Applications of microchip electrophoresis and capillary electrophoresis for screening FLT3-ITD gene mutation in acute myeloid leukemia / 中国实验血液学杂志

The purpose of the present study was to compare the reliability of microchip electrophoresis and capillary electrophoresis for screening FLT3-ITD gene mutation in acute myeloid leukemia. The FLT3-ITD mutation in the genomic DNA samples from 214 untreated AML patients were separately detected by PCR-microchip electrophoresis and PCR-capillary electrophoresis, then the DNA direct sequencing analysis was carried out. The results from PCR-microchip electrophoresis showed that there were 151 FLT3-ITD mutation negative, 58 FLT3-ITD mutation positive (58/214, 27.1%) and 5 FLT3-ITD mutation doubtful positive (5/214, 2.3%), while the outcomes from PCR-capillary electrophoresis displayed that there were 147 FLT3-ITD mutation negative and 67 FLT3-ITD mutation positive (67/214, 31.3%) without doubtful positive. In the 67 FLT3-ITD mutation positive samples detected by using PCR-capillary electrophoresis, 4 samples were detected as the negative while 5 samples were measured as the doubtful positive by using PCR-microchip electrophoresis. The followed sequencing analysis demonstrated that the above 9 samples were all FLT3-ITD mutation positive, indicating that PCR-capillary electrophoresis was more accurate and sensitive in screening the FLT3-ITD mutation, although statistic analysis showed that there were no significant differences in the detected results between PCR-microchip electrophoresis and PCR-capillary electrophoresis groups (Pearson Chi-squared Test, P > 0.05). It is concluded that both PCR-microchip electrophoresis and PCR-capillary electrophoresis were convenient and fast for screening FLT3-ITD mutation, but the accuracy of PCR-microchip electrophoresis awaits further improvement.

Expression of WT1 and PRAME gene in bone marrow and peripheral blood samples of patients with myelodysplastic syndrome / 中国实验血液学杂志

This study was aimed to explore the transcription level of WT1 and PRAME two genes in bone marrow and peripheral blood samples of patients with myelodysplastic syndrome(MDS) and their relationship with bone marrow dysplasia and karyotype. The quantitative expression of WT1 and PRAME transcripts detected by RQ-PCR in the bone marrow samples of 203 MDS patients and 19 aplastic anemia(AA), 6 other benign anemia(BA), 4 paroxysmal nocturnal hemoglobinuria(PNH) patients from July 2009 to June 2012 and 14 healthy donors, and in 92 peripheral blood samples. The results showed that WT1 and PRAME expression levels in both BM and PB samples of MDS group were higher than those in normal controls, AA, and BA patients (BM: WT1:P = 0.000, 0.000, 0.000, PRAME: P = 0.048, 0.000, 0.064; PB: WT1:P = 0.012, 0.000, 0.011, PRAME: P = 0.020, 0.004, 0.003). What is more, this expression in high risk MDS group (RAEB1, RAEB2, MDS-AML) were higher than those in low risk group (RCUD, RCMD, MDS-U) and AA and BA. The WT1 and PRAME mRNA expression levels in PB and BM were well correlated (WT1:r = 0.6028, P = 0.001; PRAME: r = 0.7628, P = 0.000), as well as the WT1 expression levels in BM samples with the Karyotype (P = 0.049). In addition, the same positive rate of WT1 or PRAME expression existed in BM and PB samples of MDS patients. It is concluded that the WT1 and PRAME gene expression levels in both BM and PB samples of MDS patients are higher than those in healthy controls, AA and other benign anemia patients, and increase with the progression of the disease. The WT1 and PRAME transcripts constitute good molecular markers for the clinical diagnosis and prognosis and monitoring minimal residual disease after treatment of MDS. What is more, when bone marrow is not so convenient to get, the transcript levels of PB samples can be detected.

