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Objective@#To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison.@*Methods@#Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated.@*Results@#①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH.@*Conclusion@#The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.
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Objective To investigate the monitoring significance of WT1 gene level in the prognosis of acute myeloid leukemia (AML) patients with normal karyotype after hematopoietic stem cell transplantation (HSCT). Methods The clinical data of 115 AML patients with normal karyotype who were treated with HSCT from July 2009 to March 2017 in the First Affiliated Hospital of Soochow University were retrospectively analyzed. The dynamic detection of bone marrow WT1 gene was carried out by using reverse transcription_polymerase chain reaction (RT_PCR). According to the relative expression level median of WT1 gene before transplantation, the whole patients were divided into the two groups (<median group and ≥median group) for survival analysis. Results There were 52 males and 63 females in 115 patients. The average age was (39± 10) years old. The median white blood cell count at initial diagnosis was 20.45×109/L [(0.5-355.9)×109/L], the ratio of blast cells in the bone marrow was 0.60±0.28, and the relative expression level median of WT1 gene was 87×104, while the median time of the follow_up was 24 (3-79) months. Among 115 patients, 19 cases relapsed. Remission group (96 cases) and relapse group (19 cases) were followed up. The WT1 gene level was monitored by using bone marrow puncture in 1 month, 3 months, 6 months, 12 months after transplantation. It was found that the WT1 gene relative expression level of relapse group was higher than that of remission group, and the differences between the two groups at 6 month_point [remission group (187±50)×104, relapse group (871±211)×104, t ＝ 2.519, P＝ 0.014] and 12 month_point [remission group (51±9)×104, relapse group (1 797±312)×104, t ＝ 4.000, P< 0.05] were statistically different. The overall survival (OS) rate of 2_year, progression_free survival rate in WT1 gene relative expression level < 87×104 group were higher than those in WT1 gene relative expression level ≥87×104 group, the relapse rate in WT1 gene relative expression level <87×104 group was lower than that in WT1 gene relative expression level ≥87×104 group, and the differences were statistically different (all P<0.05). Multivariate analysis showed that the level of WT1 gene at 12 months after transplantation was an independent factor affecting OS ( HR＝4.12, P＝0.046) and PFS ( HR＝5.95, P＝0.001). There were 19 cases of recurrence (16.5%), with a median relapsed time of 11 (1-60) months. When WT1 gene relative expression level was significantly increased in 19 patients, firstly immunosuppressive agents were reduced, of which 6 patients were not influenced by this intervention; in other 13 cases, only 5 cases were influenced by intervention. Conclusions For CN_AML patients, the expression level of WT1 gene before transplantation has a negative correlation with the prognosis. The expression level of WT1 gene at 12 months after transplantation is an independent factor for affecting the survival. The relapsed patients have a higher WT1 expression level, and clinical interventions for relapsed patients have a favorable effect.
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Objective@#To investigate the characteristics of the essential thrombocythemia (ET) cases transformed to the acute myeloid leukemia (AML) and the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of this disease.@*Methods@#The clinical and laboratory characteristics of 3 ET cases before and after transformation and after allo-HSCT were retrospectively analyzed, meanwhile the related literatures were reviewed and discussed.@*Results@#Case 1 was a male patient of 44 years old, whose PLT was 500×109/L when firstly diagnosed ET. After 3 years the disease progressed into myelodysplastic syndrome (MDS) while WT1 expression increased from 77 (first visit) to 13 171 copies/10 000 ABL copies, at the same time chromosome changed dramatically. During the period of decitabine treatment the disease processed into AML. Case 2 was a male of 58 years old whose PLT was 2 100×109/L when firstly diagnosed ET. The disease progressed to AML after 9 years, whose WT1 expression increased from 130 (first visit) to 3 222 copies/10 000 ABL copies, and he relapsed shortly after intensive chemotherapy. Case 3 was a male of 60 years old whose PLT was 900×109/L when firstly diagnosed ET. The disease progressed to AML after 5 years, whose WT1 increased from 56 (first visit) to3 696 copies/10 000 ABL copies. Moreover leukemia spread to central nervous system (CNS) during chemotherapy. Before allo-HSCT, cases 1 did not achieve remission; case 2 relapsed after a short time of remission and case 3 transferred to CNS leukemia. All of the 3 cases underwent allo-HSCT successfully, and they all achieved completely remission, whose chromosome and gene mutation recovered negative. At the same time, CNS leukemia of case 3 disappeared. The median WT1 decreased to 50 copies/10 000 ABL copies. There was no severe complication during the median time of 5 months after allo-HSCT.@*Conclusions@#The patients transformed to AML had poor prognosis, allo-HSCT was the only method that can cure the disease now.
