In Vitro Identification and Characteristics of CD34Leukemia Stem Cell in t(8;21) Acute Myeloid Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To preliminarily identify the existence of CD34leukemia stem cell (LSC) in t(8;21) acute myeloid leukemia (AML) by in vitro test.</p><p><b>METHODS</b>Bone marrow samples collected from newly diagnosed t(8;21) AML patients were tested. LinCD34CD38(abbreviation, CD34CD38), LinCD34CD38(abbreviation, CD34CD38) and LinCD34CD38CD45SSC(abbreviation, CD34"LSC") cell fractions were gated by flow cytometry after staining with fluorescent antibodies. Cells in Gphase were identified through Hoechst 33342 and pyronin Y staining. Aldefluor reagent was used to test aldehyde dehydrogenase (ALDH) activity. The above-mentioned 3 cell fractions were sorted, and mRNA levels of AML1-ETO and WT1 were measured by real-time quantitative PCR.</p><p><b>RESULTS</b>The 3 tested samples displayed the same tendency in ratio of the cells in Gphase: CD34"LSC">CD34CD38>CD34CD38. The paired t-test of 53 patients showed that frequency of ALDHcells of both CD34CD38and CD34"LSC" cell fractions was significantly higher than that of CD34CD38(P<0.01), furthermore, the ALDHcell frequency was significantly higher in CD34"LSC" than that in CD34CD38(P<0.01). AML1-ETO mRNA levels of cells sorted from 3 patients were similar among the 3 cell fractions within each patient, whereas WT1 mRNA levels were significantly higher in CD34"LSC" than that in other 2 cell fractions.</p><p><b>CONCLUSION</b>CD34LSC may exist in t(8;21) AML, and may be more primitive than CD34LSC. These results promote the necessity to perform in vivo xenogeneic transplantation mice.</p>

Immunophenotypic and clinical characteristic analysis of NPM1 mutated acute myeloid leukemia with a normal karyotype / 中国实验血液学杂志

This study was purposed to compare the immunophenotypic and clinical characteristics of NPM1 mutated acute myeloid leukemia with a normal karyotype under the similar constituent ratio of FAB subtypes. Immunophenotyping and NPM1 gene mutation type-A,B and D and other leukemic related fusion genes were detected by multiparameter flow cytometry and real time RT-PCR or PCR, respectively. 77 AML patients with a normal karyotype (NK) and mutated NPM1 gene (NPM1m(+)AML) detected by immunophenotyping assay were included in this study. 55 cases without NPM1 mutation (NPM1m(-)AML) and with normal karyotype were served as negative control. The results showed that there was significant difference between NPM1m(+)AML and NPM1m(-)AML in terms of sex, white blood count, platelet counts, blast, WT1 expression level, FLT3-ITD mutation positive rate and response to treatment. The characteristic immunophenotype is lower expression of early differentiation-associated antigens (CD34, HLA-DR, CD117, CD38), lymphocytic antigens (CD7, CD4, CD19, CD2) and higher expression of CD33 and CD123 (P < 0.05). When above features was further analyzed between the M1/2 and M4/5 subgroups in NPM1m(+)AML patients, the M1/2 cases retained a higher frequency in women and a higher WT1 expression level (P < 0.05) . Monocytic differentiation-associated antigens including HLA-DR and lymphocytic antigens CD7 were higher expressed and CD117 was lower expressed in M4/5 subgroup (P < 0.05). It is concluded that under condition of similar constituent ratio of M1/2 and M4/5 subtype and normal karyotype, NPM1m(+)AML patients have higher WT1 expression level and better response to treatment. The major immunophenotype features of NPM1m(+)AML patients are lower expression of early differentiation antigens and lymphoid lineage antigens and higher expression of CD33 and CD123. Monocytic differentiation-associated antigens only higher are expressed in M4/5 cases when compared with M1/2 cases within NPM1m(+) AML patients.

Expression of PML-RARα is up-regulated during ATRA and arsenics combined induction without influence on long-term prognosis of acute promyelocytic leukemia / 中国实验血液学杂志

The early molecular kinetics during all-trans retinoic acid (ATRA) plus arsenic-based induction therapy and its prognostic value for acute promyelocytic leukemia (APL) remain unclear. This study was purposed to investigate the molecular and cytogenetic kinetics and its clinical significance in treatment of APL with ATRA plus arsenic-based induction. The molecular and cytogenetic kinetics was assessed by real-time quantitative RT-PCR and interphase fluorescence in situ hybridization (FISH) in 32 newly diagnosed APL patients. The results showed that the median PML-RARα transcript levels (PML-RARα/ABL) were very significantly up-regulated at 14 days of induction therapy compared with that of pre-treatment (40.10% vs 57.74%, P < 0.01), and then decreased at 28 days of induction therapy and at the end of consolidation therapy (6.97% and 0%), respectively. The total of 65.62% and 31.25% patients showed up-regulation of PML-RARα transcript at 14 and 28 days after induction, as compared with pretreatment. The PML-RARα copies per APL cell before treatment, and at 14 and 28 days after induction were calculated as 0.9, 2.2, 1.4 by the formula of PML-RARA/ABL(%)×2/APL cells (%). With the median follow-up time of 22 months, 32 patients were still in continuous clinical remission and no molecular relapse occurred. Up-regulation of PML-RARa expression during the induction had no effect on outcomes of APL patients. It is concluded that up-regulation of PML-RARa expression is a common event during induction therapy with ATRA plus arsenics. Up-regulation of PML-RARa expression during induction therapy hasn't influenced the long-term prognosis of APL.

