</p><p><b>CONCLUSIONS</b>RTK mutations are common in patients with CBF-AML. c-Kit mutations frequently and JAK2V617F mutation rarely appear in CBF-AML. c-Kit mutations, especially mutKIT17 confers higher relapse risk and poorer prognosis.</p>

The incidence of TET2 gene mutation and its clinical significance in acute myeloid leukemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the prevalence of TET2 gene mutation in acute myeloid leukemia (AML) patients, and analyze their clinical characteristics and prognosis.</p><p><b>METHODS</b>Polymerase chain reaction (PCR) and direct sequencing were used to sequence exon 3 to 11 of TET2 gene.</p><p><b>RESULTS</b>Among 96 AML patients, TET2 gene mutation was detected in 13 (13.54%) patients (95%CI 6.70% - 20.38%). The median age was 54 years in mutated group and 41 years in unmutated group (P = 0.010). Mutated and unmutated patients did not significantly differ in gender, white blood cells (WBC) count at diagnosis, platelet count, PB and BM blast percentage and chromosome karyotype, excepting for hemoglobin level 84 (70 - 108) g/L in mutated group versus 70 (55 - 87) g/L in unmutated group (P = 0.032). TET2 gene mutation had no significant correlation with C-KIT, FLT3, JAK2V617F mutations, but did with NPM1 mutation. TET2 mutated patients had lower CR1 rate and 2-year overall survival than unmutated in non-M(3) patients (P < 0.05).</p><p><b>CONCLUSIONS</b>TET2 gene mutation is more prevalent in older AML patients and has a certain correlation with clinical characteristics and outcome. It may be a molecular marker for poor prognosis in AML.</p>

Detection and clinical significance of JAK2 mutation in 412 patients with chronic myeloproliferative neoplasms / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the frequency of JAK2V617F mutation in Chinese patients with chronic myeloproliferative neoplasms (MPN) and to study the relationship between JAK2V617F mutation and clinical characteristics.</p><p><b>METHODS</b>JAK2V617F mutation was screened by allele-specific polymerase chain reaction (AS-PCR).</p><p><b>RESULTS</b>JAK2V617F mutation was detected in 277 of the 412 patients with MPN. The frequency of JAK2V617F mutation was similar among essential thrombocythemia (ET), idiopathic myelofibrosis (IMF) and chronic myeloproliferative disorders-unclassified (MPD-U) (P > 0.05), but it was significantly lower than that in polycythemia vera (PV) (P < 0.05). The presence of JAK2V617F was found to be significantly correlative with advanced age at diagnosis (P < 0.01) and with higher hemoglobin levels and higher leukocyte counts (P < 0.05). Significant difference was found in complication of vascular events between JAK2V617 positive and negative patients (P < 0.05). JAK2V617F positive MPD-U patients were more prone to progress into typical MPN compared with JAK2V617F negative MPD-U patients. The association between abnormal karyotype and JAK2V617F was not found in cytogenetical analysis of 301 patients.</p><p><b>CONCLUSION</b>The presence of JAK2V617F in MPD-U is associated with the disease development. There is a correlation between JAK2V617F mutation in MPN and advanced age, higher leukocyte counts, hemoglobin level and vascular events.</p>

Organ-specific T cell receptor repertoire in target organs of murine graft-versus-host disease after haploidentical bone marrow transplantation / 中国实验血液学杂志

This study was purpused to analyze the characteristics of T cell receptor repertoire in target organs of murine graft-versus-host after haploidentical bone marrow transplantation (hiBMT) and the molecular characteristics of complementarity determining region3 (CDR3) repertoires of monoclonal T cell in liver, skin and ileum in murine after hiBMT. Murine haploidentical BMT model was established, CDR3-size spectratyping was used to study TCRBV repertoires in recipient liver, skin, ileum, spleen and a group of CDR3 molecules was obtained from GVHD-target tissues. The results showed that GVHD occurred as early as days 14 after transplantation and was proven by histology in liver, skin and ileum. A number of new monoclonal and oligoclonal T cells emerged in GVHD-target tissue. 45 CDR3 molecules had six C'-terminal motifs, which obtained from liver, skin, ileum in different times after hiBMT. It is concluded that target organs of murine graft-versus-host disease after hiBMT emerged a number of clonal or oligoclonal T cells, part of this T cell clones commonly uses some conserved CDR3 motifs and may recognize similar antigen.