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Objective@#To investigate the immune reconstruct regularity profile of KIR2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation (allo-HSCT) with KIR-AA genotype.@*Method@#75 donor-recipient pairs were performed by KIR genotying using PCR-SSP, and all donors were identified with KIR-AA genotype. Dynamic detections (including unrelated-donor on the day of transplantation and the recipient each month post allo-HSCT) of the expression of KIR2DL1/3DL1 on NK cell and mRNA level were performed in 291 cases using flow cytometry (FCM) and real-time fluorescent quantitation PCR (RT-qPCR) .@*Result@#①The median expression of KIR2DL1 in unrelated-donor on transplant’s day was 21.60%, the median expression of KIR2DL1 in recipient 1M, 2M, 3M and 3-6M after transplantation were 7.40%, 12.00%, 16.92%, 17.64% respectively. The median expression of KIR2DL1 in unrelated-donor on transplant’s day was 265.14 copies/10 000abl copies, the median expression of KIR2DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 332.17, 438.31, 723.25, 414.17, 180.76 and 234.67 copies/10 000abl copies respectively. The median expression of KIR2DL1 on NK cells and mRNA level gradually increased at all time points after transplantation, and reached the highest expression at 3 months after transplantation. But mRNA expression levels increased earlier than NK cell membrane proteins. ②The median expression of KIR3DL1 in unrelated-donors on transplant’s day was 18.56%, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M after transplantation were 23.83%, 22.57%, 23.02%, 21.60% respectively. The median expression of KIR3DL1 in unrelated-donor on transplant’s day was 572.29 copies/10 000abl copies, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 1 233.74, 1 140.42, 876.73, 1 057.07, 739.02 and 514.43 copies/10 000abl copies respectively. The median expression of KIR3DL1 on NK cells and mRNA level were higher than donors at 1 month after transplantation, and stable expression at all time points after transplantation, so mRNA and NK cell membrane proteins expression increased at the same time.@*Conclusion@#The immune reconstruct regularity of KIR2DL1 and KIR3DL1 gene were different, which provided an experimental basis for selecting the best time to detect the expressions of KIR2DL1 and 3DL1 after transplantation.
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Objective@#To investigate the overexpression frequencies of BRE and EVI1, the correlation between BRE and EVI1 expressions and their possible clinical implications in 11q23/MLL rearrangement acute leukemia.@*Methods@#Cytogenetic examination of bone marrow cells was performed by short-term culture method. R-banding technique was used for karyotype analysis. 47 patients were detected by interphase fluorescence in situ hybridization (FISH) with dual-color break apart MLL probe. The expressions of EVI1 and BRE genes were detected by real time quantitative reverse transcription polymerase chain reaction (RQ-PCR) . The correlation and prognostic significance were statistically tested.@*Results@#11q23/MLL rearrangements were confirmed by karyotyping and FISH, respectively in 47 patients. According to immunophenotypic analyses of 37 patients, 5 patients showed positive for CD19, CD79a or CD10, 1 for CD7; the others for CD33, CD13, CD14 and CD15, and 16 of them for CD34. Of the 47 patients, 18 patients showed EVI1 overexpression and most of them presented with t (6;11) and M4/M5. The EVI1 expression was high in t (6;11) or t (9;11) subgroup comparable with levels observed in normal subgroup (P=0.038, 0.022, respectively) . 15 patients showed high BRE expression, and most of them presented with t (9;11) and M4/M5. High BRE expression was found in t (4;11) , t (6;11) , t (9;11) and t (11;19) subgroups, respectively by comparing with normal subgroup. The BRE expression was higher in t (4;11) (P=0.004) or t (9;11) (P=0.012) subgroup than in t (6;11) subgroup. Patients with EVI1 overexpression had a short survival compared with those with low EVI1 expression (P=0.049) and it also did in t (9;11) subgroup (P=0.024) . Patients with t (9;11) and high BRE expression had a long survival compared with those with t (9;11) and low BRE expression (P=0.024) .@*Conclusion@#The EVI1 overexpression was significantly frequent in acute leukemia patients with 11q23/MLL rearranged, especially within t (6;11) subgroup and M4/M5, which was associated with an inferior outcome. High BRE expression was observed frequently in 11q23/MLL-rearranged acute leukemia especially within t (9;11) subgroup and M5.