The more, the less: age and chemotherapy load are predictive of poor stem cell mobilization in patients with hematologic malignancies / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Intensive treatment such as autologous peripheral blood stem cell (PBSC) transplantation is an important therapeutic strategy in many hematologic malignancies. A number of factors have been reported to impact PBSC mobilization, but the predictive factors varied from one study to another. This retrospective study assessed our current mobilization and collection protocols, and explored the factors predictive of PBSC mobilization in patients with hematologic malignancies.</p><p><b>METHODS</b>Data of 64 consecutive patients with hematologic malignancies (multiple myeloma, n = 22; acute leukemia, n = 27; lymphoma, n = 15) who underwent PBSC mobilization for over 1 year were analyzed. Four patients with response to treatment of near complete remission or better were administered granulocyte colony-stimulating factor (G-CSF) to mobilize PBSCs. Sixty patients received G-CSF followed by chemotherapy mobilizing regimens. Poor mobilization (PM) was defined as when ≤ 2.0'10(6) CD34(+) cells/kg body weight were collected within three leukapheresis procedures.</p><p><b>RESULTS</b>The incidence of PM at the first mobilization attempt was 19% (12/64). The PM group was older than the non-PM group (median age, 51 vs. 40 years; P = 0.013). In univariate analysis, there were no significant differences in gender, diagnosis, and body weight between the PM and non-PM groups. A combination of chemotherapy and G-CSF was more effective than G-CSF alone as a mobilizing regimen (P = 0.019). Grade III or IV hematopoietic toxicity of chemotherapy had no significant effect on the mobilization efficacy. Supportive care and the incidence of febrile neutropenia were not significantly different between the two groups. In multivariate analysis, age (odds ratio (OR), 9.536; P = 0.002) and number of previous chemotherapy courses (OR 3.132; P = 0.024) were two independent negative predictive factors for CD34(+) cell yield. PM patients could be managed well by remobilization.</p><p><b>CONCLUSION</b>Older age and a heavy load of previous chemotherapy are the negative risk factors for PBSC mobilization.</p>

Effect of different regimens on bone disease of multiple myeloma / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the difference of effects of two regimens (bortezomib and dexamethasone, BD; and thalidomide and dexamethasone, TD) on bone disease in multiple myeloma (MM).</p><p><b>METHODS</b>Forty patients with newly diagnosed and refractory or relapsed MM were treated with BD or TD regimens from Dec 2006 to Sep 2008. Bone pain score and X-ray examination were carried out before and after therapy. Serum levels of DKK-1, sRANKL, OPG and TRACP-5b were measured by ELISA before and 3 months after therapy.</p><p><b>RESULTS</b>Serum TRACP-5b concentration was significantly decreased in patients received TD regimen (5.94 U/L before therapy vs 4.84 U/L 3 months after therapy, P < 0.05), and so did for serum DKK-1 concentration in patients responded to BD regimen (35.11 µg/L before vs 32.03 µg/L 3 months after therapy, P < 0.05); for serum concentration of sRANKL in patients responded to BD regimen (1.05 pmol/L before vs 0.67 pmol/L 3 months after therapy, P < 0.05); and for serum concentration of TRACP-5b in responders to BD regimen (5.57 U/L before therapy vs 4.90 U/L 3 months after therapy, P < 0.05).</p><p><b>CONCLUSION</b>Bortezomib lowers levels of serum DKK-1 and RANKL in responders, thus leads to normalization of abnormal bone remodeling through the increase of bone formation and reduction of bone resorption. Thalidomide decreases bone resorption regardless of treatment response.</p>

