Clinical Characteristics and Prognosis of Acute Myeloid Leukemia Patients with inv(16)/t(16;16)(p13.1;q22) and/or CBFβ-MYH11 / 中国实验血液学杂志

OBJECTIVE@#To summarize the clinical and laboratory characteristics of patients with acute myeloid leukemia (AML) with inv(16)/t(16;16) (p13.1;q22), and to analyze the risk factors affecting the prognosis of the patients.@*METHODS@#AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFβ-MYH11+ admitted to the Department of Hematology, The First Affiliated Hospital of Soochow University from January 1, 2008 to October 30, 2019 were retrospective analyzed, the clinical and laboratory indicators, as well as treatment plans and efficacy evaluations of the patients were all recorded. Furthermore, related factors affecting the overall survival (OS) and event-free survival (EFS) of the patients were analyzed.@*RESULTS@#Among 151 AML patients with inv(16)/t(16;16) (p13.1;q22) and/or CBFβ-MYH11+, the percentage of additional chromosomal abnormalities was about 27.8%, and the most common additional chromosomal abnormality was +22 (33/151, 21.8%), followed by +8 (11/151, 7.3%). There were 112 patients with perfect NGS examination, and the result showed the most common accompanying gene mutations were KIT mutation (34/112, 30.4%) and FLT3 mutation (23/112, 20.5%). Univariate analysis showed that factors affecting EFS included: NE≤0.5×109/L (P=0.006) and combined K-RAS mutation (P=0.002); Factors affecting OS included: Age≥50 years old (P<0.001) and NE≤0.5×109/L (P=0.016). Multivariate analysis showed that NE≤0.5×109/L (P=0.019) was the risk factors affecting OS. The proportion of bone marrow eosinophilia (BME)≥10.00% (P=0.029) was the risk factors affecting EFS.@*CONCLUSION@#The prognosis for those newly diagnosed AML patients who were of advanced age, the high proportion of bone marrow eosinophils, K-RAS mutations, and agranulocytosis is poor. The treatment plans can be adjusted in the early stage to improve the prognosis of such patients.

Clinical Significance of Common Gene Mutations in 53 Patients with Acute Myeloid Leukemia Harboring 11q23/MLL Rearrangements / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical significance of AML patients with 11q23/MLL rearrangement, and to evaluate the effect of those mutations on the AML patients.@*METHODS@#53 cases involving translocations of chromosome 11q23 were identified by chromosome banding analysis. MLL rearrangements were detected by fluorescence in situ hybridization and/or multiplex nested PCR. The samples were screened for mutations in the candidate genes FLT3-ITD, FLT3-TKD, TET2, N-RAS, ASXLI, EZH2, DNMT3, C-Kit, NPM1, WT1, CEBPA by using genomic DNA-PCR and deep-sequencing.@*RESULTS@#21/53 MLL-rearranged AML cases showed at least one additional chromosomal aberrations. The most common additional aberration was +8. Gene mutations were observed in 23 cases (43.4%) and most cases showed singal mutation. N-RAS mutation was more frequent (8 cases, 15.1%), followed by WT1 mutation in 4 cases (7.5%), FLT3-ITD mutation in 3 cases, ASXL1 mutation in 2 cases, DNMT3A mutation in 2 cases, EZH2 mutation in 1 case, c-Kit17 mutation in 1 case, FLT3-TKD mutation in 1 case, and FLT3-ITD and TKD mutation coexistent in 1 case. No mutation was detected in CEBPA, NPM1, C-KIT8, TET2. Median OS for gene mutated patients was 8.5 months and 13 months for no mutated patients. Median OS for patients who received hematopoietic stem cell transplantation (HSCT) was 22.5 months and 7.5 months for patients who olny received chemotherapy.@*CONCLUSION@#A relatively high mutation frequency is observed in AML patients with 11q23/MLL rearrangements and most cases shows single mutation. The RAS signaling pathway alterations are most common. Gene mutation does not affect the OS of these patients, who show poor prognosis. A significantly higher Hb at initial diagnosis in FLT3 mutated patients is significantly higher than that in FLT3 wild-type cases. Patients who underwent HSCT show a better prognosis than those only received chemotherapy.