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Objective To investigate the characteristics of activation-induced cytidine deaminase (AID) expression level in de novo acute leukemia (AL) patients, chronic myeloid leukemia chronic phase (CML-CP), chronic myeloid leukemia blastic crisis (CML-BC) patients and leukemia cell lines. Methods The expression level of AID mRNA was measured in 89 cases of newly-diagnosed acute lymphoblastic leukemia (ALL) patients, 79 cases of de novo acute myeloid leukemia (AML) patients, 5 cases of CML-BC patients, 5 cases of CML-CP patients and leukemia cell lines NB4, THP-1, KG-1, Raji, K562 by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR), bone marrow mononuclear cells of 16 normal healthy donors were used as the control group. Results The expression levels of AID mRNA in 89 cases of ALL and 79 cases of AML were 0.006-7 463.175 and 0.005-69.107, the median expression levels were 3.785 and 1.812, the expression level of AID mRNA in the normal control group was 0.146-4.707, and the median expression level was 1.483, respectively. The AID expression levels of ALL, B-ALL, Burkitt leukemia, M4 patients and Raji cells were significantly higher than those of the normal control group (all P <0.05). Nevertheless, the AID mRNA expression levels of M3 patients and NB4, KG-1 cells were lower than those of the normal control group (all P <0.05). Furthermore, the AID mRNA expression levels of K562 cell were strikingly higher than that of the CML-CP patients (P<0.001), so were those of CML-BC, chronic myeloid leukemia myeloid blast crisis (CML-MBC), chronic myeloid leukemia lymphoblastic blast crisis (CML-LBC) patients. Conclusion AID gene shows high expression level in B-ALL, Burkitt leukemia and M4, low expression level in M3 and KG-1 cells, and obvious high expression level in CML-BC.
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<p><b>OBJECTIVE</b>To report clinical and laboratory features of 4 cases of myeloid neoplasm with t (5;12) (q33;p13).</p><p><b>METHODS</b>Cytogenetic examination of bone marrow cells obtained from patients was performed by 24 h culture method. R banding technical was used for karyotype analysis. PDGFRβ gene rearrangement was detected by FISH using dual color break apart PDGFRβ probe. ETV6-PDGFRβ fusion genes were detected by multiple-reverse transcription polymerase chain reaction (RT-PCR). Direct sequencing analysis was performed on the PCR products in case 1. Immunophenotype analysis was carried out by flow cytometry. Four cases were treated with imatinib (IM) and followed up.</p><p><b>RESULTS</b>The diagnoses included 3 MPN and 1 AML-M2. The t (5;12) (q33;p13) was a primary abnormality in 3 cases of MPN and a secondary abnormality in 1 case of AML-M2. PDGFRβ gene rearrangement and ETV6-PDGFRβ fusion genes were detected by FISH and multiple-RT-PCR in 4 cases, respectively. The immunophenotypical analysis of leukemia cells showed positive for CD13, CD33 and CD34. Two cases obtained MMR after the treatment of IM, one case complete hematologic and complete cytogenetic response. ETV6-PDGFRβ was negative detected by multiple-RT-PCR after the treatment of IM, but relapsed and died soon in case 4.</p><p><b>CONCLUSIONS</b>The t (5;12) myeloid neoplasm was a subtype with unique features. The t (5;12) maybe a primary chromosome abnormality in MPN and a secondary in AML. MPN with t (5;12) could benefit from IM, but not for AML. Dual-FISH was a reliable tool for detecting PDGFRβ rearrangement.</p>
Subject(s)
Humans , Chromosome Banding , Gene Rearrangement , Hematologic Neoplasms , Genetics , Immunophenotyping , In Situ Hybridization, Fluorescence , Karyotyping , Myeloproliferative Disorders , Genetics , Polymerase Chain Reaction , Proto-Oncogene Proteins c-ets , Genetics , Receptor, Platelet-Derived Growth Factor beta , Genetics , Remission Induction , Repressor Proteins , Genetics , Translocation, GeneticABSTRACT
Objective To investigate the differential expression of miR-155 in newly diagnosed pediatric acute myeloid leukemia(AML) and its clinical significances.Methods Fifty-two AML children and 30 non-malignant disease matched children were recruited as the controls.The preliminary AML children were divided into favorable group,moderate group and poor group according to the National Comprehensive Cancer Network(NCCN) 2013.Real-time quantitative polymerase chain reaction was applied to validate the expressions of miR-155 in bone marrow samples (the data presented by 2-△△Ct).Results By comparing expressions of miR-155 between AML patients and controls,the miR-155 expressions were significantly higher in the AML children than those in the controls (Z =-5.391,P ＜ 0.001).There were significant differences among different prognostic groups,with a significantly lower level in the favorable group compared with others (x2 =12.586,P =0.002).It was also found that differential expressions existed not only in kinds of mutation cohort,with the highest level in FLT3-ITD and the lowest one in FLT3-TKD mutation group (x2 =11.216,P =0.024),but also among fusion gene subgroups (x2 =12.254,P =0.016),with the highest level in AML-ETO group and the lowest level in PML-RARa group:meanwhile,the expressions of miR-155 were statistic different according to French-America-British (FAB) subtypes (x2 =17.814,P =0.013),which was lower in M3 patients than non-M3 patients (Z =-3.291,P =0.001).Conclusions It indicates that the expressions of miR-155 may increase sharply in preliminary AML children,and the lower expression of miR-155 is closely related to favorable prognosis.