The clinical characteristics of newly diagnosed acute myeloid leukemia patients with NPM1 mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical characteristics of newly diagnosed acute myeloid leukemia (AML) with NPM1 mutation.</p><p><b>METHODS</b>NPM1 mutation (including A, B, D mutation type) was detected in 206 patients with newly diagnosed AML by real-time quantitative RT-PCR.</p><p><b>RESULTS</b>The incidence of NPM1 mutation was 15.5% in total AML patients and 32.5% in normal karyotypes AML patients. The characteristics of 174 NPM1 wild type patients v.s. that of 32 NPM1 mutation patients was as follow, median age (46 vs 35 years old, P < 0.01), WBC counts (27 × 10(9)/L vs 8 × 10(9)/L, P < 0.01), BPC (82 × 10(9)/L vs 36 × 10(9)/L, P < 0.01), proportion of AML-M(5) (31.2% vs 5.8%, P = 0.01), incidence of normal karyotypes (92.6% vs 40.8%, P < 0.01), incidence of FLT3-ITD-positive (25.0% vs 7.5%, P < 0.01), CD34-positvie (23.3% vs 69.5%, P < 0.01), cases with fusion gene (0 vs 47.1%, P < 0.01). No statistic difference was found in sex, percentage of blasts in bone marrow, complete remission rate, overall survival between the two groups. Relapse-free survival in AML patients with NPM1-mutation and FLT3-ITD-negative tended to be higher than in those with NPM1-mutation and FLT3-ITD-positive.</p><p><b>CONCLUSION</b>It is necessary to detect NPM1 mutation and FLT3-ITD in newly diagnosed AML patients, especially in patients with high WBC and BPC, CD34-negative, normal karyotype, which might help to molecular classification and treatment.</p>

Prognostic significance of minimal residual disease detected by multiparameter flow cytometry in acute myeloid leukemia / 中国实验血液学杂志

This study was aimed to explore prognostic significance of minimal residual disease (MRD) detection in patients with acute myeloid leukemia (AML) by multiparameter flow cytometry (MCF). Leukemia-associated immunophenotype (LAIP) of newly diagnosed AML patients were determined by 4-color 5 antibody panels and patients with sensitive LAIP were chosen for MRD detection. 601 bone marrow samples from 95 patients were acquired after treatment and MRD were considered positive by the critical normal value plus twice standard deviation in normal bone marrow specimen. The patients were divided into three groups and the clinical significance was analyzed every 2 months within initial 6 months after induction treatment. The results showed that the relapse rate and relapse-free survival (RFS) rate were all significantly different between MRD positive and MRD negative patients in the three groups (p < 0.05). Patients with MRD positive had a median relapse-free survival time of 11 months, 11.5 months and 11 months at 1 - 2, 3 - 4 and 5 - 6 months respectively, while all patients with MRD negative were not observed to reach median release-free survival time (p < 0.05). Furthermore, the clinical significance was analyzed after induction and one course of consolidate treatment, the relapse rate of MRD positive and MRD negative patients were 57.14% versus 0% and 91.67% versus 2.27% respectively (p = 0.000 and p = 0.000). It is concluded that MRD detection by multi-parameter flow cytometry can predict outcome of AML patients, which should be continuously monitored after treatment.

Relationship of immunophenotypic features with minimal residual disease detection and gene types in 221 cases of acute promyelocytic leukemia / 中国实验血液学杂志

This study was aimed to investigate the relationship of immunophenotypic features with minimal residual disease (MRD) detection and gene types in APL patients. Immunophenotypes were analyzed in 221 newly diagnosed APL patients by using four-color flow cytometry. Among of them, CD123 antibody was examined in 87 patients and the fused gene pml-raralpha were detected by PCR in 196 specimens simultaneously. The results of immunophenotyping demonstrated that the positive percentages of CD123, CD33 and CD9 in newly diagnosed APL patients were 100%, 99.1% and 96.0% respectively, and mean percentages of positive cells in positive patients were all around 90%. Although the positive rates of CD117, CD13, CD38 and CD64 were all above 96%, but the mean percentages of positive cells in different positive patients were diverse and average percentages of positive cells were about 70%. CD15, CD56 and CD11b were expressed in some patients, but CD34 and HLA-DR were rarely expressed in the majority of patients, and average positive percentages were all lower. Among 196 newly diagnosed APL patients, bcr1, bcr2 and bcr3 expressions were 63.3%, 4.6% and 32.1% respectively. The results showed a strong correlation of positive expression of CD34 with bcr3 isoform. When cut-off value was chosen as 20%, the proportions of CD34 positive patients in bcr3 and bcr1 cases were 15.4% (10/65) and 3.3% (4/121) separately, which had a significant difference (p < 0.05). When cut-off value was 10%, bcr3 cases had a significantly higher percentage of CD34 positive, compared with bcr1 cases (p < 0.001), which was 47.7% (31/65) and 5.8% (7/121) respectively. However, there was no statistically significant difference on the other antigens between the two groups. Bcr3 isoform was highly indicated when CD34 was positive and non- large side scatter (NL-SSC) was shown in APL cells. It is concluded that there is a unique characteristics of immunophenotyping, and antigens such as CD123, CD33 and CD9 are more applicable to the detection of MRD in APL patients. The positive expression of CD34 and NL-SSC are associated with bcr3 isoform, and the relationship between gene type and antigen expression can be suggested more accurately when the cut-off value is chosen as 10%.