Analysis of Factors Influencing Overall Survival of MDS Patients Transplanted with HSCs / 中国实验血液学杂志

OBJECTIVE@#To analyze the effect of clinical features, routine laboratory examination and related gene mutation on the OS of patients with myelodysplastic syndrome (MDS) after hematopoietic stem cell transplantation (HSCT).@*METHODS@#121 patients diagnosed as MDS and underwent hematopoietic stem cell transplantation in the First Affiliated Hospital of Soochow University from October 2013 to August 2018 were selected. Basic information of the patients was collected, and blood cells, bone marrow blasts at initial diagnosis, chromosomal karyotypes and gene mutations of the patients were detected.The effect of different factors on overall survival (OS) was analyzed by statistical method.@*RESULTS@#Kaplan-Meier univariate analysis shows that OS was significanly different among different age groups. The 3-year OS rate of patients aged 0-29 years was (83.3±7.7) %, the 3-year OS rate in patients aged 30-49 years was (58.1±7.7 %), and the 3-year OS rate of patients aged 50-69 years was (31.0±22.6) %, which was statistically different (P＜0.05) between different groups. There were also significant differences in OS among patients with different transplantation types. 3-year OS rate: HLA-matched sibling HSCT＞unrelated HLA-matched HSCT＞haploidentical HSCT＞micro HSCT. The OS rate of patients with bone marrow blasts≥10% seems lower than blasts＜10%, but there was no statistical difference.The 3-year OS rate of patients with chromosomal karyotype complex abnormality was (47.7±11.5) %, and that of patients without complex abnormality was (80±4.2) % which was statistical difference (P＜0.05). Patients with DNMT3A, NRAS, TP53 and GATA2 mutations had shorter OS time compared with patients without mutation of these genes, which shows statistically significant (P＜0.05). COX multivariate analysis showed that age, chromosome karyotype, DNMT3A, TET2, GATA2 and NRAS were the independent factors influencing OS of patients after HSCT, with statistically significant difference.@*CONCLUSION@#age of patients, donor selection of HSCT, chromosome karyotype, DNMT3A, NRAS, TP53, GATA2 and TET2 gene mutations are all independent factors affecting the OS of patients after HSCT. Therefore, the assessment of the OS of MDS patients with transplantation requires comprehensive consideration.

Clinical Features and Prognosis of 188 Patients with Acute Myeloid Leukemia-M / 中国实验血液学杂志

OBJECTIVE@#To summarize the clinical characteristics of patients with acute myeloid leukemia-type M (AML-M) and analyze the factors affecting the prognosis.@*METHODS@#One hundred eighty-eight AML-M patients were retrospectively analyzed for the following parameters including peripheral blood, immune phenotypes, fusion genes and cytogenetics to explore their significance for the overall survival (OS) and progression-free survival (PFS). The prognostic factors were also analyzed.@*RESULTS@#Among 188 patients with AML-M, the chromosomal abnormality with t (8;21), normal chromosome and other abnormalities accounted for 37% (70/188), 41% (77/188) and 22% (41/188), respectively. For the immunopheno typing of M patients, the hematopoietic progenitor cell differentiation antigen CD117 (96.1%) were mainly expressed, CD34 (81.6%) and HLA-DR (55.9%), and myeloid-associated antigen of CD13 (90.5%) and CD33 (89.4%) were also highly expressed. There were lymphoid-associated antigens expressed in some patients, among which the positive expression rate of CD19 was highest (29.6%), and the next was CD7 (28.5%). The most common accompanied mutations was FLT3 mutation (30.2%). The univariate analysis showed that the patients at age＜50 years old, without extramedullary infiltration, with positive expression of CD19, NPM-1 (-), CEBPA double mutation(+), and HSCT were significant superior in OS and PFS (P＜0.05); the multivariate analysis showed that the patient at age＜50 years old, without extramedullary infiltration, with positive expression of CD19 and CEBPA double mutation (+) were significant superior in OS and PFS (P＜0.05). The analysis indicated that the Karytypes affected only OS (P＜0.05), while the NPM-1 gene mutation positive affected only PFS (P＜0.05). The univarate analysis of factors affecting the survival in 70 AML-M patients with t (8;21) abnormatity showed that the C-KIT gene mutation was a poor factor for OS and PFS.@*CONCLUSION@#The clinical characteristics are different between M patients with different karyotype, and prognostic analysis shows that the karytypes have an impact on overall survival; age, extramedullary infiltration, CD19 expression and CEBPA double mutation are also the main factors impacting the prognosis of patients.