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<p><b>OBJECTIVE</b>To explore the impact of ITD mutation characteristics on the overall survival (OS) and complete remission duration (CRD) in FLT3-ITD positive non-M3 acute myeloid leukemia (AML).</p><p><b>METHODS</b>Capillary electrophoresis was used to detect the FLT3-ITD characteristics after PCR amplication. Single or multiple mutations were identified by the numbers of peak. FLT3-ITD mutation burden was calculated by the peak area of mutant divided by the wild-type and mutant peak areas. Clinical data was collected and followed up in the FLT3-ITD mutation patients.</p><p><b>RESULTS</b>Multiple ITD mutations were common in patients aged 60 and above. Patients with single ITD mutation had higher percentage of blasts in bone marrow than multiple ITD mutations (0.758 vs 0.638, P=0.028). The numbers and length of FLT3-ITD mutation had no impact on prognosis. Patients with less than 10% of ITD mutation burden showed no difference with the intermediate-risk c-kit group in OS and CRD, but the two groups had longer OS and CRD than ITD mutation burden above 10% (OS: undefined, undefined, 9.9 months, P<0.05; CRD: undefined, undefined, 6.7 months, P<0.05). In patients with ITD mutation burden above 10%, cases with NPM1 or CEBPA mutation alone had markedly longer CRD than ITD mutation alone (25.0 vs 5.1 months, P=0.003), while OS were similar (11.4 vs 8.0 months, P>0.05).</p><p><b>CONCLUSION</b>Non-M3 AML patients with less than 10% FLT3-ITD mutation burden had a better prognosis than those above 10%.</p>
Subject(s)
Humans , Genotype , Leukemia, Myeloid, Acute , Mutation , Prognosis , Remission Induction , fms-Like Tyrosine Kinase 3ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of up-regulation of Rap1GAP on the invasion ability of leukemic HL-60 cells in vitro, and to establish leukemia mouse model to verify the effects in vivo.</p><p><b>METHODS</b>Quantitative RT-PCR and Western blot methods were used to detect the expression of Rap1GAP in Venus/HL-60 (vehicle control) and Rap1GAP/HL-60 cells (R1 andR2). Transwell method was used to examine the invasion ability in vitro. Quantitative RT-PCR and gelatin zymograph were used to study the expression of MMP-2 and MMP-9. Four-week-old BALB/c nu/nu mice were pre-treated and inoculated with leukemic cells from different groups, several index including survival time were then monitored.</p><p><b>RESULTS</b>Rap1GAP mRNA level of R1 and R2 increased about 16-17 folds as compared to the control cells. The invasion rate of R1 and R2 are (55 ± 5)% and (59 ± 4)%, which are significantly higher than (14 ± 4)% of the control cells. The mRNA level of MMP-9 was up-regulated about 12.0 folds in R1 and R2 cells compared to the corresponding control cells. The median survival times of R1 and R2 mice are (32.00 ± 1.85) d and (33.37 ± 2.50) d, respectively, which are shorter than (43.62 ± 2.32) d of the control group. Three mice of R1 and R2 groups showed leukemic cells infiltration in meninges tissue, and the genes of Rap1GAP and MMP-9 were amplified by PCR method.</p><p><b>CONCLUSION</b>Up-regulated expression of Rap1GAP increased the invasion ability of HL-60 cells accompanied with enhancement of MMP-9 expression in vitro, and the experiment in mouse model also confirmed that Rap1GAP enhanced the invasion of HL-60 cells in vivo.</p>
Subject(s)
Animals , Humans , Mice , GTPase-Activating Proteins , Metabolism , HL-60 Cells , Leukemia , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , RNA, Messenger , Transcriptional Activation , Up-RegulationABSTRACT