The immunophenotypic and clinical characteristics of NPM1 mutated acute myeloid leukemia patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare the immunophenotypic and clinical characteristics between NPM1 mutated acute myeloid leukemia (AML) (NPM1m(+)AML) and unmutated AML(NPM1m(-)AML) not otherwise characterized (NOS) under similar FAB subtypes constituent ratio.</p><p><b>METHODS</b>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. 104 AML patients with NPM1m(+)AML and performed immunophenotyping assay were included, 97 with NPM1m(-)AML.</p><p><b>RESULTS</b>There were significant difference between the two groups at presentation in terms of sex, white blood count(WBC), platelet counts (PLT), blast ratio, normal karyotype ratio, WT1 expression level, FLT3-ITD mutation positive rate and remission rate of first course of induction therapy (P < 0.05). On the immunophenotype, the expression of early differentiation antigens (CD34, HLA-DR, CD117, CD38), lymphocytic antigens (CD7, CD4, CD19, CD2), myeloid and monocytic differentiation-associated antigens (CD13, CD14, CD15) were lower, and that of CD33 as well as CD123 were higher in NPM1m(+)AML patients. Among them, only CD34, HLA-DR, CD7, and CD4 positive cases were significantly lower in NPM1m(+)AML group than in NPM1m(-)AML group (P < 0.05), the rest of them had significant difference in the number of positive cells (P < 0.05). Above features were further analyzed between the M1/M2 and M4/M5 subgroups. M1/M2 cases retained the women prominent and had a higher WT1 expression level (P < 0.05). The expression of monocytic differentiation-associated antigens including HLA-DR and lymphocytic antigens were higher and that of CD117 were lower in M4/M5 subtype (P < 0.05). Among them, the positive rates of HLA-DR, CD64, CD11b, CD10, CD15, and CD4 were significantly higher in M4/M5 than in M1/M2 in NPM1m(+)AML group (P < 0.05).</p><p><b>CONCLUSION</b>The most clinical characteristics in NPM1m(+)AML patients are consistent with reports, but some immunophenotype are different to the previous reports under similar FAB subtypes constituent ratio. The major immunophenotypic features of NPM1m(+)AML patients are lower expression of progenitor, myeloid and lymphoid lineage antigens. Monocytic differentiation-associated antigens are only higher expression in M4/M5 cases when comparison with M1/M2 cases within NPM1m(+)AML group.</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>

Quantitative chimerism kinetics in relapsed leukemia patients after allogeneic hematopoietic stem cell transplantation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Chimerism analysis is an important tool for the surveillance of post-transplant engraftment. It offers the possibility of identifying impending graft rejection and recurrence of underlying malignant or non-malignant disease. Here we investigated the quantitative chimerism kinetics of 21 relapsed leukemia patients after allogeneic hematopoietic stem cell transplantation (HSCT).</p><p><b>METHODS</b>A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time polymerase chain reaction (RT-PCR) to obtain the informative marker for every leukemia patient. Quantitative chimerism analysis of bone marrow (BM) samples of 21 relapsed patients and 20 patients in stable remission was performed longitudinally. The chimerisms of BM and peripheral blood (PB) samples of 14 patients at relapse were compared.</p><p><b>RESULTS</b>Twenty-one patients experienced leukemia relapse at a median of 135 days (range, 30 - 720 days) after transplantation. High recipient chimerism in BM was found in all patients at relapse, and increased recipient chimerism in BM samples was observed in 90% (19/21) of patients before relapse. With 0.5% recipient DNA as the cut-off, median time between the detection of increased recipient chimerism and relapse was 45 days (range, 0 - 120 days), with 76% of patients showing increased recipient chimerism at least 1 month prior to relapse. Median percentage of recipient DNA in 20 stable remission patients was 0.28%, 0.04%, 0.05%, 0.05%, 0.08%, and 0.05% at 1, 2, 3, 6, 9, and 12 months, respectively, after transplantation. This was concordant with other specific fusion transcripts and fluorescent in situ hybridization examination. The recipient chimerisms in BM were significantly higher than those in PB at relapse (P = 0.001).</p><p><b>CONCLUSIONS</b>This SP-based RT-PCR assay is a reliable method for chimerism analysis. Chimerism kinetics in BM can be used as a marker of impending leukemia relapse, especially when no other specific marker is available. Based on our findings, we recommend examining not only PB samples but also BM samples in HSCT patients.</p>

Clinical significance of CD34(+)CD38(+) and CD34(+)CD38(low/-) subgroups in bone marrow of patients with B lymphoblastic leukemia / 中国实验血液学杂志