Quantitative analysis for JAK2 mutation in 98 patients with essential thrombocythemia and its clinical significance / 中国实验血液学杂志

The objective of this study was to identify the frequency and types of JAK2V617F mutation in chinese patients with essential thrombocythemia (ET), to quantitatively detect the level of mutation transcripts and to investigate its clinical significance. The frequency and types of JAK2V617F mutation were detected by amplification-refractory mutation sequencing polymerase chain reaction (ARMS-PCR), the transcript level of JAK2V617F mutation was determined by using capillary electrophoresis. The results indicated that the JAK2V617F mutation was detected in 59 out of 98 patient with ET, 18 of whom were homozygous mutation. The mean age of patients with homozygous and heterozygous mutation was higher than that of patients with wild type mutation (p < 0.05). The quantitative assay using capillary electrophoresis showed that the transcript level of JAK2V617F mutation in patients with homozygous mutation was (89.9 +/- 6.7)%, which was higher than that in patients with heterozygous mutation (57.1 +/- 6.7)% (p < 0.05); the transcript level of JAK2V617F mutation in patients with age < 60 years was (62.3 +/- 16.5)%, which was lower than that in patients with age > 60 years (72.4% +/- 15.8)% (p < 0.05). The rate of thrombotic complications in patients with JAK2V617F-positive was higher than that in patients with JAK2V617F-negative in which the rate of thrombotic complication in patients with homozygous mutation was higher than that in patients with heterozygous mutation (p < 0.05). Compared with patients without thrombotic events, there were higher level of transcripts of JAK2V617F mutation in patients with thrombotic events. It is concluded that the JAK2V617F positive and negative patients with ET display the different clinical features, therefore, the analysis of mutation types and detection of transcript levels not only helps to identify the disease status and progression, but also guides the treatment of ET patients.

5-azacytidine treatment induces tumor suppressor gene XAF1 expression and inhibits proliferation in myeloma cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of 5-azacytidine on XAF1 expression in myeloma cell lines RPMI8226 and XG-7 and the in vitro anti-myeloma activity of 5-azacytidine.</p><p><b>METHODS</b>XAF1 mRNA and protein expression was detected by semi-quantitative reverse transcriptase PCR and Western blot, respectively. Methylation specific PCR (MSP) was used to detect methylation status of XAF1 promoter CpG islands. RPMI8226 and XG-7 cells were treated with 0-5 micromol/L of 5-azacytidine and Cell Counting Kit-8 colorimetric assay was used to evaluate the growth inhibitory effect. Cell apoptosis was determined with Annexin V-PE/7-AAD staining by flow cytometry.</p><p><b>RESULTS</b>Untreated RPMI8226 cells expressed XAF1 mRNA isoforms 1 and 2, and untreated XG-7 cells had no XAF1 expression. Hypermethylation of XAF1 promoter CpG islands was detected in both the cell lines. After treated with 2.5 micromol/L 5-azacytidine for 72 h, both the cell lines expressed full-length XAF1 transcript and protein. 5-azacytidine treatment led to XAF1 promoter CpG islands hypomethylation and showed anti-myeloma activity in a time- and concentration-dependent manner with IC50 of 2.4 micromol/L and 2.6 micromol/L at 48 h for RPMI8226 and XG-7 cell lines, respectively.</p><p><b>CONCLUSIONS</b>Lack of XAF1 expression and abnormal expression of XAF1 in myeloma cell lines are associated with XAF1 gene promoter CpG islands hypermethylation. 5-azacytidine treatment can induce XAF1 mRNA and protein expression and exerts anti-myeloma activity via apoptosis at clinically achievable concentrations.</p>

