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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 monitor the WT1 mRNA level and its dynamic changes in patients with myelodysplastic syndromes (MDS) after hypomethylating agents (HMA) , as well as to assess the significance of WT1 mRNA levels and its dynamic changes in evaluating the efficacy of HMA and distinguishing the disease status of heterogeneous patients with stable disease (SD) .@*Methods@#Bone marrow or peripheral blood samples of 56 patients with MDS who underwent hypomethylating agents (≥4 cycles) from November 2009 to March 2018 were tested by real-time quantitative polymerase chain reaction (PCR) to detect the expression of WT1 mRNA, and to observe the correlation between the dynamic changes of WT1 mRNA expression and clinical efficacy and prognosis of patients.@*Results@#WT1 mRNA expression levels of MDS patients decreased significantly after 3 cycles of hypomethylating agent treatment. Besides, the WT1 mRNA expression levels of patients increased significantly after diseases progression. According to the dynamic changes of WT1 mRNA expression levels during SD, 45 cases could be further divided into increased group and non-increased group. In those SD patients with increased WT1 mRNA expression level, the ratio of suffering disease progression or transformation to AML was 95.65% (22/23) , whereas the ratio turned to be 9.09% (2/22) for the non-increased group (χ2=33.852, P<0.001) . Compared with those SD patients reporting no increase in WT1 mRNA expression level, the overall survival[17 (95%CI 11-23) months vs not reached, P<0.001] and progression-free survival [13 (95%CI 8-18) months vs not reached, P<0.001] of those SD patients reporting increase in WT1 mRNA expression level were significantly shorter.@*Conclusion@#WT1 mRNA expression level is a useful indicator to assess the efficacy of hypomethylating agents in MDS patients. Especially in patients with SD, detection of the changes in WT1 mRNA expression level is able to predict disease progression and help to make clinical decision.
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Objective@#To investigate the effects of artesunate treatment on chronic graft-versus-host disease (cGVHD).@*Methods@#Recipient BALB/c mice received 8 × 106 bone marrow cells with 8×106 spleen cells from B10D2 mice. Artesunate solubilized in acetone was injected intraperitoneally every day at the dose of 1 mg/kg at Day 28 after BMT. The clinical scores, survival and histopathological damage were analyzed. The frequency of Th17 and Tregs in PB and spleens from the mice were evaluated by flow cytometry. In addition, CD4+ T cells from the spleens of mice were cultured in vitro, then stimulated with artesunate, the frequency of Th17 and Tregs in these splenocytes were evaluated by flow cytometry.@*Results@#Artesunate administration diminished clinical and histopathological damage, and improved the survival of cGVHD mice[(46.57±7.83)% vs (55.71±6.99)%, χ2=5.457, P=0.020]; Artesunate contributed to Tregs development [(4.45±0.04)% vs (8.40±0.23)%, t=15.679, P<0.001; (6.62±0.24)% vs (10.48±0.48)%, t=6.587, P=0.003] while decreased Th17 cells [(1.51±0.18)% vs (0.58±0.19)%, t=7.233, P<0.001; (1.48±0.38)% vs (0.71±0.18)%, t=3.653, P=0.011] expressions in both PB and spleens, and decreased the Th17/Treg ratio (0.34±0.05 vs 0.09±0.03, t=7.621, P=0.002; 0.19±0.03 vs 0.06±0.02, t=6.993, P=0.002). Moreover, artesunate suppressed the Th17 cells expressions [(0.82±0.37) % vs (3.39±1.22) %, t=4.044, P=0.007] and contributed to Tregs development [(34.63±1.29) % vs (14.28±1.69) %, t=19.119, P<0.001], and also decreased the Th17/Treg ratio (0.24±0.09 vs 0.02±0.01, t=4.780, P=0.003) in vitro.@*Conclusions@#Artesunate suppressed the Th17 cells expressions and contributed to Tregs development, which provided new sights into the development of a novel drug for cGVHD, e.g., artemisinin.
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AIM:To investigate the expression and potential role of complement 5a receptor (C5aR) in chro-nic graft-versus-host disease (cGVHD).METHODS:The expression of C5aR on lymphocytes and the frequency of CD4+CD25+ Foxp3+ regulatory T cells (Tregs) in 20 cGVHD patients and 9 healthy donors was detected by flow cytometry.The correlation between the expression of C5aR and the percentage of Tregs in the cGVHD patients was analyzed.In addition, the splenocytes from the mice were cultured in vitro, and stimulated these splenocytes with recombinant mouse C5a protein (rmC5a).The peripheral blood mononuclear cells (PBMCs) from cGVHD patients were cultured in vitro, which was inhibited by C5aR antagonist (C5aRA).The frequency of Tregs in these splenocytes and the PBMCs were evaluated by flow cytometry.RESULTS:The expression of C5aR on the lymphocytes was significantly increased in the cGVHD patients compared with the healthy donors, while the percentage of Tregs was markedly lower in the cGVHD patients.The expression of C5aR was negatively correlated with the percentage of Tregs.Furthermore, the development of Tregs was suppressed by rmC5a stimulation, but was promoted by C5aRA in vitro.CONCLUSION:C5aR elevation is associated with Treg reduction in cGVHD, indicating that C5aR may play a potential role in suppressing Tregs, resulting in the incidence of cGVHD.