Arsenic trioxide downregulates the expression of annexin II in bone marrow cells from patients with acute myelogenous leukemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Most patients with acute myelogenous leukemia (AML) suffer from disordered hemostasis. We have previously shown that annexin II (Ann II), a high-affinity co-receptor for plasminogen/tissue plasminogen activator, plays a central role in primary hyperfibrinolysis in patients with acute promyelocytic leukemia (APL). The expression of Ann II in cells from patients with major subtypes of AML and the effect of arsenic trioxide (As2O3) on Ann II expression in AML cells were investigated to determine whether As2O3-mediated downregulation of Ann II could restore hemostatic stability.</p><p><b>METHODS</b>A total of 103 patients (48 females and 55 males; age, 19 - 58 years) were included. Plasma samples were collected before and after treatment as well as after complete remission. Ann II and plasminogen activation were measured in leukemic cells during treatment with 1 micromol/L As2O3.</p><p><b>RESULTS</b>Before As2O3 treatment, Ann II mRNA expression (real-time PCR) was the highest in M3 cells (P < 0.05), higher in M5 cells than that in M1, M2, M4, and M6 cells (P < 0.001), and positively correlated with Ann II protein expression (flow cytometry) (r = 0.752, P < 0.01). Exposure for up to 120 hours to As2O3 (1 micromol/L) had no significant effect on Ann II protein in M1 and M2 leukemic cells, but decreased Ann II protein expression twofold within 48 hours of exposure in M3 cells (P < 0.05) and twofold within 96 hours in M5 cells (P < 0.05). The rate of plasmin generation was higher in APL, M5, and M4 cells than in M1, M2, and M6 cells.</p><p><b>CONCLUSIONS</b>As2O3 may reduce hyperfibrinolysis in AML by downregulation of Ann II. Furthermore, As2O3 affects more than one form of AML (APL, M4 and M5), suggesting its potential role in their management.</p>

PRAME mRNA expression in newly diagnosed acute myeloid leukemia patients and its application to monitoring minimal residual disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression level of preferentially expressed antigen of melanoma (PRAME) mRNA in newly diagnosed acute myeloid leukemia (AML) patients and evaluate its usefulness for detecting minimal residual disease (MRD).</p><p><b>METHODS</b>PRAME mRNA levels were detected in bone marrow samples from 142 newly diagnosed AML patients (72 of them didn't express any specific fusion gene) by TaqMan based real-time quantitative PCR methods, and were serially monitored in 60 bone marrow samples from 9 follow-up patients (2 of them without specific fusion gene), including 3 in continuous complete remission, 6 in hematological relapse. Bone marrow samples from 22 bone marrow donors (NBM) were served as normal controls. Samples from 7 AML1-ETO (+) M2 patients were detected for AML1-ETO mRNA simultaneously. abl was selected as control gene, PRAME and AML1-ETO mRNA levels were expressed by their copies/abl copies in percentage.</p><p><b>RESULTS</b>All NBM samples expressed PRAME mRNA and the upper limit was 0.28%. For all newly diagnosed AML patients, median PRAME mRNA level was 3.97% (0.00%-714.97%), 76.8% of them was higher than 0.28%, 54.9% had over 1-log increasing and 26.1% had over 2-log increasing. For patients without specific fusion gene, median PRAME mRNA level was 0.60% (0.00%-408.72%), 56.3% of them was over 0.28%, 32.4% and 11.3% had over 1-log and 2-log increasing, respectively. There was a significant difference in PRAME mRNA levels between subtypes of AML patients (P<0.01). AML1-ETO (+) M2 patients expressed the highest levels (all P<0.01), followed by acute promyelocytic leukemia patients with S type PML-RAR alpha fusion gene. PRAME and AML1-ETO mRNA levels of follow up patients displayed similar kinetic patterns, and correlated well in 43 follow up samples (r=0.88, P<0.01). PRAME mRNA levels in 3 hematological relapsed patients increased above 0.28% 1-4 months ahead relapse, and in other 3 relapsed patients the levels never decreased to normal range even in remission.</p><p><b>CONCLUSIONS</b>PRAME mRNA could be used to monitor MRD for AML patients with higher than normal levels, and it increases over or persistently higher than normal range predicts hematological relapse.</p>

Detection of PML/RARalpha gene transcripts in 46 newly diagnosed acute promyelocytic leukemia patients by real-time quantitative reverse-transcription polymerase chain reaction / 中国实验血液学杂志