A quantitative assay for JAK2 mutation in 135 patients with chronic myeloproliferative neoplasms / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the frequency and mutational status of JAK2 mutation in Chinese patients with chronic myeloproliferative neoplasms (MPN) and study the relative quantitation and clinical implications of mutated JAK2 transcript.</p><p><b>METHODS</b>JAK2 mutation and the mutational status were screened with amplification-refractory mutation sequencing polymerase chain reaction (ARMS-PCR), the relative quantity of mutated JAK2 mRNA by using capillary electrophoresis.</p><p><b>RESULTS</b>JAK2V617F mutation was detected in 95 of 135 MPN patients, including 37 (97.4%) of 38 polycythemia vera (PV), 56 (59.6%) of 94 essential thrombocythemia (ET) and 2 of 3 idiopathic myelofibrosis (IMF) patients; the difference between the mutations in PV and ET was significant (P<0.05). Of 95 JAK2V617F patients examined, 18/38 PV patients (47.3%) and 17/94 (18.1%) ET patients and 1 IMF patient were homozygotes, and ET patients showed lower prevalence of homozygote (P<0.05). In 95 MPN patients, the mutated mRNA ratio was higher in homozygote than in heterozygote patients. PV heterozygote patients showed higher levels of mutated JAK2 mRNA than ET heterozygote patients (P<0.05). The levels of JAK2V617F mRNA in patients over 60 years of age were significantly higher than that in those less than 60 years of age (P<0.001). Higher leukocyte counts were observed in PV and ET patients with higher levels of mutated JAK2 mRNA (P<0.05). The presence of JAK2V617F was found to be significantly associated with higher hemoglobin level in ET patients. Cytogenetic analysis was performed in 101 of the 135 patients, the association between abnormal karyotype and JAK2V617F was not found.</p><p><b>CONCLUSION</b>The ARMS-PCR technique can be used to detect the frequency and mutational status of JAK2V617F mutation, and along with capillary electrophoresis, the estimation of minimal residual disease becomes possible.</p>

Detection of JAK2V617F mutation and its clinical significance in 80 patients with M2 acute myelogenous leukemia / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the prevalence and prognostic significance of JAK2V617F gene mutation in acute myelogenous leukemia M2 (AML-M2) patients.</p><p><b>METHODS</b>Allele specific polymerase chain reaction (PCR) was used to detect JAK2 gene mutation.</p><p><b>RESULTS</b>Of 80 de novo AML-M2 patients, 6 at the time of first diagnosis and 1 at relapse were found to have JAK2V617F gene mutation (8.8%, 7/80). Morphologically, the whole blood and bone marrow of the 7 AML-M2 patients with JAK2V617F gene mutation presented a picture of acute leukemia instead of myeloproliferative disorders. Immunophenotypically, bone marrow samples showed myelogenous linage expression. Complete remission was obtained in 4 of 5 AML-M2 patients with JAK2V617F mutation who received treatment, while one patient had no response to the treatment. Follow-up was performed in all the 5 patients, with a median survival of 18.5 months in 4 patients.</p><p><b>CONCLUSION</b>JAK2V617F gene mutation, as a type-1 mutation, might not be an initial event in the pathogenesis of acute myelogenous leukemia, and its presentation does not mean a poor prognosis in de novo AML patients.</p>