All-Trans Retinoic Acid and Decitabine Synergistically Induce Anti-Leukemia Effect on U937 Cell Line and Newly Diagnosed Elder AML Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of all transretinoicacid(ATRA) combined with decitabine (5-Aza-2'-deoxycytidine;DAC) on DNA methylation and gene expression of p16INK4a (p16) and retinoic acid receptor β (RARβ), and to explore their combined anti neoplastic effect on U937 cells and newly diagnose delder acute myeloid leukemia(AML) patients.</p><p><b>METHODS</b>The expression levels of p16 and RARβ were determined by qRT-PCR and Western blot. Methylation-specific PCR was used to analyze their methylation status. WST-1 and flow cytometry were performed to detect growth inhibition, differentiation, apoptosis and cell cycle of U937 cells respectively.</p><p><b>RESULTS</b>The expression p16 and RARβ was down-regulated by promoter hypermethylation in newly diagnose delder AML patients and U937 cells. Combination treatment of ATRA and DAC induced DNA hypomethylation as well as gene expression of p16 and RARβ, which contributed to the growth inhibition, differentiation, apoptosis and cell cycle arrest of U937 cells. In addition for elder AML patients intolerable to standard chemotherapy, the combination regimen of ATRA and DAC showed antineoplastic activity accompamied by up-regulation of p16 and RARβ expression and decrease of bone marrow blast, moreover the parients showed good tolerence to the reginen.</p><p><b>CONCLUSION</b>The regimen of ATRA combined with DAC as the combination therapeutic strategy for inducing differentiation and demethylation possesses the anti-AML potency, and contributes to optimizing the therapeutic strategy for elder AML patients and promoting the clinical prognosis.</p>

Effect of Additional Chromosomal Abnormalities on the Outcome of CML-CP Patients Receiving TKI Therapy / 中国实验血液学杂志

<p><b>OBJECTIVES</b>To explore the effect of additional chromosomal abnormalities on the prognosis and outcome of CML-CP patients receiving imatinib therapy.</p><p><b>METHODS</b>The clinical and genetic data of 589 CML-CP patients receiving imatinib treatment between May 2009 and October 2014 in the 1st Affiliated Hospital of Soochow University were analyzed, the 589 patients were divided into 5 groups according to the karyotypes at the initial diagnosis. The OS(overall survival), PFS (progression-free survival), EFS (event-free survival), Cumulative MMR (major molecular remission) and Cumulative CCyR (complete cytogenetic remission) were calculated by using the Kaplan-Meier method and compared by using the log-rank text by Graphpad 6.0. The χ test was used to compare the frequency of optimal molecular response at 3, 6, 12 months among the 5 groups.</p><p><b>RESULTS</b>There was significant difference about the frequency of optimal molecular response at 3 and 6 months between CML-CP patients with additional chromosomal abnormalities and those with classic t(9;22) ［50%（12/24） vs. 73.94%(261 /353), P<0.05; 50%(10 /20) vs. 72.05%(232 /322) (P<0.05)］, and the same significant difference was found at 6 months between the group with variant translocations and that with classic t(9;22) ［53.3% (16 /30) vs. 72.05%(232 /322) (P<0.05)］. The P values of cumulative CCyR (P<0.05) and EFS (P<0.01) for 4 years were statistically significant between CML-CP patients with additional chromosomal abnormalities and the other 4 groups. Compared one to another, there was the significant difference in cumulative CCyR and EFS for 4 years between CML-CP patients with additional chromosomal.abnormalities and those with classic t(9;22) (47.25% vs. 84.01%)(P<0.05); (75.03% vs. 90.01%)(P<0.01).</p><p><b>CONCLUSION</b>The additional chromosomal abnormalities influence the outcome of CML-CP patients receiving imatinib treatment, which make poor prognosis.</p>