This study was purpose to investigate the biological characteristics of B lymphoblastic leukemia (B-ALL) between CD34 positive CD38 positive (CD34(+)CD38(+)) and CD34(+)CD38(low/-) subgroups and their clinical significance. Immunophenotyping of B cells in bone marrow of 54 patients with newly diagnosed CD34(+)B-ALL were analyzed by 4 color multiparametric flow cytometry (FCM). According to the different expression of CD38, the newly diagnosed patients with B-ALL were divided into two groups: CD34(+)CD38(+) subgroup and CD34(+)CD38(low/-) subgroup. BCR-ABL, TEL-AML1 fusion genes and WT1 gene were detected by real time RT-PCR simultaneously. After chemotherapy, minimal residual disease (MRD) was monitored by one tube of 7 color FCM. The average follow-up time was 12 months (range 1 - 28), the average follow-up interval was 2 months (range 1 - 5). The results showed that there was no significant differences such as WBC, Plt count and Hb level between the two groups at diagnosis, the positive rate of BCR-ABL, TEL-AML1 and WT1 gene was also no significantly different. After clinical complete remission (CR), MRD positive (MDR(+)) case rates were 28.57% (10/35) in CD34(+)CD38(+) subgroup and 68.42% (13/19) in CD34(+)CD38(low/-) subgroup (P < 0.01). The relapse rate between the two groups was 5.71% (2/35) in CD34(+)CD38(+) subgroup (relapse time at 94 and 245 d respectively) and 36.84% (7/19) in CD34(+)CD38(low/-) group [median relapse time was 263 d (range 46 - 468), P < 0.01]. The age distribution was analyzed in these two subgroups (> 16 or ≤ 16 years old), there was 8 (8/35) adult patients (> 16 years old) in CD34(+)CD38(+)group and 10 (10/19) adult patients in CD34(+)CD38(low/-) group (P < 0.05). It is concluded that CD34(+)CD38(low/-) phenotype is more often presented in adult patients and the CD34(+)CD38(low/-) patients with B-ALL are more likely to have MRD(+)and relapse after treatment.

Quantified research about the effects of sleep quality on attention in class and acadamic achievements in primary school children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the adverse effects of sleep problems and sleep insufficiency on attention in class and pupils' acadamic achievements.</p><p><b>METHODS</b>A total of 1138 students from four primary schools at ages of 6-12 years were randomly sampled from four districts of Changsha city, Hunan Province June 2009 to April 2010. The inquired items included sleep problems, sleep time, sleep quality, attention in class and academic achievements. Teachers and parents observed the pupils according to the unified requirements for 3 months and then filled out the questionnaires.</p><p><b>RESULTS</b>The total valid inquiry tables were 1091 with the answering rate of 95.87%, including 549 boys and 542 girls. The sleep quality was more poor in children with sleep problems or sleep insufficiency than in children with normal sleep. The sleep quality was reduced and aggravated along with the increasing sleep problems and the reducing sleep time. The attention in class and academic achievements in children with sleep problems or sleep insufficiency were more poor than in children with normal sleep. The sleep quality index was negatively correlated with attention in class and academic achievements. The attention in class was positively correlated with academic achievements.</p><p><b>CONCLUSIONS</b>The decline in sleep quality directly affects the attention in class and reduce the academic achievements in primary school children.</p>

Evaluating the feasibility of nucleated cells isolated by lysis method for real-time quantitative RT-PCR / 中国实验血液学杂志

This study was aimed to investigate whether difference exists between real time RT-PCR results of nucleated cells isolated by lysis method and mononuclear cells isolated by gradient concentration method. 14 bone marrow samples from leukemia patients (7 samples of AML-M(2), 1 of AML-M(4), 1 of AML-M(4)EO, 1 of AML-M(6), 1 of APL and 3 of CML) were collected. Each sample was divided into 2 parts, and was used to isolate mononuclear cells by Ficoll-Hypaque gradient centrifugation, and other was used to isolate nuclear cells by lysis method. The RNA extraction and detection of internal reference gene ABL for all of samples were performed by RT-PCR, and mRNA expression levels in 3 samples of BCR/ABL, 6 of AML/ETO, 1 of CBFβ-MHY11, 1 of WT1 and 6 PRAME were detected by RT-PCR. The results showed that ABL copies of all samples were over 3 × 10(4), and there was significant difference in ABL copies between each pair of samples by lysis and gradient centrifugation method (p > 0.05). There was also no significant difference in every detected mRNA levels between 14 pair samples (p > 0.05). It is concluded that the lysis method may be useful one for extracting RNA from nuclear cells, and can adopt as a routing detection method for simultaneously extracting RNA from many samples to detect leukemia specific mRNA by using RT-PCR.