Relationship between CMV reactivation and KIR haplotype/HLA-Cw genotype in patients after unrelated-donor hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the relationship between CMV reactivation and KIR haplotype or HLA-Cw genotype in patients after unrelated-donor hematopoietic stem cell transplantation (HSCT).</p><p><b>METHODS</b>From January 2003 to December 2008 the HLA-Cw/KIR genotype of 48 patient-donor pairs were determined by polymerase chain reaction with sequence specific primers (PCR-SSP) and sequence specific nucleotide (PCR-SSOP). Posttransplant CMV reactivation was performed by immune histochemically assay.</p><p><b>RESULTS</b>Of 48 patients, 15 were transplanted from unrelated donors with an antigen mismatch for HLA Cw and 33 patient-donor pairs were matched for HLA-Cw. The CMV reaction rate was 66.7% for HLA-Cw mismatch group and 48.5% for HLA-Cw match group (chi(2) = 1.39, P = 0.2375). Thirty-seven donor-patients pairs belonged to group C1 and 11 to group C2, and CMV reaction rate was 64.9% in group C1 and 18.2% in group C2 (chi(2) = 18.13, P < 0.0001). Twenty-six patients received graft from KIR haplotype A (group A donor) and 22 from KIR haplotype B donors (group B donor) and CMV reaction rate was 57.7% in group A donor and 50.0% in group B donor (chi(2) = 0.28, P = 0.5941). The number of donor activating KIRs (aKIRs) was less than that of recipient aKIRs in 34 patient-donor pairs in which the CMV reaction rate was 70.6%, and the number of donor aKIRs was more than that of recipient aKIRs in 14 patient-donor pairs in which the CMV reactivation was 14.3%. There was a significan difference between the two group (chi(2) = 12.44, P = 0.0004).</p><p><b>CONCLUSION</b>KIR and HLA-Cw genotypes influence the rate of CMV reactivation following non-T cell deleted unrelated donor hematopoietic cell transplantation.</p>

Experimental study on IL-2- and IL-15 application in allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the impact of IL-2- and IL-15-activated donor natural killer (NK) cell infusion on graft-versus-host-disease (GVHD) and graft-versus-leukemia (GVL) effect post allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>The C57BL/6 mice splenic NK cells were selected by microbeads, and then expanded in the media containing IL-2 and IL-15. The killing activity of NK cells was detected. In the leukemia mouse model, recipients (BALB/c) were intravenously inoculated with EL9611 leukemia cells 8 days before transplantation. Lethally irradiated BALB/c recipient mice were transplanted with 5 x 10(6) bone marrow cells (BMCs), or 5 x 10(6) BMCs plus 1 x 10(7) splenocytes with or without 1 x 10(7) activated NK cells. Additionally, NK cell infusion group mice were intraperitoneally injected with a mixture of IL-2 and IL-15 post transplant. Survival time, GVHD occurrence, lineage chimerism, TRBV spectra-typing were observed post transplant.</p><p><b>RESULTS</b>The purity of isolated splenic NK cells was 95.7% - 97.1%. The killing activity of NK cells after activation was increased by 3 times. GVHD did not occurred in allogeneic BMCs infusion group, whereas did from 1 week after transplant in allogeneic BMCs + splenocytes infusion group. The severity of GVHD in total body irradiation (TBI) experimental group was significantly lower than in splenocytes infusion group (P < 0.05). The survival time was 9.5 - 14.0 d in TBI alone conditioning group. In leukemia mouse model, 100 day survival rate was 10% the rest of them were died of leukemia while in experimental group, the more than 100 days survival rate was 80% (P < 0.01). PB NK cells at 2 week post-transplant were 4.8% in experimental group and 2.8% in control group. NK cells recovery in experimental group was earlier than that in control group (P < 0.05). TRBV reconstitution was faster in experimental group than in control group, moreover, the number of TRBV family expression was more in experimental group than in control group which mainly expressed monoclone or oligo-clone.</p><p><b>CONCLUSIONS</b>Donor alloreactive NK cells can be efficiently expanded and activated with IL-2 and IL-15. Donor activated NK cell infusion and IL-2, IL-15 treatment can promote immune reconstitution, mitigate GVHD and reduce leukemia relapse.</p>