In order to explore the application of real-time quantitative reverse-transcription polymerase chain reaction (Q-PCR) for detecting PML/RARalpha gene transcripts in patients with acute promyelocytic leukemia (APL), the bone marrow samples from 46 newly diagnosed APL patients were collected for analysis. Three plasmids containing cDNA fragments of the bcr1-, bcr3-form PML/RARalpha and ABL control gene were constructed respectively. The ABI Prism 7500 Sequence Detection System using Taqman fluorogenic probes was used to quantify target gene. PML/RARalpha mRNA was detected by Q-PCR in 46 APL patients and 40 non-APL patients. The normalized quotient (NQ) of PML/RARalpha mRNA was calculated as followings: NQ = PML/RARalpha mRNA copy numbers/ABL mRNA copy numbers. Immunophenotype of acute promyelocytic leukemia was determined by four-color flow cytometry. The results showed that the coefficients of variation (CV) of inter-day assay and intra-day assay by using Q-PCR were 1.58% and 0.88% respectively. Q-PCR could detect reproducibly 5 copies of target gene per 100 ng RNA and no pseudopositive results were found. The median NQ of PML/RARalpha mRNA was 0.450 (0.084 - 1.082) in 46 APL patients. There was no indication of any correlation of PML/RARalpha mRNA type with age, sex, hemoglobin, platelet count, percentage of promyelocytes in bone marrow detected by morphology or flow cytometry, PML/RARalpha NQ, or signs of clinically diagnosed coagulation/bleeding disorders. Compared with bcr1-form cases, bcr3-form cases had more M(3v) phenotype (42.9% vs 9.4%, P = 0.015) and higher WBC count (9.35 x 10(9)/L vs 2.15 x 10(9)/L, P = 0.038). APL cells could be classified into large side scatter population (L-SSC) and non-large side scatter population (NL-SSC) in CD45/SSC histogram of flow cytometry. 87.50% patients with bcr1-form showed L-SSC phenotype and 64.29% patients with bcr3-form showed NL-SSC phenotype. It is concluded that a sensitive Q-PCR method is established. The median NQ of PML/RARalpha mRNA was 0.450 in newly diagnosed APL patients. There was no significant difference about PML/RARalpha mRNA expression of both bcr3-form and bcr1-form APL patients. Type of PML/RARalpha transcripts is related with the morphology and immunophenotype.

Detection of common fusion transcript levels in untreated leukemia patients by real-time quantitative RT-PCR technique / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate levels of common specific fusion transcripts M-bcr-abl, m-bcr-abl, TEL-AML1, AML1-ETO, PML-RAR alpha, CBF beta-MYH11 in untreated leukemia patients.</p><p><b>METHODS</b>Specific fusion transcript levels were detected by TaqMan-based real-time quantitative RT-PCR technique in a total of 208 samples, including 195 bone marrow samples from 50 M-bcr-abl(+) chronic phase-chronic myeloid leukemia (CML-CP), 10 M-bcr-abl(+) acute lymphoblastic leukemia (ALL), 19 m-bcr-abl(+) ALL, 11 TEL-AML1(+) ALL, 30 AML1-ETO(+) acute myeloid leukemia (AML), 58 PML-RAR alpha(+) acute promyelocytic leukemia (APL) and 17 CBF beta-MYH11(+) AML patients and 13 peripheral blood samples from 13 M-bcr-abl(+) CML-CP patients. abl was chosen as internal control gene. Fusion transcript level was calculated as fusion transcript copies/abl transcript copies in percentage.</p><p><b>RESULTS</b>Bone marrow and peripheral blood samples of CML-CP patients had similar M-bcr-abl fusion transcript levels (median 30% vs 35%, P > 0.05). M- and m-bcr-abl (median 64% vs 54%) levels were similar in ALL patients (P > 0.05), M-bcr-abl level was significantly higher in ALL than CML-CP patients(P < 0.001). Median TEL-AML1 level was 228% in ALL patients. Among AML patients, AML1-ETO level was significantly higher than CBF beta-MYH11 and PML-RAR alpha levels (median 388% vs 145%, 388% vs 47%, all P < 0.001), CBF beta-MYH11 level was significantly higher than PML-RAR alpha level (P < 0.001). Fusion transcript levels of L-, V- and S-type PML-RAR alpha were 45%, 44% and 55%, respectively. L-type was significantly lower than S-type (P = 0.04).</p><p><b>CONCLUSIONS</b>Fusion transcript levels in untreated leukemia patients were different and patient-to-patient variations did exist. Detection of fusion transcript levels in untreated leukemia patients not only provides baseline for minimal residual disease monitoring and treatment evaluation but also enable the comparison in inter-laboratory data.</p>

Correlation of class II transactivator with HLA-DR antigen and its implications / 中国实验血液学杂志