Quantitative assessment of hematopoietic chimerism by quantitative real-time polymerase chain reaction of sequence polymorphism systems after hematopoietic stem cell transplantation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT.</p><p><b>METHODS</b>A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally.</p><p><b>RESULTS</b>Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation <1.85%), which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method.</p><p><b>CONCLUSION</b>This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.</p>

Abnormally lower expression of cmtm5 gene in bone marrow cells from patients with multiple myeloma / 中国实验血液学杂志

This study was aimed to detect the expression level of cmtm 5 (CKLF-like MARVEL transmembrane domain containing member 5) gene in the bone marrow cells from patients with multiple myeloma (MM), and to investigate the correlation between the expression level of cmtm5 and various clinical characteristics. Real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) was used to measure the expression levels of cmtm5 gene in the bone marrow cells collected from MM patients, and the MM cell lines, namely, RPMI8226 and CZ1 cells. The normal donor marrow specimens were used as the reference. The ratio of cmtm5 copy number to abl (Abelson murine leukemia viral oncogene homolog) gene copy number was used for indicating the expression level. The results showed that the expression level of cmtm5 gene was significantly down-regulated in bone marrow cells of 51 untreated or relapsed/refractory MM patient as compared to those of normal donor marrow cells (0.047+/-0.062 for the untreated or relapsed/refractory MM patients versus 0.255+/-0.333 for the normal, p<0.01). According to the International Staging System (ISS), the cmtm5 expression level in marrow cells of patients in ISS III stage was significantly lower than that in patients in ISS I stage (0.034+/-0.034 for the ISS III stage versus 0.103+/-0.109 for ISSI stage, p<0.01). Similarly, lower expression levels of cmtm5 gene were also found in two human MM cell lines (0.014+/-0.009 for RPMI8226 cells and 0.004+/-0.006 for CZ1 cells). After the MM patients were effectively treated, their expression levels of cmtm5 gene significantly increased (0.020+/-0.005 for the untreated patients versus 0.227+/-0.038 for the effectively treated patients, p<0.01). A significant negative correlation was observed between the expression level of cmtm5 gene and the number of bone marrow plasma cells (r=-0.307, p<0.05). However, the correlation was not found between the expression level of cmtm5 gene and the clinical characteristics, such as gender, age, hemoglobin level, or M-protein level, etc. It is concluded that the expression level of cmtm5 gene is abnormally lower in the bone marrow cells from the MM patients, and are associated with ISS stages. Furthermore, the expression level of cmtm5 gene is negatively correlated with the number of bone marrow abnormal plasma cells in MM patients, which suggests that the abnormally lower expression of cmtm5 may be involved in the pathogenesis of the MM patients.

Abnormal expression of programmed cell death 5 gene in multiple myeloma patients / 中国实验血液学杂志

The aim of this study was to investigate the gene expression of programmed cell death 5 (pdcd5) in plasma and bone marrow cells from patients with multiple myeloma (MM). Enzyme liked immunosorbent assay (ELISA) and real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) were used to examine pdcd5 gene expression in plasma and marrow cells in 45 MM patients and 20 normal controls. The results showed that serum levels of PDCD5 protein in 45 MM patients were lower significantly compared with the normal controls and 20 responsive patients after chemotherapy, their plasma levels were (16.91 +/- 0.28) ng/ml, (19.11 +/- 0.29) ng/ml and (17.94 +/- 0.154) ng/ml respectively (p < 0.05). The pdcd5 gene expression levels detected by RQ-RT-PCR in 45 MM patients were lower significantly compared with the normal controls, their pdcd5 gene expression levels were 0.64 +/- 0.47 and 1.28 +/- 1.21 respectively (p < 0.05). It is concluded that the PDCD5 protein expression levels are low in patients with MM. These findings suggest that abnormal expression of pdcd5 may be involved in the pathogenesis of MM.