Prevalence and clinical significance of FLT3 mutations in acute promyelocytic leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the prevalence of Fms-Like tyrosine kinase 3 (FLT3) mutations including internal tandem duplication (ITD) of juxtamembrane region and point mutation in the second tyrosine kinase domain (TKD) in acute promyelocytic leukemia (APL) and its clinical significance.</p><p><b>METHODS</b>Bone marrow mononuclear cells from 160 newly diagnosed APL patients were analyzed. Polymerase chain reaction (PCR) was used to detect FLT3-ITD mutations, FLT3-ITD positive samples were further analyzed for the ITD allelic ratio (ITD-AR, mutant-wild type ratio). The FLT3-TKD mutation was analyzed by PCR amplification of exon 20 followed by EcoR V digestion and sequencing.</p><p><b>RESULTS</b>Out of 160 patients, 30 (18.75%) patients were FLT3-ITD positive, 17 (10.62%) were FLT3-TKD positive, 2 had both of mutations. The initial WBC count and the ratio of short type PML-RAR alpha isoforms in FLT3-ITD positive and FLT3-TKD positive patients were all higher than that in patients with wild-type FLT3 (FLT3-wt) (P < 0.05). For FLT3-ITD positive patients, the incidences of retinoic acid syndrome (RAS) and disseminated intravascular coagulation (DIC) were 41.7% and 65.4%, respectively, being higher than that of FLT3-wt patients, while their complete remission (CR) rate was lower (69.2% vs 90.3%, P < 0.05). For FLT3-TKD positive patients, the incidence of RAS, DIC and CR rate were not significantly different from that of FLT3-wt patients (P > 0.05). FLT3-ITD positive patients had a shorter overall survival (OS) (P < 0.05), but not disease-free survival (DFS) (P > 0.05) as compared with FLT3-wt patients. There was no significant difference in either OS or DFS between FLT3-TKD positive and FLT3-wt patients. The ITD-AR of 30 FLT3-ITD positive patients varied from 0.11 to 6.55 with a median of 1.0. The initial WBC count, incidence of RAS and DIC, CR rate were not significantly different between the patients with ITD-AR greater than 1.0 and lower than 1.0 (P > 0.05).</p><p><b>CONCLUSIONS</b>FLT3 mutations (FLT3-ITD or FLT3-TKD) are frequently identified in patients with newly diagnosed APL, both mutations are associated with higher initial WBC and short type PML-RAR alpha isoforms. FLT3-ITD mutation is more frequent than FLT3-TKD mutation, and predicts a poorer prognosis, whereas FLT3-TKD mutation does not show the same unfavorable prognostic effect on APL patients.</p>

Study on relationship between pretransplantation host thymic recent output function and prognosis in HLA-matched sibling hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the relationship between pretransplantation host thymic recent output function and prognosis in HLA-matched sibling bone marrow transplantation (MSD-BMT) and determine whether pretransplantation host thymic recent output function can act as a marker for predication of prognosis after HSCT.</p><p><b>METHODS</b>T-cell receptor excision circle (TREC) in DNA of pretransplantation peripheral blood mononuclear cells from 64 patients underwent MSD-BMT was detected by real-time quantitative PCR. The content of TREC in 70 normal donors was detected as well. All clinical data of patients after HSCT were collected and studied. Survival rates of patients after HSCT were estimated with Log-rank test. Univariate and multivariate analysis of prognostic factors were carried out by COX's proportional hazard regression model.</p><p><b>RESULTS</b>The mean value of TREC in normal donors was (3351 +/- 3711) copies/10(5) cells. There was an inverse correlation between TREC and age in the donor groups. Before transplantation, all patients were detected TREC, with a mean TREC number of (180 +/-332) copies/10(5) cells being significantly lower than that of normal donors. The results of univariate analysis showed that the counts of pre-HSCT TREC were closely, correlated with long term survival and chronic graft versus host disease (cGVHD) (P < 0.05) and with CMV infection (P = 0.084) but not with acute graft versus host disease (aGVHD). The results of multivariate analysis showed the same thing as that of univariate analysis.</p><p><b>CONCLUSION</b>Pretransplantation host thymic recent output function is closely correlated with prognosis in MSD-BMT and can be a factor for predicting the outcome of HSCT.</p>