The present study was purposed to investigate the relation and difference of expression phase between class II transactivator (CIITA) and HLA-DR antigens after IFN-gamma induction, and the inhibition of CIITA and HLA-DR by STAT1-alpha antisense oligonucleotides (STAT1-alpha AS); and to explore the potential effect and significance of CIITA and STAT1-alpha AS in transplantation immunity. T lymphocytes from peripheral blood of healthy subjects were incubated with IFN-gamma at different doses. RT-PCR was used to detect CIITA mRNA and Western blot was used to analyze HLA-DR antigen. Then the optimum dose of IFN-gamma was chosen for the experiment. CIITA mRNA and HLA-DR antigen were detected at various time points. Different doses of STAT1-alpha AS and sense oligonucleotides (STAT1-alpha S) were added to T lymphocytes followed by IFN-gamma. After incubation with IFN-gamma, the expression of CIITA mRNA and HLA-DR was detected once again. The results showed that CIITA mRNA was detectable at 5 hours after IFN-gamma incubation and reached the peak at 14 hours, then declined, but the CIITA mRNA was still found at 23 hours. HLA-DR antigen was detectable at 28 hours after IFN-gamma incubation and reached a peak at 52 hours, then declined. CIITA mRNA expression was positively correlated to HLA-DR expression, and was earlier than the latter. The expression of CIITA mRNA in the AS groups was significantly lower than that in the control group after 5 micromol/L, 10 micromol/L and 20 micromol/L STAT1-alpha AS treatment (P < 0.01). The expression of CIITA mRNA in the S groups was higher than that in the AS groups (P < 0.01), but there was no significant difference between the S group and the control group. The expression of HLA-DR antigen was significantly inhibited by STAT1-alpha AS, and the expression level of HLA-DR protein in the AS group was about 64.3% of that in the control group (P < 0.01), while there was no significant difference in HLA-DR expression between the S group and the control group. The changes in HLA-DR expression were similar to those in CIITA expression after STAT1-alpha AS treatment. It is concluded that CIITA expression is positively correlated with HLA-DR expression, and was detectable earlier than that of latter after IFN-gamma incubation. Stat1-alpha antisense oligonucleotides may have a sequence-specific inhibiting effect on the expression of CIITA and HLA-DR antigen after IFN-gamma incubation in vitro culture, and can prevent T lymphocyte activation. CIITA may play an important role in pathogenesis of transplantation immunity.

Detecting PML-RARalpha transcript in acute promyelocytic leukemia using real-time quantitative RT-PCR / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Real-time quantitative RT-PCR (RQ-PCR) assay has become a vital tool to monitor residual disease of leukemia. However, the complexity and standardization of RQ-PCR should never be overlooked and the results should be interpreted cautiously in clinical conditions. We aimed to assess the methodology of RQ-PCR and its clinical applications in monitoring molecular kinetics of 36 newly diagnosed cases of acute promyelocytic leukemia patients with t (15; 17) from October 2004 to December 2005.</p><p><b>METHODS</b>All the TaqMan probe-based RQ-PCR reactions and analysis were performed on an ABI-PRISM 7,500 platform. The quantitation of PML-RARalpha transcripts was represented by the normalized quotient, that is, PML-RARalpha transcript copies divided by ABL transcript copies. According to induction therapy, the patients were classed into two groups: group 1 (n = 23), three-drug combination including arsenics, all-trans retinoic acid and mitoxantrone; and group 2 (n = 13), two-drug combination from all-trans retinoic acid, arsenics and mitoxantrone.</p><p><b>RESULTS</b>The sensitivity of RQ-PCR was 1 per 10(5) cells and 5 copies of the PML-RARalpha transcript could be reproducibly detected. No false positive results occurred in 40 non-acute promyelocytic leukemia samples. Optimal amplification efficiency could be attained, which was determined by the slope of the standard curves (slope: -3.2 - -3.7). The inter-assay and intra-assay variation coefficients of the method were 1.01% and 0.56% respectively. Although the time to attain hematological complete remission was similar in both groups, the time to achieve molecular remission of group 1 was significantly shorter than that of group 2 (61 days vs 75 days, P = 0.034). The rate of molecular remission within 70 days was higher in group 1 than in group 2 (75.00% vs 38.46%, P = 0.036). Compared with pretreatment, median reduction of the PML-RARalpha transcript before first consolidation therapy differed significantly between group 1 and group 2 (log scale, 3.15 vs 2.31, P = 0.024). Interestingly, we found that PML-RARalpha transcript levels temporarily increased in bone marrow (7 patients) and peripheral blood (22 patients) samples of patients during induction therapy in both groups.</p><p><b>CONCLUSIONS</b>The RQ-PCR assay is reliable for the detection of PML-RARalpha transcripts. Arsenics, all-trans retinoic acid and mitoxantrone triad induction treatment of acute promyelocytic leukemia is superior to two-drug combination induction therapy in terms of the molecular response.</p>