Quantitative analysis of dendritic cell subsets in bone marrow of patients with acute myeloid leukemia / 中国实验血液学杂志

In order to study the quantity of dendritic cell (DC) subsets of bone marrow in patients with acute myeloid leukemia (AML), the bone marrow aspirate were collected from 77 newly diagnosed AML patients and from 30 healthy persons. The quantity of DC subsets (myeloid dendritic cells, mDC and plasmacytoid dendritic cells, pDC) were detected by flow cytometry and analysed by 3-color and 4-color cytometric gate. Based on the conventional 3-color panel, mDC were identified by Lin-HLA-DR+CD11c+ and pDC were identified by Lin-HLA-DR+CD123+. Based on the 4-color panel, mDC were identified by Lin-HLA-DR+CD11c+ BDCA-1+ and pDC were identified by Lin-HLA-DR+CD123+BDCA-2+. The results showed that a reduction of mDC was found in 74.0% (57/77) and 58.4% (45/77) patients, a reduction of pDC was found in 90.9% (70/77) and 46.8% (36/77) patients respectively by 3-color and 4-color cytometric analysis. Meanwhile an expansion of mDC was showed in 19.5% (15/77) and 22.1% (17/77) patients, an expansion of pDC was showed in 1.3% (1/77) and 27.3% (21/77) patients respectively by 3-color and 4-color cytometric analysis. In subtypes of AML-M2, AML-M3 or AML-M4/5, 81.4%, 100% and 42.1% patients showed mDC decrease and 88.4%, 100% and 89.5% patients showed pDC decrease respectively by 4-color cytometric analysis. It is concluded that the 4-color cytometric gate is better method for detection of mDC and pDC from bone marrow of newly diagnosed AML patients as compared with 3-color cytometric gate, the majority of AML patients showed reduction of mDC and pDC. The percentages of patients with mDC normal or mDC increase in AML-M4/5 subtypes are more than that in AML-M2/3 subtypes, while the pDC does not show difference between AML subtypes.

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%.

Expression of 5 genes in CD7 positive acute myeloid leukemia stem/progenitor cells from bone marrow / 中国实验血液学杂志

This study was aimed to investigate abca5, mdr-1, kdr, dapk and irf-1 expressions in leukemia stem/progenitor cells (LSC) from CD7 positive acute myeloid leukemia, the expression of these 5 genes in mononuclear cells (MNC) from 15 normal bone marrow (NBM) and 16 AML patients bone marrow (AML BM) specimen were detected by real-time quantitative PCR (RQ-PCR). CD34(+)CD38(+) progenitor cells and CD34(+)CD38(-)Lin(-) stem cells were sorted by flow cytometry (FCM) from the MNCs of 10 NBM and 21 AML BM specimen. These 5 gene expressions in the sorted cells were detected by small amount cell RQ-PCR. The results showed that these 5 genes above mentioned all expressed in NBM-MNC, in which the expression levels of irf-1 and dapk were highest with the relative expression levels 4.08 and 3.86, the expression levels of abca 5 and mdr-1 were in the middle with the relative expression 0.49 and 0.84 respectively, the kdr expression was lowest with the relative expression level 0.02. In CD34(+)CD38(+) progenitor cells, the expression level of kdr increased dramatically (p < 0.05) while irf-1 and dapk dramatically decreased (p < 0.05). There was no obvious change of expression in the rest 2 genes. In CD34(+)CD38(-) stem cells the expression level of these 5 genes all increased nearly 2 times as much as that in CD34(+)CD38(+) progenitor cells, but kdr increased 3 times as much, and the increase of kdr and irf-1 expressions was of statistical significance (p < 0.05). Compared with the NBM, expression levels of 5 genes in AML-MNC decreased, and out of them abca 5, mdr-1, kdr and dapk were decreased most remarkably (p < 0.05). Comparison between AML CD34(+)CD38(+) cells and AML MNC showed that the expression level of irf-1 and dapk were decreased dramatically (p < 0.05) while the rest 3 genes increased their expression with statistical significance (p < 0.05). The expression levels of these 5 genes were higher in CD34(+)CD38(-) cells than those in CD34(+)CD38(+) stem cells, and the increase of kdr and irf-1 expressions showed statistical difference (p < 0.05). These 5 genes expression levels were all higher than those in CD34(+)CD38(+) cells whether in AML CD34(+)CD38(-)CD7(+) cells or CD34(+)CD38(-)CD7(-) cells. The increase of kdr expression in CD34(+)CD38(-)CD7(+) cells as well as kdr and irf-1 expressions in CD34(+)CD38(-)CD7(-) cells were all of statistical significance (p < 0.05). In conclusion the expression level of kdr in NBM was highest in stem cells while dapk and irf-1 were highest in differentiated cells. The expression levels of these 5 genes in CD34(+)CD38(-)Lin(-) stem cells were higher than those in CD34(+)CD38(+) progenitor cells. The gene expressions in AML CD34(+)CD38(-)CD7(+) cells and CD34(+)CD38(-)CD7(-) cells are in accordance with the characteristics of stem cells.