CCL2 Protein Regulates Migration and Invasion of THP-1 cells by Autosecreting Inflammatory Chemokines / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects and mechanism of CCL2 on the migration and invasion of human leukemia cell THP-1.</p><p><b>METHODS</b>The CCL2 gene was recombined with the transfer plasmid-PLVX and transfected into THP-1 cells. The CCL2 expression at RNA level was detected by RT-PCR, the CCL2 expression at protein level was determined by Western blot and ELISA, the influence of overexpression of CCL2 recombinant protein and THP-1 cells on the migration and invasion ability of THP-1 cells was analyzed by transwell migration and invasion tests, the PCR-array of migration-related cytokines was used to clarify the patential mechanism.</p><p><b>RESULTS</b>With the Trans-Matrigel assay, the concentration of CCL2 in THP-1 transfected with CCL2 in the upper cells was higher than that in the lower cells, meanwhile, the invasion ability of CCL2-transfected THP-1 cells decreased. Increasing recombinant protein of CCL2 (rpCCL2) in the lower cells promoted migration of THP-1 cells. Migration RT2 profiler PCR array showed that the cells treated with rpCCL2 had higher levels of expression of CCL2, EPX, SPP1, CX3CL1 and CXCL13, as compared with control group.</p><p><b>CONCLUSION</b>CCL2 affects the migration and invasion of THP-1 by autosecreting a series of inflammatory chemokines.</p>

Over-expression of C3AR1 Promotes HL-60 Cell Migration and Invasion / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of C3AR1 on migration and invasion of HL-60 cells and its mechanism.</p><p><b>METHODS</b>The HL-60 cells overexpresing C3AR1 was constructed, and the HL-60-C3AR1 cell line was validated by real-time PCR and Western blot. The migration and invasion assay were performed by using transwell system, the PCR Array and flow cytometry were employed to reveal the potential mechanisms.</p><p><b>RESULTS</b>C3AR1 gene was significantly expressed in AL cell lines, especially in acute myeloid leukemia(AML cell lines). C3a and SDF-1 together promoted migration and invasion of C3AR1 over-expressed HL-60 cells. The PCR array detection found that 16 genes (including CCL2) were significantly upregulated and 4 genes were significantly down-regulated. However, the expression of CXCR4 did not significantly change after treated by C3a and SDF-1 together.</p><p><b>CONCLUSION</b>Over-expression of C3AR1 shows the effects on the migration and invasion of HL-60 cells under the treatment of C3a and SDF-1 together, which may be mediated by the regulation of CCL2 and other genes related to chemotactic factors.</p>

Prognostic Significance of the Percentage of Blasts with CD34/CD38/CD123Phenotype in Acute Myeloid Leukemias / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the percentage of blasts with the CD34/CD38/CD123phenotype in de novo acute myeloid leukemia (AML) patients and analyse its correlation with prognosis.</p><p><b>METHODS</b>The percentage of CD34/CD38/CD123cells in the blast population of 148 newly diagnosed patients with AML was determined by using flow cytometry and its correlation with complete response, disease-free survival and overall survival were evaluated.</p><p><b>RESULTS</b>The median percentage of CD34/CD38/CD123cells in newly diagnosed patients was 2.8% (ranged from 0.01 to 67%). The high expression of CD34/CD38/CD123in AML patients positively correlated with the NPM1 wild-type (χ=5.194,P<0.05), but did not relate with the positive FLT3-ITD mutations (χ=0.418,P>0.05). Further multivariable analysis showed that the higher expression of the CD34/CD38/CD123was associated with lower complete remission (P<0.05), worse disease-free survival(P<0.01) and shorter overall survival(P<0.01) in AML patients.</p><p><b>CONCLUSION</b>The percentage of CD34/CD38/CD123cells at diagnosis significantly correlates with the response to treatment and survival. This prognostic marker may be used to rapidly identify the risk of treatment failure in clinical practice.</p>

Autophagy Activity of CD34+ Cells in MDS Patients and Its Clinical Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the autophagy activity of CD34+ cells in bone marrow of MDS patients and its clinical significance.</p><p><b>METHODS</b>The activity of autophagy in bone marrow CD34+ cells from 20 MDS patients, 20 non-malignant anemia patients and 5 AML patients admitted in our hospital from October 2012 to March 2014 was detected by flow cytometry (FCM).</p><p><b>RESULTS</b>The autophagy activity in low risk MDS patients and non-malignant anemia patients were both significantly higher than that in both high risk MDS and AML patients (P<0.05), and more interestingly, the autophagy activity in MDS negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r=-0.877) .</p><p><b>CONCLUSION</b>The autophagy activity CD34+ cells in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores, indicating that the decrease of autophagy activity maybe accelerate the genesis and development of MDS and relate with the prognosis of MDS patients.</p>

