[Detection of BCR-ABL Fusion Gene in Chronic Myeloid Leukemia by Novel Digital PCR].

OBJECTIVE: To establish a new digital polymerase chain reaction (dPCR) system for the detection of BCR-ABL fusion gene in patients with chronic myeloid leukemia (CML), and explore its analytical performance and clinical applicability in the detection of BCR-ABLp190/210/230. METHODS: A new dPCR system for detecting BCR-ABLp190/210/230 was successfully developed, and its sensitivity difference with qPCR and improvement of drug side effects in patients with CML during drug reduction or withdrawal were compared. RESULTS: Among 176 samples, qPCR and dPCR showed high consistency in the sensitivity of detecting BCR-ABL (82.39%), and the positive rate of dPCR was about 5 times higher that of qPCR (20.45% vs 3.98%). During follow-up, blood routine (25% vs 10%), kidney/liver/stomach (25% vs 20%) and cardiac function (10% vs 0) were significantly improved after drug reduction or withdrawal in patients with initial dPCR negative compared with before drug reduction or withdrawal. CONCLUSIONS: This new dPCR detection system can be applied to the detection of BCR-ABLp190/210/230. It has better consistency and higher positive detection rate than qPCR. Drug withdrawal or dose reduction guided by dPCR has a certain effect on improving drug side effects.

Anthracycline dose optimisation in patients with diffuse large B-cell lymphoma: a multicentre, phase 3, randomised, controlled trial.

BACKGROUND: Anthracycline dose optimisation in the treatment of diffuse large B-cell lymphoma has rarely been tested. We aimed to find out whether R-CEOP70 was non-inferior to R-CHOP50 with less cardiotoxicity, and whether R-CEOP90 had a superior efficacy to R-CHOP50 or R-CEOP70 with acceptable toxic effects. METHODS: In this multicentre, phase 3, randomised, controlled study (NHL-001), patients with newly diagnosed diffuse large B-cell lymphoma or follicular lymphoma grade 3B were enrolled from 20 centres of the Multicenter Hematology-Oncology Programs Evaluation System in China. Young patients (16-60 years) were randomly assigned 1:1:1 (block size of six) to six courses of R-CHOP50, R-CEOP70, or R-CEOP90, and older patients (61-80 years) were assigned 1:1 (block size of four) to R-CHOP50 or R-CEOP70. Patients were randomly assigned using computer-assisted permuted-block randomisation. Investigators and patients were not masked to treatment assignment. In the R-CHOP50 group, patients were given rituximab 375 mg/m2 intravenously on day 0, cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, and vincristine 1·4 mg/m2 (maximum dose 2 mg) intravenously on day 1, and prednisone 60 mg/m2 (maximum dose 100 mg) orally from day 1-5; in the R-CEOP70 group, epirubicin 70 mg/m2 replaced doxorubicin; and in the R-CEOP90 group, high dose epirubicin 90 mg/m2 replaced doxorubicin. All patients received two additional courses of rituximab 375 mg/m2 intravenously every 21 days. Consolidation radiotherapy was given to patients with bulky disease at diagnosis or residual disease at the end of treatment. The primary endpoint was 2-year progression-free survival. The non-inferiority margin for R-CEOP70 versus R-CHOP50 was defined by hazard ratio [HR] as the upper limit of its 95% CI being no greater than 1·50. Analysis of efficacy and safety were of the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01852435. FINDINGS: From May 15, 2013, to March 16, 2016, a total of 648 patients were enrolled, including 404 (62%) young patients (R-CHOP50 [n=135], R-CEOP70 [n=134], or R-CEOP90 [n=135]), and 244 (38%) older patients (R-CHOP50 [n=122] or R-CEOP70 [n=122]). Four patients were excluded from the study for consent withdrawal and one patient for misdiagnosis before treatment. The 2-year progression-free survival in the R-CHOP50 group was 72·5% (95% CI 66·6-77·6) and in the R-CEOP70 group was 72·4% ([66·5-77·5]; HR 1·00 [0·73-1·38]; p=0·99). The non-inferiority was met and adverse events were similar between the two groups. Fewer patients in the R-CEOP70 group (14 [13%] of 110) presented with over 10% decrease in left ventricular ejection fraction (LVEF) than those in the R-CHOP50 group (31 [29%] of 108) at 3 years after remission. For young patients, the 2-year progression-free survival in the R-CEOP90 group was 88·8% (82·1-93·1) and was significantly improved compared with the R-CHOP50 group (75·9% [67·7-82·3]; 0·44 [0·25-0·76]; p=0·0047) and the R-CEOP70 group (77·4% [69·4-83·7%]; 0·49 [0·27-0·86]; p=0·017). Grade 3-4 neutropenia occurred more frequently in the R-CEOP90 group (97 [72%] of 134) than in the R-CHOP50 group (87 [65%] of 133) and R-CEOP70 group (84 [63%] of 133) in young patients but without further increase of clinically significant infections. Fewer patients in the R-CEOP70 group (7 [11%] of 66) and in the R-CEOP90 group (10 [13%] of 79) presented with more than 10% decrease in LVEF than those in the R-CHOP50 group (17 [26%] of 66) at 3 years after remission. INTERPRETATION: R-CEOP70 could serve as an alternative regimen to R-CHOP50 with mild long-term cardiotoxicity. Young patients with diffuse large B-cell lymphoma might benefit from high-dose epirubicin. Epirubicin is an alternative drug to doxorubicin in regular R-CHOP with mild long-term cardiotoxicity. FUNDING: National Natural Science Foundation of China, National Key Research and Development Program, Shanghai Commission of Science and Technology, Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant Support, Multicenter Clinical Research Project by Shanghai Jiao Tong University School of Medicine, Clinical Research Plan of Shanghai Hospital Development Center, and Chang Jiang Scholars Program.