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 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>

Adenovirus-mediated PDCD5 gene transfer sensitizes apoptosis of K562 cells induced by etoposide / 中国实验血液学杂志

This study was purposed to investigate the effect of adenovirus-mediated transfer of PDCD5 gene on apoptosis of K562 cells induced by etoposide. Recombinant adenovirus PDCD5 (Ad-PDCD5), control vectors Ad-null and Ad-eGFP were constructed by AdMax vector system respectively. After K562 cells were transfected by Ad-PDCD5, Ad-null or Ad-eGFP with different multiplicity of infection (MOI), the expression level of the PDCD5 gene was examined by RQ-RT-PCR assay. The effects of etoposide in combination with Ad-PDCD5 on the proliferation and apoptosis of K562 cells were measured by using MTT assay and flow cytometry with Annexin-V-FITC/PI dual labeling technique, respectively. The results showed that the transfection efficiencies of Ad-eGFP in K562, Jurkat and CEM cells ranged from 60% to 86%. Expression level of PDCD5 gene in K562 cells was evidently increased following transfection with Ad-PDCD5. The Ad-PDCD5 synergistically enhanced the apoptotic percentage of K562 cells induced by VP-16, as compared with that of Ad-null + VP16 and VP-16 alone respectively. It is concluded that Ad-PDCD5 may be a potential agent for enhancing the chemotherapy effect.

Analysis of immunophenotype and leukemia associated immunophenotype in 610 patients with acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze the immunophenotype and leukemia associated immunophenotype (LAIP) of leukemia cells from patients with acute myeloid leukemia (AML) in minimal residual disease (MRD) detection.</p><p><b>METHODS</b>Four-color multiparametric flow cytometry (FCM) with CD45/SSC gating was used to determine the expression of the following antibodies of CD7, CD117, CD33, CD34, CD10, CD19, CD56, CD38, CD13, CD14, CD64, CD9, CD16, CD2, CD5, CD11b, CD123, HLA-DR in 610 AML patients and 20 normal bone marrow (NBM) samples.</p><p><b>RESULTS</b>The mean percentages of CD34+ and CD117+ side scatter low (SSC(low)) cells in NBM mononuclear cells (BMMNCs) were (0.35 +/- 0.15)% and (0.76 +/- 0.31)%, respectively. The majority (84% -95%) of CD34+ SSC(low) cells co-expressed CD13, CD33, CD38, CD117 and HLA-DR. 33% and 20% of CD34+ SSC(low) cells were CD15+ and CD9, respectively. Only a small proportion (< 10%) of CD34 SSC(low) cell co-expressed CD11b, CD56, CD19 and CD64, while co-expressed CD7 was 12%. In CD117 SSC(low) cells, the relative proportions of CD19+, CD11b+, CD56+ and CD7+ were less than 10%, while CD9+ was 14%. CD38+ and HLA-DR+ were 87% and 91%, respectively. The expressions of CD15, CD34, CD13 and CD33 on CD117 SSC(low) cells were between 30% and 50%. In AML patients, most cases were CD117+ (95.08%), CD38+ (94.74%), CD9+ (84.93%), CD33+ (84.60%), HLA-DR+ (77.23%) and CD13 (75.25%). The proportions of CD64+ and CD34+ were 64.41% and 59.51%, and that of CD15 and CD11b+ were 43.06% and 22.07%, respectively. 86.39% of AML patients were found to have at least one LAIP, the highest incidence being in AML-M1 and M3 subtypes and the lowest in AML-M4Eo subtype. The cross-lineage antigen and asynchronous antigen expression were the most frequent aberrant phenotypes. CD7, CD19 and CD56 expressing on CD34+ cells were major cross-lineage antigen. For asynchronous antigen expression, CD34+ CD64+, CD117+ CD11b+ , CD34+ CD38(-/dim) and CD34+ HLA-DR(-/dim) were seldom expressed on normal BMMNCs (about 0.01%), and the logarithm difference between AML and NBM was larger than 3.0, being the more sensitive LAIP.</p><p><b>CONCLUSION</b>MRD detection by multiparameter flow cytometry can be applied to more than 80% of AML patients.</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.