Quantitative assay for Janus kinase 2 (JAK2) mutation in Chinese patients with chronic myeloproliferative disorders.

The Janus kinase 2 (JAK2) V617F mutation has considerably helped understanding of the molecular pathogenesis of chronic myeloproliferative disorders (MPD), hence this study investigated for the first time the mutational status and relative quantitation of JAK2 V617F mRNA in Chinese patients with chronic MPD. The study cohort comprised 123 chronic MPD patients (35 with polycythaemia vera [PV], 85 with essential thrombocythaemia [ET], three with idiopathic myelofibrosis [IMF]). Blood samples examined by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and capillary electrophoresis showed that the prevalence of the JAK2 V617F mutation was 100%, 62.4% and 66.7% in PV, ET and IMF patients, respectively. The proportion of JAK2 V617F mutated mRNA was 89.5% in homozygotes and 57.9% in heterozygotes; 18 PV heterozygous patients showed significantly higher mutated JAK2 mRNA levels than 36 heterozygous ET patients. Six of 93 patients exhibited abnormal karyotypes, but specific chromosomal abnormality was not found. The combination of ARMS-PCR and capillary electrophoresis enables quantitative assay of JAK2 V617F mutation, which helps in chronic MPD diagnosis and estimation of minimal residual disease.

[An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels].

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.

[Allogeneic hematopoietic stem cell transplantation for the treatment of acute myeloid leukemia with primary thrombocytosis: three cases report and literatures review].

Objective: To investigate the characteristics of the essential thrombocythemia (ET) cases transformed to the acute myeloid leukemia (AML) and the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the treatment of this disease. Methods: The clinical and laboratory characteristics of 3 ET cases before and after transformation and after allo-HSCT were retrospectively analyzed, meanwhile the related literatures were reviewed and discussed. Results: Case 1 was a male patient of 44 years old, whose PLT was 500×10(9)/L when firstly diagnosed ET. After 3 years the disease progressed into myelodysplastic syndrome (MDS) while WT1 expression increased from 77 (first visit) to 13 171 copies/10 000 ABL copies, at the same time chromosome changed dramatically. During the period of decitabine treatment the disease processed into AML. Case 2 was a male of 58 years old whose PLT was 2 100×10(9)/L when firstly diagnosed ET. The disease progressed to AML after 9 years, whose WT1 expression increased from 130 (first visit) to 3 222 copies/10 000 ABL copies, and he relapsed shortly after intensive chemotherapy. Case 3 was a male of 60 years old whose PLT was 900×10(9)/L when firstly diagnosed ET. The disease progressed to AML after 5 years, whose WT1 increased from 56 (first visit) to3 696 copies/10 000 ABL copies. Moreover leukemia spread to central nervous system (CNS) during chemotherapy. Before allo-HSCT, cases 1 did not achieve remission; case 2 relapsed after a short time of remission and case 3 transferred to CNS leukemia. All of the 3 cases underwent allo-HSCT successfully, and they all achieved completely remission, whose chromosome and gene mutation recovered negative. At the same time, CNS leukemia of case 3 disappeared. The median WT1 decreased to 50 copies/10 000 ABL copies. There was no severe complication during the median time of 5 months after allo-HSCT. Conclusions: The patients transformed to AML had poor prognosis, allo-HSCT was the only method that can cure the disease now.

[Clinical significance of expressions of EVI1 and BRE genes in 47 acute leukemia patients with MLL rearrangement].

Objective: To investigate the overexpression frequencies of BRE and EVI1, the correlation between BRE and EVI1 expressions and their possible clinical implications in 11q23/MLL rearrangement acute leukemia. Methods: Cytogenetic examination of bone marrow cells was performed by short-term culture method. R-banding technique was used for karyotype analysis. 47 patients were detected by interphase fluorescence in situ hybridization (FISH) with dual-color break apart MLL probe. The expressions of EVI1 and BRE genes were detected by real time quantitative reverse transcription polymerase chain reaction (RQ-PCR) . The correlation and prognostic significance were statistically tested. Results: 11q23/MLL rearrangements were confirmed by karyotyping and FISH, respectively in 47 patients. According to immunophenotypic analyses of 37 patients, 5 patients showed positive for CD19, CD79a or CD10, 1 for CD7; the others for CD33, CD13, CD14 and CD15, and 16 of them for CD34. Of the 47 patients, 18 patients showed EVI1 overexpression and most of them presented with t (6;11) and M(4)/M(5). The EVI1 expression was high in t (6;11) or t (9;11) subgroup comparable with levels observed in normal subgroup (P=0.038, 0.022, respectively) . 15 patients showed high BRE expression, and most of them presented with t (9;11) and M(4)/M(5). High BRE expression was found in t (4;11) , t (6;11) , t (9;11) and t (11;19) subgroups, respectively by comparing with normal subgroup. The BRE expression was higher in t (4;11) (P=0.004) or t (9;11) (P=0.012) subgroup than in t (6;11) subgroup. Patients with EVI1 overexpression had a short survival compared with those with low EVI1 expression (P=0.049) and it also did in t (9;11) subgroup (P=0.024) . Patients with t (9;11) and high BRE expression had a long survival compared with those with t (9;11) and low BRE expression (P=0.024) . Conclusion: The EVI1 overexpression was significantly frequent in acute leukemia patients with 11q23/MLL rearranged, especially within t (6;11) subgroup and M(4)/M(5), which was associated with an inferior outcome. High BRE expression was observed frequently in 11q23/MLL-rearranged acute leukemia especially within t (9;11) subgroup and M(5).