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AIM:To investigate the role of mesenchymal stem cell-induced regulatory dendritic cells ( MSC-DCregs) in mouse acute graft-versus-host disease( aGVHD) model.METHODS: Bone marrow cells from BALB/c ( H-2 d ) mice were isolated and were induced to differentiate into DCs.The DCs were selected by flow cytometry, and after 10 d co-culture with MSCs, they were induced to be MSC-DCregs.Male 8-week-old C57BL/6 (H-2b) mice were used as do-nor mice.The female 8-week-old BALB/c (H-2d) mice, who had received 100 cm source-skin distance, 30 cm ×30 cm radiation field, 700 cGy total body irradiation (TBI) pretreatment were used as recipient mice.The recipients were divided into 5 groups:control group, TBI group ( injected with medium only) , bone marrow transplantation group ( injected with 1 ×107 bone marrow cells), aGVHD group (injected with 1 ×107 bone marrow cells and 1 ×107 spleen cells), and MSC-DCregs group (injected with 1 ×107 bone marrow cells, 1 ×107 spleen cells and 1 ×106 MSC-DCregs).The white blood cell count, recipients’ chimerism, clinical evaluation of aGVHD, survival analysis and pathological changes were deter-mined.RESULTS:Hematopoieic recovery was seen at 10 d after transplantation.The recipients’ chimerism was parallel to the donors’ at 30 d.The median survival time of the mice in aGVHD group and MSC-DCregs group was 27 d and 33 d, and the survival rates at 30 d were 20% and 100% (P<0.01), respectively.The clinical scores of the mice in MSC-DCregs group were lower than those in aGVHD group ( P<0.01) .Moreover, the pathological changes in the skin and liver of the mice in MSC-DCregs group were less serious than those in aGVHD group.CONCLUSION: The MSC-DCregs in-duce an aGVHD tolerance in vivo, and further research of its mechanism is still in great necessary.
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<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
<p><b>OBJECTIVE</b>To investigate the accuracy and consistency of the detection of BCR-ABL tyrosine kinase domain point mutation among different laboratories.</p><p><b>METHODS</b>Every one of 6 laboratories prepared 10 cDNA samples from tyrosine kinase inhibitors resistant BCR-ABL (P210 or P190) positive patients'bone marrow or peripheral blood. Each cDNA sample was divided into 6 aliquots and delivered to the laboratories. All 6 laboratories tested BCR-ABL point mutations of 60 samples according to their own protocols. Peking University People's Hospital analyzed the comparison results based on both the reports and sequencing chromatogram from all laboratories.</p><p><b>RESULTS</b>All laboratories reported the same nucleotide and corresponding amino acid mutations in 37 samples (61.7%）. Of 60 samples, 53 had confirmed mutation types, and a total of 23 types were included; 1 had no mutation; mutation types of 6 samples could not be determined because of the big differences among chromatograms from different laboratories. Low percentages of mutants were significantly related to results inconsistency (P=0.008). Inconsistent result of one sample was caused by the unique chromatogram of the mutant L248V, and one by the non-coverage amplification of PCR product from different laboratories. Amplification was failed in 3 samples. Testing or sequencing mistakes occurred in 7 samples. The differences in the mutant percentages among laboratories were less than 20% in the 80.6% of samples with confirmed results. Low internal control gene copies (ABL<10 000) were significantly related to both failed amplification and big differences among chromatograms from different laboratories (P=0.005 and <0.001, respectively).</p><p><b>CONCLUSION</b>Problems in the clinical routine detection of BCR-ABL point mutation could be exposed and improvement could be achieved by sample exchange and comparison. Low percentage of mutant is the main reason which causes the discrepancy of BCR-ABL point mutation results among different laboratories.</p>
Subject(s)
Humans , Bone Marrow , DNA Mutational Analysis , Fusion Proteins, bcr-abl , Genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Point Mutation , Polymerase Chain ReactionABSTRACT
<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
Objective To analyze the T- cell receptor excision DNA circles (TRECs) level and evaluate the thymic recent output nave T cells function in patients with acute promyelocytic leukemia (APL). Methods Quantitative detection of TRECs in DNA of peripheral blood mononuclear cells (PBMC) from 9 cases with APL were preformed by real- time PCR (TaqMan) analysis. The TRECs- number was related to the number of T- cells by determination of the number of CD-3 positive cells. 14 normal individuals served as controls. Results In comparison with normal individuals [ (4.10?3.65) copies/1000 PBMC and (6.36?5.28) copies/1000 CD+3 cells], a dramatic reduction of TRECs values in patients with APL [ (0.13?0.22) copies/1000 PBMC and (0.77?1.60) copies/1000 CD+3 cells] could be found (P =0.0004, P =0.0005). Conclusions This is, to our knowledge, the first description of TRECs level which was markedly reduced in APL patients.
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Objective:To investigate the gene expression of TCR V? subfamily T cells and clonality in peripheral blood CD4+ and CD8+ cells from patients with chronic myelogenous leukemia-chronic phase(CML-CP).Methods:The complementarity determining region 3(CDR3)of TCR V? subfamily genes in peripheral blood mononuclear cells(PBMCs),sorted CD4+ and CD8+ cells from 19 CML-CP patients were amplified using RT-PCR,the products were further by genescan analysis to identify the clonality of T cells.Results:Only 1 to 21 V? subfamilies were detected in peripheral blood CD4+ and CD8+ cells from patients with CML.The most frequent expression of V? subfamily was V?13 followed by V?9.Clonal expanded T cells in some V? subfamilies could be identified in both CD4+ and CD8+ cells,especially in CD8+ cells.The clonal expanded of V?21 subfamily in CD4+ cells and V?11 subfamily in CD8+ cells were identified most frequently.Conclusion:The restricted distribution of TCR V? repertoire has been found in CD4+ and CD8+ cells in patients with CML.Clonal expanded CD4+ cells and CD8+ cells have been identified which may relate to host immune response to CML antigen and may play a important role in anti-CML effect.