Analysis of NPM1 gene mutations in acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the prevalence of nucleophosmin (NPM1) gene exon 12 mutations in adults with acute myeloid leukemia (AML) and its clinical characteristics.</p><p><b>METHODS</b>Genomic DNAs from 101 AML adults were screened by PCR and sequencing or capillary electrophoresis (CE) for NPMI mutations.</p><p><b>RESULTS</b>NPM1 exon 12 mutations were present in 31.7% of the overall cohort, including 1/1 (100%) of M0, 9/17(52.9%) of M1 , 7/25 (28.0%) of M2, 0/23(0%) of M3, 2/7 (28.6%) of M4 and 13/25 (52.0% ) of M5. NPM1 gene mutations were more prevalent in patients with normal karyotype (27/59, 45.8%) compared with that in those with karyotypic abnormalities (5/42, 11.9% ) (P < 0.001). NPM1 mutant cases were significantly associated with old age (P < 0.05), high peripheral white cell count (P < 0.05) and low expression of CD34 (P < 0.05) and CD17 (P<0.05). Sequence analysis of these NPM1 mutant cases revealed 5 known mutations (type A, B, D, N(M), and P(M)) and 1 novel variant (named as type S).</p><p><b>CONCLUSIONS</b>NPM1 exon 12 mutations occur with a considerable percentage in AML patients with normal karyotype, M1/M5 subtype and older age, and are associated with higher peripheral white cell count and lower expression of CD34 and CD117.</p>

The molecular characteristics of T-cell immune reconstitution in leukemia patients after allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the molecular characteristics of CDR3 repertoires of T cell receptor beta chain variable region (TCRBV) of T lymphocytic clones in leukemia recipients after allogeneic hematopoietic stem cell transplantation ( allo-HSCT).</p><p><b>METHODS</b>RT-PCR was used to amplify 24 subfamily genes of TCRBV from peripheral blood (PB) lymphocytes in twenty-four leukemia patients underwent three kinds of allo-HSCT and in five normal donors as control. The PCR products were further analyzed by genescan to evaluate the clonality of BV subfamily and characteristics of CDR3 and calculate usage rate of BV subfamily. The monoclonal bands which associated with GVHD and CMV infection were obtained by denaturation polyacrylamide gel electrophoresis and sequenced. Comparison of the sequences of TCRBV CDR3 with other CDR3 sequences which associated with GVHD or CMV infection was reported.</p><p><b>RESULTS</b>2 approximately 19 months after transplantation, there were 6 approximately 14 BV subfamilies expressed and the polyclonal expression reached 33% in nine patients underwent haploidentical bone marrow transplantation(HI-BMT). In five patients underwent matched unrelated peripheral blood stem cell transplantation ( MU-PBSCT), there were 10 approximately 15 BV subfamilies expressed of which 45% were poly-clones. In 10 patients underwent matched sibling bone marrow transplantation(MS-BMT), 10 approximately 16 BV subfamilies were expressed and more than 48% of them were poly-clones. Monoclones and oligo-clones existed in 24 BV subfamilies but no common one monoclone BV subfamilies was found. Immune reconstitution in patients underwent HI-BMT was later than that in other two groups. In 2 patients TCRBV was detected in 2m and 3m after allo-HSCT and found that there was a tendency of increasing usage of BV subfamilies and increasing expression of CDR3 polymorphism. Twenty three TCRBV CDR3 molecules associated with GVHD and CMV infection were compared each other by bioinformatics and found that different cases of the same BV subfamilies may share similarity in amino acid motif, while in different BV subfamilies none appeared to share the same amino acid motif.</p><p><b>CONCLUSION</b>In 1.5 years after allo-HSCT, the usage of TCRBV subfamilies still restricted. Immune reconstitution in patients underwent HI-BMT was later than that in other two groups. TCRBV CDR3 molecules associated with GVHD and CMV infection showed that different cases of the same BV subfamilies may share similarity in amino acid motif, while in different BV subfamilies none of clones appeared to share the same amino acid motif.</p>