[Immune reconstruct regularity profile of KIR2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation].

Objective: To investigate the immune reconstruct regularity profile of KIR2DL1 and KIR3DL1 in unrelated-donor allogeneic hematopoietic stem cell transplantation (allo-HSCT) with KIR-AA genotype. Method: 75 donor-recipient pairs were performed by KIR genotying using PCR-SSP, and all donors were identified with KIR-AA genotype. Dynamic detections (including unrelated-donor on the day of transplantation and the recipient each month post allo-HSCT) of the expression of KIR2DL1/3DL1 on NK cell and mRNA level were performed in 291 cases using flow cytometry (FCM) and real-time fluorescent quantitation PCR (RT-qPCR) . Result: ①The median expression of KIR2DL1 in unrelated-donor on transplant's day was 21.60%, the median expression of KIR2DL1 in recipient 1M, 2M, 3M and 3-6M after transplantation were 7.40%, 12.00%, 16.92%, 17.64% respectively. The median expression of KIR2DL1 in unrelated-donor on transplant's day was 265.14 copies/10 000abl copies, the median expression of KIR2DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 332.17, 438.31, 723.25, 414.17, 180.76 and 234.67 copies/10 000abl copies respectively. The median expression of KIR2DL1 on NK cells and mRNA level gradually increased at all time points after transplantation, and reached the highest expression at 3 months after transplantation. But mRNA expression levels increased earlier than NK cell membrane proteins. ②The median expression of KIR3DL1 in unrelated-donors on transplant's day was 18.56%, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M after transplantation were 23.83%, 22.57%, 23.02%, 21.60% respectively. The median expression of KIR3DL1 in unrelated-donor on transplant's day was 572.29 copies/10 000abl copies, the median expression of KIR3DL1 in recipient 1M, 2M, 3M, 3-6M, 6-9M, 9-12M after transplantation were 1 233.74, 1 140.42, 876.73, 1 057.07, 739.02 and 514.43 copies/10 000abl copies respectively. The median expression of KIR3DL1 on NK cells and mRNA level were higher than donors at 1 month after transplantation, and stable expression at all time points after transplantation, so mRNA and NK cell membrane proteins expression increased at the same time. Conclusion: The immune reconstruct regularity of KIR2DL1 and KIR3DL1 gene were different, which provided an experimental basis for selecting the best time to detect the expressions of KIR2DL1 and 3DL1 after transplantation.

[Critical roles of matrix metalloproteinases secreted by leukemic cells in the pathogenesis of central nervous system leukemia].