[Expression of CXCR7 in Acute Monocytic Leukemia and Its Effects on THP-1 Cell Functions].

OBJECTIVE: To study the expression of stromal cell derived factor-1α (SDF-1α) receptor CXCR7 in acute monocytic leukemia (AML-M5), and its effects on proliferation, apoptosis, invasion of acute monocytic leukemia cell line THP-1. METHODS: CXCR7 protein and mRNA expression levels in THP-1 cells and peripheral blood mononuclear cells (PBMNC) from the newly diagnosed AML-M5 patients and normal individuals were detected by flow cytometry, Western blot and RT-PCR respectively. CCK8, Annexin V/PI double staining and Transwell assay were used to observe the effects of CXCR7 on the proliferation, apoptosis, and invasion of THP-1 cells in vitro. RESULTS: The expression of CXCR7 on immature cell surface of the newly diagnosed AML-M5 patients was significantly higher than that in the control group (P<0.05). CXCR7 was also highly expressed on THP-1 cells surface. The CXCR7 protein and mRNA levels in THP-1 cells and PBMNC of AML-M5 patients were significantly higher than those in the control group (P<0.05). The THP-1 cell proliferation activity was higher in SDF-1α-treated group, but this activity could be inhibited by CXCR7 antibody (P<0.01). CXCR7 antibody did not affect THP-1 cell apoptosis (P>0.05). CXCR7 antibody could inhibit SDF-1α -induced THP-1 cell invasiveness (P<0.01). CONCLUSION: CXCR7 highly expresses in AML-M5 patients and THP-1 cells, and involves in cell proliferation and invasion. The blocking CXCR7 expression can reduce the risk of AML-M5 cell infiltration.

[Value of Neutrophil/Lymphocyte Ratio and Platelet/Lymphocyte Ratio for Prognostic Evaluation of Diffuse Large B-cell Lymphoma].

OBJECTIVE: To investigate the predictive value of neutrophil/lymphocyte ratio (NLR) and platelet/lymphocyte ratio (PLR) for the patients with diffuse large B-cell lymphoma (DLBCL). METHODS: The clinical data of 57 DLBCL patients admitted in the First Affiliated hospital of Anhui Medical University were analyzed retrospectively. According to ROC curve, the cut-off value for NLR and PLR was deterimined, and the patients were divided into high and low NLR/PLR groups before first chamotherapy. Then the relation of NLR and PLR with overall survival (OS) and progression-free survival (PFS) was analyzed by univariate and multivariate COX regression. RESULTS: The optimal cut-off value for NLR and PLR was 2.915 and 270.27, respectively. NLR at the diagnosis was found to be an independent predictor for OS and PFS by univariate and multivariate analysis, while the PLR was an independent predictor for PFS, but did not affect the OS. CONCLUSION: NLR and PLR may provide additional prognostic information for DLBCL patients.

[Influence of Anticoagulants on Detection of ITP Platelet-Specific Autoantibodies and Relationship of Autoantibody Types with Glucocorticoid Efficacy].