Detection of phosphotyrosine in bcr-abl-positive cells with PY20 antibody and its clinical applications / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the specificity of anti-phosphotyrosine monoclonal antibody PY20 in bcr-abl+ cells and its possible clinical applications.</p><p><b>METHODS</b>Bcr-abl cell lines( K562, MEG-01) and bcr-abl- cells lines( Jurkat, MCF-7 )were stained with PY20. Phosphotyrosine protein of K562 and MEG-01 cells was detected by flow cytometry before and after treatment with imatinib. Phosphotyrosine protein in bone marrow cells from 49 patients with chronic myeloid leukemia (CML), Ph+ acute lymphoblastic leukemia(Ph(+) -ALL), Ph- ALL, acute myeloid leukemia (AML-M1, M2, M3, M5, FAB classification), chronic lymphocytic leukemia (CML) and 3 normal donor. Positive cells over 5% of total cells was considered positive cases for phosphotyrosine protein. The level of tyrosine phosphorylation was determined by median fluorescence intensity (MFI).</p><p><b>RESULTS</b>Bcr-abl cell lines and marrow cells from 10 CML patients and 8 ALL patients were all PY20-positive, while bcr-abl- cell lines and marrow cells from 18 leukemia patients and 3 normal donor were all PY20-negative. MFI decreased remarkably after blocked by imatinib in K562 cells and MEG-01 cells. The positive cell percent of marrow cells from 10 newly diagnosed CML patients and 9 imatinib-sensitive CML patients was (54.20 +/- 19.82)% and (14.84 +/- 6.17)% (P < 0.05), while that of 2 cases of imatinib-resistant was 64.3% and 57.2%. There was significant difference of MFI between imatinib-resistant patients and imatinib-sensitive patients (99.42 +/- 4.87 vs 46.41 +/- 4.67, P < 0.01).</p><p><b>CONCLUSION</b>PY20 monoclonal antibody is highly specific for bcr-abl+ cells. It might be useful in rapid detection of bcr-abl+ cells and sensitivity to imatinib of CML patients.</p>

Monitoring bcr-abl mRNA levels by real-time quantitative RT-PCR in chronic myeloid leukemia patients after hematopoietic stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the value of real time quantitative RT-PCR (Q-PCR) for monitoring bcr-abl mRNA levels in chronic myeloid leukemia (CML) patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>Quantification of bcr-abl mRNA was performed on 316 bone marrow samples from 112 patients with CML after HSCT by Q-PCR using the TaqMan probe system. The bcr-abl mRNA level was normalized by control gene abl. Cytogenetic response was evaluated with fluorescent in-situ hybridization (FISH).</p><p><b>RESULTS</b>The reproducible sensitivity of Q-PCR was 5 copies. The coefficients C(T) of interassay and intraassay variation for abl and bcr-abl were all below 2.0%. 289 bone marrow samples were collected from 101 CML patients who achieved a sustained complete cytogenetic response (CCyR) one month post allo-HSCT in a period of 6 - 60 months (median 12 months) at different intervals. In general, the median bcr-abl levels gradually decreased with the prolongation of time after HSCT: the median bcr-abl levels were 0.035% (0 - 0.406%) at 1 month post allo-HSCT (+ 1 month), 0.006% (0 - 0.683%) at +3 month, 0% (0 - 0.225%) at +6 month and remained 0% till +24 months. The highest level in CCyR patients detected at + 6 month was 0.068%. The bcr-abl mRNA level was decreased by 3 log in sustained CCyR patients at + 1 month compared with the newly diagnosed CML-CP patients (33.0%, data unpublished). On the contrary, Q-PCR results ranged from 0.12% to 13.45% in 8 cytogenetic non-responders or relapsed patients post allo-HSCT. Among them, 5 patients' samples were collected 1 - 2 months before cytogenetic relapse, the results were ranged from 0.09% to 3.42%. If 0.09% was assumed 0.09% as a threshold, 9 sustained CCyR patients (8.9%) were tested once higher than that within 6 month after HSCT but decreased to 0% eventually. 2 blast crisis patients achieved CCyR within 1.6 and 3 months after HSCT, but hematological relapse occurred after 1 and 1.5 months, and their bcr-abl mRNA levels increased dramatically from 0% and 0.14% to 46.9% and 75.9% respectively.</p><p><b>CONCLUSIONS</b>Q- PCR is a sensitive, precise and reliable technique, and can be used to monitor CML patients post allo-HSCT regularly. Patients in blast phase of CML should be monitored more frequently.</p>

Application of CD123 in detection of minimal residual disease in acute promyelocytic leukemia / 中国实验血液学杂志