<p><b>OBJECTIVE</b>To revalidate the conversion factor（CF）for the conversion of BCR-ABL （P210）transcript levels to the international scale（BCR- ABLIS）in chronic myeloid leukemia（CML） which validated before.</p><p><b>METHODS</b>Peking University People's Hospital（PKUPH）prepared the exchange samples for revalidation of CFs of 15 laboratories which validated nine or eighteen months ago. The fresh BCR-ABL（P210）（+）bone morrow or peripheral blood nucleated cells were diluted with BCR-ABL （P210）（-）cells to achieve different BCR- ABL levels, totally 16 sets and 24 samples per set were prepared. TRIzol reagent was added in each tube. Each laboratory tested BCR-ABL transcript levels of one set of samples. Agreement between BCR-ABLIS of each laboratory and PKUPH was assessed by the Bland- Altman method. For laboratories which did not meet the criteria of revalidation, linear regression equation was derived after the samples with maximum BCR-ABL deviation were removed until R²>0.98, then new CF was calculated.</p><p><b>RESULTS</b>10 laboratories met the revalidation criteria with both bias within ±1.4 fold and 95% limits of agreement within ±6 folds, and their CFs still could be used for accurately conversion of BCR-ABLIS. New CFs were recalculated as of 1.8-6.3 folds of their previous CFs in 5 laboratories not met the criteria.</p><p><b>CONCLUSION</b>Revalidation of CF by sample exchange among laboratories was necessary for accurate and continuous application of BCR-ABLIS, which not only tested the validity of CF acquired before but also calculated new available CFs for those with invalid CFs.</p>
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Humans , Bone Marrow Cells , Fusion Proteins, bcr-abl , Genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Diagnosis , GeneticsABSTRACT
Background:Galectin-3 is a member of the galectin family that participates in a variety of physiological and pathological events including cell growth and apoptosis,cell adhesion,angiogenesis,as well as tumor invasion and metastasis,and has been reported to be overexpressed in many human cancers.Aims:To investigate the effect of galactin-3 targeted RNA interference on proliferation,apoptosis and chemosensitivity of human gastric cancer cell line SGC-7901. Methods:Galectin-3 targeted siRNA was constructed and transfected into SGC-7901 cells.Efficacy of RNA interference was evaluated by real time PCR and Western blotting,while cell proliferation was assessed by CCK-8 assay and cell apoptosis by flow cytometry.Results:The transfection efficiency at 24 hours after transfection was 83.8%;expression of galectin-3 in SGC-7901 cells was significantly inhibited at mRNA and protein levels with a decreasing of 87.8% and 90.4%,respectively (P <0.01).Proliferation inhibition rates of SGC-7901 cells in galectin-3 siRNA group at 24,48 and 72 hours after transfection were 15.57% ±1.45%,32.90% ±0.76% and 57.35% ±1.05%,respectively,and the apoptosis rate at 72 hours after transfection was 46.17% ±2.39%;all were significantly higher than those in blank control,liposome control and negative siRNA control groups at the same time points (P <0.01).Proliferation inhibition of SGC-7901 cells induced by oxaliplatin,a chemotherapeutic agent,was also markedly increased in galectin-3 siRNA group (P <0.01).Conclusions:Expression of galectin-3 in SGC-7901 cells can be inhibited successfully by RNA interference;cell proliferation is decreased,cell apoptosis is increased and sensitivity to chemotherapeutic agent is augmented,which indicates that galectin-3 is a promising target for gastric cancer gene therapy.
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Objective To investigate the mechanism of AS2 O3 inducing the apoptosis of myelodysplastic syndrome(MDS) cell line SKM-1 .Methods SKM-1 cells were incubated with AS2 O3 ,and then the cellular morphology was observed ,flow cytometry was used to determine the apoptosis ,RT-PCR was used to detect the expressions of Bcl-2 ,Bax and caspase-3 mRNA .Results 0 .25、0 .50 μmol/L AS2 O3 could not markedly induce the apoptosis of SKM-1 cells (P>0 .05) .But 2 .00、8 .00、32 .00 μmol/L of AS2 O3 could obviously promote the apoptosis of SKM-1 cells .With the increase of the acting time and concentration of AS2 O3 ,the apoptosis rate increased ,too(P<0 .01) ,the expressions of anti-apoptotic gene Bcl-2 mRNA decreased (P<0 .01) ,the expressions of promoting apoptosis gene Bax and caspase-3 mRNA increased (P<0 .01 ,P<0 .05) .Conclusion 2 .00、8 .00、32 .00 μmol/L of AS2O3 may promote the apoptosis of SKM-1 cells through down-regulating the expression of Bcl-2 gene and up-regulating the ex-pressions of Bax and caspase-3 genes .