OBJECTIVE: To investigate the influence of divalent cation chelator EDTA and heparin sodium on the detection of ITP platelet-specific autoantibodies by the modified monoclonal antibody immobilization of platelet antigen assay (MAIPA) and to explore the relationship between types of platelet specific autoantibodies and glucocorticoid efficacy. METHODS: The samples were obtained from EDTA- and heparin- anticoagulant ITP patients, respectively, so as to detect the platelet-specific autoantibodies (GPIIb/IIIa and GPIbα) in 140 ITP samples by modified MAIPA, then the differences between these two methods were compared. RESULTS: Out of 140 cases in EDTA group, 55 cases were positive for GPIIb/IIIa, while 76 cases in heparin group were positive for GPIIb/IIIa, 42 cases in both group were repeatable; among them 13 cases were positive in EDTA group and negative in heparin group, while 34 cases were positive in heparin group and negative in EDTA group, there was significant difference between them (x (2) = 9.38, P < 0.05), 62 cases in 140 cases of EDTA group were positive for GPIba, while 51 cases in heparin group were positive for GPIba, 42 cases in both group were repeatabe; among them 20 cases were positive in EDTA group and negative in heparin group, while 9 cases were positive in heparin group and negative in EDTA group, there was no significant difference between them (x (2) = 3.44, P > 0.05). A total of 320 cases received a standard glucocorticoid treatment, out of them 143 cases were positive for GPIbα with effective rate 39.9%, 177 cases were negative for GPIbα with effective rate 79.7%, there was statisticalty significant difference between them (x (2) = 53.115, P < 0.05). CONCLUSION: EDTA anticoagulant (a divalent cation chelator) has a significant influence on detection of ITP platelet-specific autoantibodies (GPIIb/IIIa) by a modified MAIPA method and the GPIbα antibody positive may be one of the important factors that results in un-sensitivity of ITP patients to glucocorticoid treatment.

[Correlation of the Desialylation of Platelets with Efficacy of the First-line Therapy for ITP].

OBJECTIVE: To detect desialylation of platelets in primary immune thrombocytopenia(ITP) patients with FITC-labelled ECL and RCA-1, and compare the correlation of the desialylation level and the efficacy of first-line therapy for ITP. METHODS: Before treatment, 48 ITP patients were selected and their levels of ECL and RCA-1 were detected with flow cytometry. RESULTS: The desialylation level in the different efficacy groups by using the first-line therapy of corticosteroids and (or) intravenous immunoglobulin G (IVIG) had a statistically significant difference (P<0.05). The correlation analysis showed negative relation of the therapeutic efficacy with desialylation level, that is to say, the more high of desialylation level, the more poor therapeutic efficacy of the first-line therapy. CONCLUSION: The desialylation level of platelets in ITP patients is related with the first-line therapeutic efficacy, the efficacy for patients with high desialylation level is poor, suggesting that the FcR-independent pathway exists in clearance of platelets in ITP patients. Therefore, the desialylation level of platelets may suggest the first-line therapeutic efficacy for ITP patients to a certain degree, and may be used as a potential target for the treatment of refractory ITP.

[Analysis of plasma trough level of imatinib in Chinese CML patients].

OBJECTIVE: To evaluate the relationship between plasma trough level of imatinib and clinical outcomes in Chinese CML patients. METHODS: Plasma trough levels in 416 CML patients who received imatinib orally in six general hospitals were assessed. The correlations of imatinib plasma trough level with baseline characteristics including age, weight and BSA, and clinical response were evaluated. RESULTS: (1) Effects of age, body weight and BSA on imatinib plasma trough levels were not to be clinically significant. (2) Median imatinib plasma trough levels was 1271 (109-4329). Imatinib plasma trough level was related to dose of imatinib administration. Plasma trough levels at imatinib of dose < 400, 400 and > 400 mg were (969 ± 585), (1341 ± 595) and (1740 ± 748) µg/L (P < 0.01), respectively. (3) There was no statistic difference in imatinib plasma trough level with complete cytogenetic response [CCyR (1337 ± 571) µg/L vs no CCyR (1354 ± 689) µg/L, P = 0.255]. (4) Imatinib plasma trough level might be important for a good clinical response in some CML patients. CONCLUSION: There was a large interpatient variability in imatinib plasma concentration in Chinese CML patients. No correlation of imatinib plasma trough level with CCyR was observed. However, higher doses of imatinib were shown to attain greater trough plasma concentration, suggesting that imatinib plasma trough level might be important for a good clinical response in some CML patients.

[Mechanism of apoptosis in human leukemia NB4 cells induced by total astragalosides].