The study was aimed to investigate the role and significance of CD123 with other immunological markers in detecting minimal residual disease (MRD) of APL patients. The immunophenotypes of 186 newly diagnosed APL patients and the percentages of cells identical with APL cell immunophenotypes in 20 normal bone marrow samples were analyzed using four-color flow cytometry. MRD in 172 specimens were monitored by mainly using CD34/CD117/CD123/HLA-DR four-color antibody panels, meanwhile 18 specimens were analyzed with the second antibody combination: CD9/CD117/CD34/CD33, simultaneously and the results were compared with real-time PCR. One hundred and sixteen of 172 bone marrow (BM) or peripheral blood (PB) specimens were from follow-up 19 newly diagnosed APL patients and the rest 56 samples were from 47 patients treated 3 to 24 months later. Among them, 117 samples and 55 samples were collected after achieving morphologic complete remission (mCR) and before achieving mCR respectively. The results of immunophenotyping demonstrated that except CD9, CD33 and CD117 were high-expressed and CD34 and HLA-DR were rarely expressed, the CD123 was expressed in 30/30 (100%) APL patients. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) and CD117(+)CD34(-)CD9(+)CD33(+) cells in nucleated cells were 0.066% +/- 0.012% and 0.089% +/- 0.066% in 20 normal bone marrow samples. The median time of achieving morphology complete remission in 19 APL patients was 4 weeks (3 - 6 weeks). The median time of FCM and PCR results turned to be negative in 13 APL patients was 7.5 weeks (5 - 11) and the median time of PCR results turned to be negative in 11 APL patients was 8 weeks (5 - 12). 41/117 (35.04%) samples were MRD positive by FCM after achieving mCR. The ratio of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells was < 5% in 33 specimens, but > 5% in another 8 specimens, their median percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells were 0.48% (range 0.02% - 4.70%) and 9.02% (range 5.26% - 18.14%) respectively. The median relative percentages of CD123(+)HLA-DR(-) cells in CD117(+)CD34(-) population were 63.59% (range 15.11% - 98.36%) and 86.77% (range 63.29% - 92.62%) respectively. In FCM MRD positive samples, 95.9% (93/97) were PCR positive, the false positive rate of FCM and the false negative rate of PCR were 4.1% (4/97) and 8.75% (7/93) respectively. In FCM negative samples, 92% (69/75) were PCR negative and 8% (6/75) were PCR positive. The percentages of CD117(+)CD34(-)CD123(+)HLA-DR(-) cells in 116 consecutive specimens and 117 specimens of mCR were related to PML/RARalpha quantified by real-time PCR (r = 0.824, P < 0.001 and r = 0.754, P < 0.001 respectively). It is concluded that the detection of APL patients by means of two sets of antibody panels is simple and suitable, which is complementary to PCR in monitoring MRD of APL patients.

Leukemia-associated immunophenotypes in 415 childhood and adult patients with B lineage acute lymphoblastic leukemia by multiparametric flow cytometry analysis / 中国实验血液学杂志

To evaluate the significance of FCM in minimal residual disease (MRD) detection, the immunophenotyping and leukemia-associated immunophenotypes (LAIP) of leukemia cells from 273 adult and 142 childhood patients with B lineage acute lymphoblastic leukemia (B-ALL) were detected by four to six antibody combinations of 4-color CD45/SSC gating multiparametric flow cytometry (FCM). The results showed that the B-ALL patients could be classified into 4 subtypes based on different expression CD34 and CD10: subtype I (CD34(+)/CD10(-)), subtype II (CD34(+)/CD10(+)), subtype III (CD34(-)/CD10(+)), subtype IV (CD34(-)/CD10(-)). The LAIP was observed in 100% and 92% patients of subtype I and subtype II, respectively, whereas only 79.2% in subtype III. The incidence of LAIP in total B-ALL cases was 90% by using the antibodies detected in this investigation. There was no significantce different for incidence of LAIP between adult and pediatric patients. LAIP was observed in 77.6% of patients by labeling only CD34/CD10/CD19/CD45 4-color antibody combination. It is concluded that in 90% of childhood and adult B-ALL patients LAIP can be found, which suits MRD detection by multiparameter flow cytometry.

Improvements of lentiviral vector and its application in gene therapy of hematological diseases / 中国实验血液学杂志

As lentiviral vector holds the characteristics of higher transfection to non-dividing cells, larger capacity of transfer gene fragments, long-term expression of therapeutic gene and lower rate of immunological response, therefore it becomes potential viral vector in gene therapy. Improvements of lentiviral vector, human immunodeficiency virus type-I as example, and its application in gene transfer for gene therapy of hematological diseases are emphasized in this review.

Specific anti-leukemic cell effect mediated by dendritic cells pulsed with chronic myelogenous leukemia lysate antigen in vitro / 中国实验血液学杂志

To investigate the specific antileukemia effect of dendritic cells (DC) pulsed with chronic myelogenous leukemic lysate antigen (CLA), dendritic cells from patients with chronic myelogenous leukemia (CML) were pulsed by CLA, and then cocultured with cytokine-induced killer (CIK) cells from CML patients (CIK + CLA-DC group). The cytotoxic activity in vitro was measured by using a lactate dehydrogenase release assay, and compared with CIK + DC, CIK and CIK + CLA groups. The results showed that under an effector-target ratio of 25:1, the cytotoxic activity of CIK + CLA-DC, CIK + DC, CIK and CIK + CLA groups against autologous CML cells was (68.8 +/- 14.2)%, (52.5 +/- 9.4)%, (20.6 +/- 7.5)% and (24.2 +/- 8.7)%, respectively. CIK + CLA-DC group displayed a strongest cytotoxic activity. When K562 and Raji cells acted as target cells and CIK as effectors, the cytotoxic activity against autologous CML cells in CIK + CLA-DC group (68.8 +/- 14.2)% was much higher than that against K562 cells (14.6 +/- 6.2)% and Raji cells (12.7 +/- 10.2)%, respectively. In conclusion, coculture of CIK cells with DC led to a significant increase in cytotoxic activity. The cytotoxicity could be further increased by DC pulse with CML cell lysate antigen, and cytotoxicity mediated by CML lysate antigen possess stronger specificity.