Differentiation of K562 Cells Induced by Pulsatilla Saponin A into Erythroid Lineage / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the differentiation-inducing potentiality of Pulsatilla saponin A on K562 cells.</p><p><b>METHODS</b>Pulsatilla saponin A of different concentrations was used to treat K562 cells; the benzidine staining and the hemoglobinometry were applied to measure the change of hemoglobin content; the flow cytometry (FCM) was used to detect the expression of CD71 and GPA on K562 cells.</p><p><b>RESULTS</b>K562 cells treated with 4 µg/ml pulsatilla saponin A differentiated into the erythroid lineage. With the treatment of pulsatilla saponin A, the hemoglobin content in K562 cells increased significantly; CD71 and GPA expression on the K562 cell surface were up-regulated.</p><p><b>CONCLUSION</b>Pulsatilla saponin A can induce K562 cells to differentiate into erythroid lineage.</p>

Clinical and Laboratorial Characteristics of Primary Acute Myeloid leukemia with Philadelphia Chromosome and Inversion 16 / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To summarize the clinical characteristics as well as diagnosis and treatment in 1 case of acute myeloid leukemia(AML) with coexpression of Ph and inv(16).</p><p><b>METHODS</b>A series of clinical tests, the cellular morphological, immunological, cytogenetic and molecular biological examinations of leukemia cells were performed.</p><p><b>RESULTS</b>The clinical characteristics of this patient were very common. The cellular morphology is similar to the AML with inv(16). The leukemia cells were stained positively for CD13, CD33, CD34, CD117 and HLA-DR. Karyotypic analysis showed a complex chromosome abnormality including inv(16) and Ph, and the FISH analysis showed that the percentage of rearrangement of CBFβ allele was over that of the BCR-ABL fusion signals. The obvious adverse events did not occur in this patient within 3 years.</p><p><b>CONCLUSION</b>Ph as secondary aberration of inv(16) rarely occures in primary AML cases, and so far there have not been the clear criteria of diagnosis and treatment. The cytogenetic and molecular biology could provide the basis for diagnosis. Moreover, autologous hematopoietic stem cell transplantation combined with imatinib probably is one of the effective treatment methods.</p>

Correlation between expression of SIL-TAL1 fusion gene and deletion of 6q in T-cell acute lymphoblastic leukemia / 中国实验血液学杂志

The present study was designed to investigate the prevalence and clinical significance of SIL-TAL1 rearrangements in T-cell acute lymphoblastic leukemia (T-ALL). The incidence of SIL-TAL1 rearrangements was analyzed by nest real-time quantitative polymerase chain reaction (RT-PCR) in 68 patients with T-ALL. Karyotypic analysis was performed by conventional R-banding assay and array-based comparative genomic hybridization (array-CGH). The results showed that SIL-TAL1 rearrangements were identified in 10/26 (38.5%) pediatric and 2/42 (4.8%) adult T-ALL cases, which indicate a pediatric preference for SIL-TAL1 rearrangements in T-ALL. Two different transcripts were detected in 6/12(50%) T-ALL samples. Abnormal karyotypes were detected in 6 out of 11 cases (54.5%) and a deletion of the long arm of chromosome 6 was observed in 4 cases. Array-CGH results of 2 T-ALL cases with SIL-TAL1 rearrangement revealed that this fusion gene was resulted from a cryptic deletion of 1p32, and the overlap region of 6q deletion was 6q14.1-16.3. These cases with SIL-TAL1 fusion had a higher white blood cell (WBC) count and higher serum levels of lactate dehydrogenase (LDH) than cases without SIL-TAL1 fusion. It is concluded that SIL-TAL1 rearrangements are associated with loss of heterozygosity of chromosomal 6q, and SIL-TAL1-positive patients are younger than SIL-TAL1-negative patients. In contrast to the cases without SIL-TAL1 fusion, there are many adverse prognostic factors in the cases with SIL-TAL1 fusion, such as higher WBC count and higher LDH levels.

Frequently ABL kinase domain G:C→A:T mutation and uracil DNA glycosylase abnormal expression in TKI-resistant acute lymphoblastic leukemia of Chinese population / 中国实验血液学杂志