OBJECTIVE: To investigate the effect of total astragalosides (TA) on proliferation and apoptosis in human leukemia NB4 cells in vitro. METHODS: The NB4 cells were treated with TA at different concentrations for 48 h in culture. Growth inhibition rates were measured by CCK-8 method. Flow cytometry was used to explore the cell apoptosis and the activity of NF-κB and Akt during apoptosis. RESULTS: TA at different concentrations (200, 400, 600, 800 mg/L) inhibited proliferation of NB4 cells in a dose-dependent manner (P < 0.05), and the inhibitory rates of TA on NB4 cells were (14.54 ± 3.20)%, (24.79 ± 3.98)%, (57.28 ± 4.71)% and (88.28 ± 4.65)%, respectively. In terms of the induction of apoptosis, there was a significant difference between the TA group and blank control [(1.80 ± 1.24)%, P < 0.05]. At TA doses of 200, 400 and 600 mg/L, the apoptotic rates of NB4 cells were (10.03 ± 3.31)%, (14.87 ± 3.65)%, (23.45 ± 1.90)%, respectively. Besides, TA induced apoptosis of NB4 cells in a dose-dependent manner in the groups of 200 mg/L, 400 mg/L, 600 mg/L (P < 0.05). But there was no significant difference in apoptotic rates between the groups of 800 mg/L and 600 mg/L [(23.45 ± 1.90)%, P > 0.05]. In the group of 800 mg/L, the necrotic cells increased highly and the necrotic rate reached (45.65 ± 3.16)%. After TA treatment of NB4 cells at different concentrations (200, 400, 600 mg/L), the expression of NF-κB protein was significantly decreased compared with that of the blank control (9.79 ± 0.95, P < 0.05), while Akt protein was not significantly decreased (P > 0.05). CONCLUSION: TA can inhibit the growth of NB4 cells and induce apoptosis in NB4 cells through an Akt-independent NF-κB signaling pathway.

[Correlative study between JAK2 mutation and thrombosis in patients with myeloproliferative neoplasm].

OBJECTIVE: To investigate the frequency and clinical implication of JAK2 mutation in patients with myeloproliferative neoplasm(MPN)and the correlation between the mutation and thrombosis. METHODS: The clinical and laboratory data of 107 MPN patients was retrospectively analyzed. JAK2 mutation were detected with allele-specific polymerase chain reaction (AS-PCR) and sequencing. The significance of the mutation in disease diagnosis and molecular pathogenesis and the correlation between the mutation and thrombosis was analysed. RESULTS: JAK2 mutation was detected in 71 (66.4%) and thrombosis in 34 (31.8%) of the 107 MPN patients. Thrombosis occurred in 34.8% (16/46) of polycythemia vera (PV), 32.6% (14/43) of essential thrombocythemia (ET), and 22.2% (4/18) of primany myelofibrosis (PMF) patients. The difference among the 3 groups was not significant (χ(2) = 0.96, P > 0.05). The frequency of thrombosis in JAK2(+) MPN patients (82.4%, 28/34) was higher than that in JAK2(-) MPN patients (17.6%, 6/34) (χ(2) = 5.71, P < 0.05). The frequency of thrombosis in MPN patients > 60 years was higher (41.5%, 27/65) than that in patients < 60 years (16.7%, 7/42) (χ(2) = 7.28, P < 0.01). CONCLUSION: JAK2 V617F mutation occurs in significant percentage of Chinese patients with MPN. Patients with JAK2 mutation and older age are more succeptible to thrombosis. JAK2 mutation screening in patients with unknown thrombosis is helpful to reveal the underlying latent-MPN.

[Detection of ABL kinase domain point mutations in chronic myeloid leukemia patients receiving imatinib treatment].

This study was purposed to evaluate ABL tyrosine kinase point mutations in imatinib-treated chronic myeloid leukemia (CML) patients and their clinical significance. 51 bone marrow samples from 28 imatinib-resistant patients and 10 newly diagnosed CML patients were collected. ABL kinase domain of bcr-abl allele was amplified by nested reverse transcription-polymerase chain reaction, followed by purifying, directly sequencing and sequence homology analysis of amplified products in order to determine the existence and type of point mutation. The results showed that the point mutations were found in 12 of 38 patients, and all the 12 ones progressed to advanced disease or death. 2 patients showed Met351Thr mutation, 7 patients showed Glu252His, 2 patients showed Glu279Lys, the other types were Glu255Val and Glu355Gly, each of which was tested in one patient. The incidence of the point mutation was 17.6%, 45.5% and 44.4% in chronic, accelerated and blast phase respectively. The incidences of point mutation in hematologically and genetically resistant patients were 50% (5/10) and 44.4% (8/18), and the 95% confidence interval (CI) was 12.3% - 87.7% and 19% - 69.9% respectively. It is concluded that ABL kinase point mutation is an important mechanism of imatinib resistance, monitoring the ABL kinase domain point mutation is helpful to estimating the prognosis and adjusting the therapeutic strategy.