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Objective To evaluate the expression of miR-196b in newly diagnosed pediatric acute myeloid leukemia (AML) and its clinical signiifcance. Methods Fifty-two AML children were enrolled in this study and 30 non-leukemia com-pared children were selected as controls. The expressions of miR-196b were detected in bone marrow samples by real-time quan-titative PCR (q-RT-PCR) and the results were expressed in 2-??Ct. Results miR-196b expressions were signiifcantly higher in M4-5 and lower in non-M4-5 of AML children than those in control (P<0.01), with a lowest level in t (15;17) and a highest level in MLL subtypes (P<0.01). The miR-196b expressions were signiifcantly different among different prognosis groups (P<0.01) and the level in the favorable prognostic group was lower than in poor prognosis group. It was also found that miR-196b expres-sion was lower in remission group than that in no-remission group after the ifrst induction remission therapy (P<0.05). Mean-while, the expression of miR-196b in the children with WBC≥100×109/L were statistically higher than that in the children with WBC<100×109/L (P<0.01), and miR-196b level was positively correlated with the platelet counts (r=0.302, P=0.030). Conclu-sions miR-196b expression is increased in poor prognosis group of AML children, and high expression of miR-196b is related with low response rate and poor prognosis. miR-1966 is expected to become a new target for the treatment of AML.
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Objective To sudy the changes in mTOR signaling pathway in childhood aplastic anemia(AA) by detecting the expression levels of the molecules of mTOR signaling pathway in T cells,and to explore immunologoical pathogenesis of AA in children from T cell intracellular signal transduction pathway.Methods Peripheral blood samples were collected from 16 newly diagnosed severe AA(SAA) patients and 8 patiens treated with effective immunosuppressive therapy,and the findings were compared with those of 17 healthy children (normal controls) and CEM cells (positive controls).The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,p-p70S6K in CD3 + T cells in peripheral blood were detected by flow cytometry(FCM).Results 1.The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,pp70S6K of the newly diagnosed SAA group were higher than those of the normal control group (P ＜ 0.05),but were lower than the postive control group (CEM group) (P ＜ 0.05).The mean fluorescence intensity (MFI) of p-Akt of three groups was 8.04 ± 3.78,2.59 ± 1.01 and 20.23 ± 8.98 respectively ;p-TSC2 was 49.73 ± 19.49,16.10 ± 8.04 and 101.05 ± 29.78 respectively ; p-mTOR was 13.90 ± 9.32,2.92 ± 1.09 and 34.3 ± 19.03 ;p-4EBP1 was 142.69 ± 53.36,26.91 ± 13.70,256.01 ± 53.79 ; p-p70S6 K were 17.67 ± 10.48,3.69 ± 2.22,31.73 ± 12.85 respectively.2.The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,p-p70S6K of the effective treatment groups were lower than those of the newly diagnosed SAA group (P ＜ 0.05) ; the expressions of p-Akt,p-TSC2,p-mTORC1,p-p70S6K were similar to those of the normal control group(P ＞ 0.05),but the expressions of p-4EBP1 were higher(P ＜ 0.05).The MFI was followed by 3.28 ± 1.27,16.50 ± 10.91,3.54 ± 1.66,74.89 ± 49.69 and 4.21 ± 1.69.Conclusions 1.The expressions of p-Akt,p-TSC2,p-mTORC1,p-4EBP1,p-p70S6K were increased in the newly diagnosed SAA patients,the mTOR signaling pathway was activated in SAA patients.2.The expressions of p-Akt,p-TSC2,p-mTORC1,p4EBP1,p-p70S6K were lower than those of the newly diagnosed SAA patients.The degree of activation of mTOR signaling pathway was associated with disease status.The signaling pathways may be involved in the T cells of AA of the immune abnormalities.