Most Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) patients often show rapid recurrence and development of ABL kinase domain (KD) mutation after tyrosine kinase inhibitor (TKI) treatment. To further investigate the mechanism of Ph(+) ALL fast relapse after TKI treatment, ABL KD mutation in 35 Chinese Ph(+) ALL with TKI resistance was detected by direct sequencing. The results showed that 77.1% (27/35) Ph(+) ALL patients with TKI resistance had ABL KD mutation and 55.6% (15/27) Ph(+) ALL patients with ABL KD mutation had T315I. Interestingly, 77.8% (21/27) Ph(+)ALL showed ABL mutation G: C→A:T, including T315I, E255K and E459K. Furthermore, all the Ph(+) ALL patients with two or more ABL KD mutations collaborated with complex chromosome abnormality and all the TKI-resistant Ph(+) ALL patients, whose karyotype progressed from simple t (9;22) into complex, developed ABL KD mutation. Moreover, the expression level of uracil-DNA glycosylase UNG2, which inhibits G:C→A:T transition in genomic DNA, decreased in Ph(+) ALL with TKI-resistance compared to that in newly diagnosis Ph(+) ALL. It is concluded that there is a high frequent ABL KD G:C→A:T mutation and a high genomic instability in Chinese TKI-resistant Ph(+) ALL. In addition, the decreased UNG2 expression in TKI-resistant Ph(+) ALL probably contributes to their high rate of ABL KD G:C→A:T mutation.

Clinical significance of common leukemia gene mutations in patients with acute promyelocytic leukemia / 中国实验血液学杂志

This study was aimed to explore whether multiple common gene mutations of leukemia synergistically involved in acute promyelocytic leukemia (APL) pathogenesis, and to investigate their relevance to clinical features, cytogenetics and molecular risk stratification. 84 specimens of admitted de novo APL patients from February 2005 to October 2010 were collected, the gene mutations of bone marrow mononuclear cells and clinical features of mutation-positive patients were analyzed by genomic DNA-PCR. The results indicated that the prevalence of mutations was 60.7% (51/84), in which the mutations with the highest incidence were found as FLT3-ITD, reaching 27.4% (23/84). Next, there were 12 cases WT1 mutation, 9 for FLT3-TKD, 7 for TET2, 5 for N-RAS, 4 for ASXL1, 2 for EZH2 mutation and 1 positive case in MLL-PTD, IDH1 and CBL mutation respectively. No mutation was found in other JAK1, DNMT3, c-Kit, NPM1, IDH2, RUNX1 and JAK2 (V617F) common leukemia-related genes. Combined analysis with clinical data demonstrated that the patients with FLT3-ITD mutation displayed higher white blood cell counts, while the patients with N-RAS mutation showed lower platelet counts. Overall survival of these patients was obviously shorten as compared with patients with wild-type. This difference between mutant and wild-type of all above mentioned cases was statistically significant (P < 0.05). The difference between APL with simple t (15;17) and additional abnormal karyotype was not statistically significant. It is concluded that the FLT3-ITD mutation is recurrent genetic change in APL, and together with N-RAS mutation indicates poor prognosis. Additional abnormal karyotype does not associate with prognosis of APL.

The different characteristics of ABL kinase domain mutation in the Chinese Han nationality imatinib resistant Philadelphia chromosome-positive acute lymphoblastic leukemia and chronic myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To identify the distribution and differentiation of ABL kinase domain mutation in the Chinese Han nationality imatinib resistant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL).</p><p><b>METHODS</b>Bone marrow or peripheral blood samples of 112 imatinib resistant CML patients and 21 Ph(+)ALL patients were obtained from the first affiliated hospital of Soochow university according to local law. Total RNA was extracted from the mononuclear cells using a TRIzol reagent. ABL kinase domain (KD) mutation was detected by direct sequencing.</p><p><b>RESULTS</b>Of the 112 imatinib resistant CML patients, 54.46%(61 cases) had ABL KD mutation. Twenty-three mutants were identified in 20 amino acid sites and 23.21% (26 cases) ABL KD mutations were in P-loop region. ABL KD mutations were also detected in 71.43% (15 cases) imatinib resistant Ph(+)ALL patients, with 10 mutations in 8 amino acid sites. The most frequent mutation was T315I (28.57%), followed by E255K/V (19.05%) and Y253F/H (14.29%). The frequency of T315I was much higher in imatinib resistant Ph(+) ALL than that in imatinib resistant CML (P = 0.001). Ph(+)ALL with additional chromosomal aberrations also had a higher rate of ABL KD mutation than that of CML (P = 0.010). Ph(+)ALL gained ABL KD mutation faster than CML (P < 0.010).</p><p><b>CONCLUSION</b>Chinese imatinib resistant CML and Ph(+)ALL patients had different characteristics in ABL KD mutation. The rate of ABL KD mutation in Ph(+)ALL with additional chromosomal aberrations was much higher than that of CML with additional chromosomal aberrations.</p>