Objective: To observe the effects of matrix metalloproteinases (MMP)-2 and MMP-9 secreted by leukemic cells on tight junction proteins ZO-1, claudin-5 and occluding and the permeability of the blood-brain barrier (BBB) and explore the mechanisms of MMP-2 and MMP-9 in leukemic cell infiltration of the central nervous system (CNS). Methods: The mRNA expressions of MMP-2 and MMP-9 in leukemic cell lines SHI-1, HL-60 and U937 were detected by quantitative RT-PCR. The MMP inhibitor GM6001 was used to inhibit the secretion of MMP-2 and MMP-9. RNA interference (RNAi) was used to knock down the expression of MMP-2 and MMP-9. Zymography was used to analyze the secretion of MMP-2 and MMP-9 in the supernatant of different leukemia cell lines treated or untreated with drugs, as well as the RNAi-treated cells. An in vitro BBB model composed of human brain microvascular endothelial cells (BMVECs) was developed on a Matrigel-based insert. Cell invasion through a barrier of Matrigel-based human basement membrane and the BMVECs-based human BBB barrier was assayed to measure the invasive capacity and the capacity to breakdown the BBB of different leukemia cell lines treated or untreated with drugs, as well as the RNAi-treated cells. The morphologic changes of BMVECs after co-culture with different leukemia cell lines treated or untreated with drugs, as well as the RNAi-treated cells in vitro BBB models were observed by invert microscopy and tight junction proteins in these BMVECs were analyzed with a laser-scanning confocal microscope. Results: ①The mRNA expression in different leukemic cell lines shown a pronounced transcription of MMP-2 and - 9, and the transcriptional level in SHI-1 cells was the highest among all leukemic cell lines tested (P<0.01). The data of activities of MMP-2 and -9 were consistent with the results of mRNA expression and SHI-1 displayed higher capacity of invasion (P<0.01). ②After incubation 24h with different leukemic cells, the BMVECs disrupted to loss cell-cell contacts and grew in single cell. Confocal imaging showed down-regulations of ZO-1, claudin-5 and occluding accompanied by the disruption of BBB in vitro models. SHI-1 cells had stronger alterations to BMVECs, tight junction proteins and the permeability of the BBB than HL-60 and U937 cells. However, GM6001 and the knock-down of MMP-2 and MMP-9 altered the responses of BBB. They reduced the degradation of three tight junction proteins with a decreased permeability of BBB. Conclusion: MMP-2 and MMP-9 secreted by leukemic cells could disrupt the BBB by degrading the tight junction proteins ZO-1, claudin-5 and occluding, which contributed the infiltration of leukemic cell into CNS.

[EVI1 expression, clinical and cytogenetical characteristics in 447 patients with acute myeloid leukemia].

Objective: To investigate EVI1 expression and its associated clinical and cytogenetic characteristics in 447 acute myeloid leukemia (AML) patients. Methods: EVI1 expressions were measured in 447 AML cases from Jan. 2007 to Apr. 2015 to couple with clinical, cytogenetic and mutations' characteristics to summarize the features of AMLs with high EVI1 expression. Results: 17.9% of AML were high EVI1 expression (EVI1 +), and the remainder low EVI1 expression (EVI1-). No significant differences between the two groups in terms of age, sex, hemoglobin level, white blood cell count and platelet count were observed. More M0, M5 and M6 subtypes were observed in EVI1+ group (P= 0.027, 0.004 and 0.011, respectively). Cytogenetic abnormalities of 11q15, 11q23/MLL, 3q26, -7/7q- and t (9;11) were observed more frequently in EVI1 + group (P<0.001, <0.001, <0.001, <0.001, =0.014, respectively). Normal karyotype, inv (16), t (8;21) were observed more frequent in EVI1- group (P=0.001, 0.009, 0.002, respectively). EVI1 + was more observed in high risk cytogenetics. Mutation of NPM1 was more observed in EVI1- group (P <0.001). Remission rate in EVI1 + group was significantly lower than EVI1- group (P<0.001). Leukemia-free survival was improved in EVI1 + AML patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Conclusions: High EVI1 expression was more observed in FAB subgroup M5, harbored more cytogenetic abnormalities of 11p15, 11q23/MLL, 3q26 rearrangement, -7/7q- and t (9;11). Remission rate of high EVI1 expression AML was lower, which could be improved by allo-HSCT.

Overexpressed miR-155 is associated with initial presentation and poor outcome in Chinese pediatric acute myeloid leukemia.

OBJECTIVE: Acute myeloid leukemia (AML) is the second leading leukemia in children. There is growing evidence that microRNAs (miRNAs) are crucial regulators involved in leukemogenesis. This study aimed to investigate the role of miR-155 in Chinese pediatric AML by evaluating its diagnostic and prognostic significance. PATIENTS AND METHODS: The expression of miR-155 and miR-25 in bone marrow specimens from 83 AML and 29 non-malignancies children were analyzed by TaqMan probe-based real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: The expression level of miR-155 was significantly higher in AML patients than in controls. Besides, a lowest miR-155 level was found in favorable prognosis group and t (15; 17)/M3 subgroup compared to the rest, while a higher level in C-Kit/FLT3-ITD mutation and relatively lower level existed in "Negative" mutation group. Moreover, miR-155 level was positively associated with the white blood cell (WBC) count, serum lactate dehydrogenase (LDH) and C-reaction protein (CRP) value in peripheral blood (PB), as well as miR-25/miR-196b expression levels. Survival analysis showed a statistically negative association with overall survival (OS) in the expression of miR-155 in chemotherapy group. CONCLUSIONS: These finding suggested that miR-155 expression cannot only be promising biomarker for the early detection of pediatric AML but also predict poor outcome. MiR-155 would be a novel biomarker for diagnosis, prognosis and therapy in pediatric AML.