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<p><b>BACKGROUND</b>MMPs and TIMPs play important roles in tumor angiogenesis and invasion. Studies have shown that TIMP-2 has two roles in tumor invasion. However, its role in leukemic infiltration has not been well investigated. This study explored the roles of TIMP-2 in extramedullary infiltration of acute monocytic leukemic SHI-1 cells both in vitro and in vitro.</p><p><b>METHODS</b>A retroviral vector carrying the human TIMP-2 cDNA was constructed and transfected into the monocytic leukemic cell line SHI-1. The expression of TIMP-2 in the positive clones was determined. The proliferation of SHI-1 cells was examined by MTT assay. Trans-Matrigel invasion assays were used to investigate the infiltration ability in vitro. SHI-1 cells were intravenously injected into pre-treated nu/nu mice to investigate the infiltration ability feature in vitro.</p><p><b>RESULTS</b>The expression of TIMP-2 on the cell membrane was significantly elevated in SHI-1/TIMP-2 cells. Over-expression of TIMP-2 promoted the cells proliferation and the invasions in vitro. The SHI-1/TIMP-2 cells demonstrated higher infiltration ability when intravenously injected into nu/nu mice.</p><p><b>CONCLUSION</b>Over-expression of TIMP-2, especially on the cell membrane, may play important roles in promoting the proliferation and infiltration of SHI-1 leukemic cells.</p>
Subject(s)
Adult , Animals , Humans , Male , Mice , Cell Line , Cell Proliferation , Physiology , Leukemic Infiltration , Mesenchymal Stem Cells , Metabolism , Physiology , Mice, Inbred BALB C , Tissue Inhibitor of Metalloproteinase-2 , Genetics , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To compare the curative effect of imatinib and allogeneic hematopoietic stem cell transplant (allo-HSCT) in the treatment of chronic myeloid leukemia (CML).</p><p><b>METHODS</b>292 CML patients received imatinib, and 141 patients underwent allo-HSCT. The clinical data of these patients were retrospectively analyzed to compare event- free survival (EFS) and overall survival (OS) between these two groups of patients in chronic and advanced (including accelerate and blast) phases.</p><p><b>RESULTS</b>(1) EFS, OS, expected 5- year EFS and OS of imatinib group (278 patients in chronic phase) were all statistically higher than of allo-HSCT group (120 patients in chronic phase) (88.5% vs 70.0%, 93.2% vs 80.0%, 84.0% vs 75.0% and 92.0% vs 79.0%, respectively, all P values < 0.01). (2) EFS and OS of imatinib group (14 patients in accelerate and blast phases) were 42.9% and 42.9%, respectively. Meanwhile EFS and OS of allo-HSCT group (21 patients in accelerate and blast phases) were 47.6% and 57.1%, respectively. There were no significant differences in terms of EFS and OS between the two groups (P values>0.05).</p><p><b>CONCLUSION</b>EFS and OS of imatinib group were significantly higher than of allo-HSCT group for CML patients of in chronic phase. Imatinib and allo-HSCT had the similar efficacy for CML patients in accelerate and blast phases.</p>
Subject(s)
Adolescent , Adult , Aged , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Young Adult , Benzamides , Therapeutic Uses , Disease-Free Survival , Hematopoietic Stem Cell Transplantation , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Therapeutics , Piperazines , Therapeutic Uses , Protein Kinase Inhibitors , Therapeutic Uses , Pyrimidines , Therapeutic Uses , Retrospective Studies , Transplantation, HomologousABSTRACT
<p><b>OBJECTIVE</b>To validate the conversion factor (CF) for the conversion of BCR-ABL (P210) transcript levels to the international scale in chronic myeloid leukemia (CML).</p><p><b>METHODS</b>In 2012, the international reference laboratory in Adelaide, Australia (IMVS) sent two batches of RNA samples, 30 samples per batch, to Peking University People's Hospital (PKUPH). By comparing BCRABL (P210) transcript levels reported by the two laboratories, CF of PKUPH was calculated and validated by IMVS. In 2013, PKUPH prepared the exchange samples for validation of CF of 9 hospitals who have calculated CFs before. The fresh BCR-ABL (P210) (+) cells were serially diluted by BCR-ABL (P210) (-) cells to prepare 22 kinds of samples with different BCR-ABL transcript levels, each kind had 10 parallel samples. Trizol reagent was added in each tube. Ten hospitals tested BCR-ABL transcript levels of one set of 22 samples. Agreement between BCR-ABL transcript levels of each laboratory and PKUPH was assessed by the Bland-Altman method.</p><p><b>RESULTS</b>PKUPH successfully validated its CF with bias 1.1 fold and 95% limits of agreement between -4.7 and 4.9 fold. Of 9 hospitals whose validation performed by sample exchanges with PKUPH, 6 hospitals successfully validated their CF with bias ≤±1.4 fold and 95% limits of agreement within ±6 fold.</p><p><b>CONCLUSION</b>Validation of CF examined the stability of the detection of BCR-ABL (P210) transcript levels, which was necessary for the valid conversion of BCR-ABL (P210) transcript levels to the international scale in CML.</p>