A multicenter comparison study on the quantitative detection of bcr-abl (P210) transcript levels in China / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the comparability of bcr-abl (P210) transcript levels detected in different hospitals.</p><p><b>METHODS</b>Ten hospitals in China took part in the four times of sample exchange and comparisons from April, 2010 to August, 2011. The exchange samples were prepared by Peking University People's Hospital. Firstly, the BCR-ABL (P210)(+) cells from a newly diagnosed chronic myeloid leukemia patient were 10-fold serially diluted by BCR-ABL (P210)(-) cells and they covered 4 magnitudes. Then, TRIzol reagents were thoroughly mixed with cells in each tube. Every 12 samples (three samples per magnitude) were sent to the other 9 hospitals. The cell number of each sample was 8×10(6). The detection of bcr-abl transcript levels by real-time quantitative PCR were performed in every hospital according to their own protocols. Conversion factors (CF) were calculated using regression equation.</p><p><b>RESULTS</b>Differences in bcr-abl transcript levels did exist among results of 10 hospitals in each comparison. In general, the results of the most of hospitals were in line with the dilutions of cells. CF of every hospital fluctuated. Three hospitals had relatively stable CF, and their ranges were 2.8 - 5.2, 1.2 - 2.8 and 2.2 - 6.8, respectively; two hospitals had unstable CF with ranges 0.76 - 7.0 and 2.1 - 18.7; three hospitals couldn't be calculated CF one or two times because of the significant deviation of the results from the actually bcr-abl transcript levels, and their ranges of CF which could be calculated were 1.9 - 19.2, 3.6 - 7.6 and 0.18 - 14.7; One hospital only had two CF (3.3 and 5.0) because of the replacement of an important reagent during the period of comparisons.</p><p><b>CONCLUSIONS</b>Comparability of bcr-abl (P210) transcript levels between different hospitals could be achieved through CF which acquired by sample exchange and comparison. The stable and reliable detection system is the premise to acquire correct CF.</p>

Transfection of HL-60 cells by Venus lentiviral vector / 中国实验血液学杂志

In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.

Effect of AML1-ETO fusion protein on the expression of BCL-2 / 中国实验血液学杂志

This study was aimed to investigate the effect of AML1-ETO fusion protein on the anti-apoptotic gene BCL-2 in leukemic cells and to explore its role in leukemogenesis. The apoptotic levels of U937-WT, U937-Mock and U937-A/E1-4 cells were examined by flow cytometry. And cleaved caspase-3 protein expression was detected by Western blot. BCL-2 gene expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and BCL-2 promoter in AML1-ETO positive leukemia cell line. The results indicated that in U937-A/E cells but not in U937-WT or U937-Mock cells, apoptotic cells statistically significantly increased, and AML1-ETO expression also significantly enhanced activation of caspase-3. AML1-ETO-expressing cell subclones displayed significantly low levels of BCL-2 mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, levels of BCL-2 mRNA were markedly lower as compared with other acute myeloid leukemias lacking this translocation. The enriched regions in transfected cells were located within BCL-2 promoter. It is concluded that BCL-2 is the direct target gene of AML1-ETO. AML1-ETO can down-regulate the expression of BCL-2.

Expression of autophagy related gene Beclin1 in myelodysplastic syndrome patients and its significance / 中国实验血液学杂志

This study was aimed to explore the contribution of autophagy associated gene Beclin1 in the prognosis of myelodysplastic syndrome (MDS) by detecting the expression level of Beclin1 in bone marrow mononuclear cells (BMNC) from 40 MDS patients, 14 non-malignant anemia patients and 25 AML patients. The expression of Beclin1 mRNA was detected by real-time quantitative polymerase chain reaction (qRT-PCR). At the same time, the Western blot was used to analyze the expression of Beclin1 proteins. The results showed that the expression of Beclin1 in low risk MDS patients and non-malignant anemia patients was both significantly higher than that in acute myeloid leukemia patients (P < 0.01). And more interestingly, the Beclin1 mRNA expression in MDS group was negatively correlated with World Health Organization classification-based prognostic system (WPSS) score (r = -0.495). It is concluded that the expression of Beclin1 in the patients with MDS is higher than that in AML patients, and negatively correlated with WPSS scores. Beclin1 is a potential biomarker for predicting prognosis of the patients with MDS.