Subject(s)
Humans , Fusion Proteins, bcr-abl , Genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Reference Standards , Transcription, GeneticABSTRACT
<p><b>OBJECTIVE</b>To explore the clinical and laboratory features of chronic myeloid leukemia (CML) with atypical e14a3 and e19a2 BCR-ABL fusion gene subtypes.</p><p><b>METHODS</b>We retrospectively analyzed a cohort of CML patients with Ph chromosome positive confirmed by cytogenetic and FISH but classical e13a3(b2a2), e14a2(b3a2)and e1a2 fusion transcripts negative identified by conventional real-time quantification RT-PCR (RQ-PCR). Further RQ-PCR was done with the forward primer and reverse primer designed to detect rare atypical BCR-ABL fusion genes including e14a3 and e19a2 transcripts. Direct sequencing analysis was performed on the PCR products and mutations in the BCR-ABL kinase domain were detected. The clinical data of patients were retrospectively analyzed.</p><p><b>RESULTS</b>Six CML patients were found to carry t(9;22) abnormality and BCR-ABL rearrangement confirmed by FISH but classical BCR-ABL fusion genes negative detected by RQ-PCR. Further RQ-PCR and sequencing analysis confirmed the fusion of BCR exon 14 and ABL exon 3 in five CML patients (case 1-5) and the fusion of BCR exon 19 and ABL exon 2 in one CML patient (case 6). E255K and I293T IM-resistant mutations were detected in case 1 and 2, respectively. Among five cases with e14a3 transcripts, four were CML-CP, one CML-AP. Four patients were male and one was female. The median age was 48 years. The patient (case 6) with e19a2 transcripts was 40-year-old female with a diagnosis of CML-CP and PLT count was more than 1 000×10⁹/L. Imatinib (IM) therapy was administer in case 1, 2, 3, 4 and hematopoietic stem cell transplantation (HSCT) was undergone in case 5 after hydroxyurea (Hu) or interferon failure. Case 1 who had E255K IM resistant mutation, responded poorly to IM but obtained a complete cytogenetic remission (CCyR) after a substitution of dasatinib for IM. Case 2 and 3 achieved CCyR 6 months later after IM treatment and had been maintained well with IM despite I293T mutation in case 2. Case 4 attained CCyR 3 months later after IM treatment but relapsed and died soon. Case 5 was still in CCyR after HSCT. Case 6 with e19a2 transcripts got complete hematologic response after Hu treatment and CCyR was achieved soon after IM therapy.</p><p><b>CONCLUSION</b>Incidence of CML with atypical transcripts is extremely low. They could benefit from tyrosine kinase inhibitors or HSCT. Rare and atypical BCR- ABL fusion gene subtypes could be missed by conventional RQ-PCR.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Fusion Proteins, bcr-abl , Genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Classification , Diagnosis , Genetics , Retrospective StudiesABSTRACT
Objective To detect the gene expression of PCA3 and PSA in peripheral blood and urine simultaneously to investigate whether PCA3 combining PSA gene could become new markers for diagnosis of Pca. Methods From June 2009 to December 2009,the initial urine after prostatic massage and the peripheral blood specimens were collected from 37 patients with PCa and 68 patients with BPH that were pathologically confirmed,g patients with urinary stone were used as normal control,the expression of PCA3 and PSA mRNA of mononuclear cells in urine sediments and peripheral blood were detected by fluorescence real-time quantitative PCR,with β-actin mRNA as internal control. Results The sensitivity and specificity of the expression of PCA3 mRNA in peripheral blood for diagnosis of prostate cancer were 48.6％ and 100％ respectively.ROC curve analysis was performed for the PCA3 score and the area under the ROC curve was 0.908.Using 64.6 as the cutoff,the sensitivity was 81.1％ and the specificity was 86.8％.In group with serum tPSA value ＜4 pg/L,the positive rate and negative rate of urinary PCA3 score for diagnosing prostate cancer were 80％ (4/5) and 89.4％ (20/22) respectively.In group with serum tPSA value 4 - 10 μg/L,the positive rate and negative rate of urinary PCA3 score were 66.7％ ( 2/3 ) and 84.2％(16/19) respectively.In group with serum tPSA value ＞ 10 μg/L,the positive rate and negative rate of urinary PCA3 score were 82.8％ (24/27) and 81.5％ (22/27) respectively.The sensitivity of simultaneous detection of PCA3 mRNA in peripheral blood and urinary PCA3 score was 86.5％. Conclusions The expression of PCA3 mRNA in peripheral blood was a specific marker for the diagnosis of PCa.The simultaneous detection of PCA3 mRNA in peripheral blood and urinary PCA3 score could increase the sensitivity for the diagnosis of PCa.