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Objective:This study has investigated the value of detecting cerebrospinal fluid (CSF) MyD88L265P mutation and interleukin-10 (IL-10) levels in the prognosis of PCNSL.Methods:We retrospectively analyzed the clinical data, CSF characteristics (including cytology, cell counting, total protein, and the level of cytokine IL-10) and treatment process of 39 PCNSL patients newly diagnosed by surgery and pathology (18 males and 21 females, aged 40-73 years) from August 2013 to December 2016 in Hua Shan Hospital North. MyD88L265P mutation was detected by digital PCR in 39 paraffin-embedded tissues and 35 cerebrospinal fluid samples. Log-rank test was used for univariate analysis and Cox regression for multivariate analysis to establish the prognosis model of PCNSL which might be related to PCNSL first progress-free survival (PFS) and overall survival (OS).Results:The median age of the 39 PCNSL patients was 59 years old, with 30.8% (12/39) intraocular involvement. The mutation rate of MyD88L265P in tissues and cerebrospinal fluid was 74.4% (29/39) and 40.0% (14/35), respectively. 51.9% (14/27) patients were observed with MyD88L265P mutation in both tissues and CFS. Univariate analysis showed that intraocular involvement, high level of IL-10 in CFS (≥45 pg/ml), and MyD88L265P mutation in CFS are factors significantly influencing median progression-free survival (mPFS) of patients ( P<0.05). Patients with intraocular involvement had shorter OS than those without involvement which was statistically significant ( HR=6.5,95% CI 1.7-47.3, P<0.05). And multivariate analysis showed that intraocular involvement ( HR=2.4, 95% CI 1.3-7.8, P<0.05) and CFS MyD88L265P mutation ( HR=2.1, 95% CI 1.1-5.7, P<0.05) were independent prognostic factors for PFS. Conclusion:The presence of intraocular involvement and MyD88L265P mutation in CFS indicated poor prognosis of PCNSL patients. High CSF IL-10 level was not an independent factor affecting prognosis.
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Objective@#To investigate the occurrence and influencing factors of anemia in lymphoma patients and its effect on treatment.@*Methods@#A total of 501 lymphoma patients, who were hospitalized in four general hospitals in Shanghai from January 2017 to June 2018, were followed up for six months. The clinical data about anemia were collected and statistically analyzed.@*Results@#Among all the enrollment patients, there were 178 patients (35.5%) had anemia. Among 289 patients whom were initially treated for lymphoma, there were 99 patients (34.3%) with anemia. In the following-up time, there were 136 new cases (42.1%) of anemia. The total prevalence of anemia was 62.7%. The univariate analysis indicated that the anemia incidence for initially treated lymphoma patients was significantly related to their age, pathological type, bone marrow infiltration, IPI scores and Ann Arbor stage. The response to initially treatment in lymphoma patients with anemia was inferior to those without anemia. The multivariate analysis indicated that IPI scores 3-5 points (P<0.001, OR=4.230, 95%CI 2.339-7.650) and chemotherapy treatment (P<0.001, OR=11.049, 95%CI 5.149-23.711) were the independent influential factors to the emerging anemia incidence. PS score used to evaluate patient physical condition was obviously related to the anemia occurrence.@*Conclusion@#Lymphoma patients have a high prevalence and incidence of anemia. The occurrence and severity of anemia are closely related to the efficacy and physical condition of lymphoma patients.
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Objective To investigate the expressions of BAP1 and TET2 proteins in bone marrow of patients with chronic myelomonocytic leukemia (CMML) and their relationship with the prognosis of CMML. Methods The bone marrow paraffin specimens of 41 cases from 41 adult CMML patients diagnosed by Shanghai Sino-US Joint Leukemia Coordination Group from September 2003 to May 2007 were collected. The immunohistochemistry was used to detect the expressions of BAP1 and TET2 proteins in 41 CMML patients. The expressions of TET2 and BAP1 proteins were also detected by the same method in 40 adult patients with acute myelomonocytic leukemia (AMML) and 20 patients with iron deficiency anemia (IDA) diagnosed at the same time as the comparison. The clinical data of 41 CMML patients were analyzed by using retrospective cohort study. The count data were compared by using chi-square test. The correlation between expressions of BAP1 and TET2 proteins was analyzed by using Pearson correlation analysis. Kaplan-Meier method was used to calculate the survival time, and Log-rank test was used for single factor analysis. Results In 41 CMML patients, the positive expression rate of BAP1 was 31.7％ (13/41), including 28.6％ (8/28) in CMML-1 patients and 38.5％ (5/13) in CMML-2 patients; the positive expression rate of TET2 was 41.5％ (17/41), including 39.3％ (11/28) in CMML-1 patients and 46.2％ (6/13) in CMML-2 patients. In 40 AMML patients, the positive expression rate of BAP1 was 32.5％ (13/40), and the positive expression rate of TET2 was 35.0％ (14/40). In 20 IDA patients, the positive expression rate of BAP1 was 5.0％ (1/20), and TET2 had no positive expression. There was no significant difference in the expressions of BAP1 and TET2 proteins between CMML-1 and CMML-2 patients (χ 2 = 0.40, P = 0.53; χ 2 = 0.17, P = 0.68). There was no significant difference in the expressions of BAP1 and TET2 proteins between CMML and AMML patients (χ 2 = 0.01, P = 0.94; χ 2 = 0.36, P = 0.64). There were significant differences in the positive expression rate of BAP1 and TET2 proteins between hematological neoplastic disease (CMML+AMML) and hematological non-neoplastic disease (IDA) (χ 2 = 6.01, P < 0.05; χ 2 = 11.04, P < 0.01). Pearson correlation analysis showed that there was no correlation between expressions of BAP1 and TET2 proteins (r = 0.35, P = 0.27). Univariate analysis showed that anemia (Hb < 60 g/L), mature monocyte count ≥ 2.0×109/L, neutrophil count (1.5×109/L), abnormal karyotype were associated with poor prognosis for CMML. Protein expressions of BAP1 and TET2 were not related with the prognosis of CMML (χ 2 = 0.28, P = 0.600; χ 2 = 0.53, P = 0.460). Conclusion Both BAP1 and TET2 proteins have high positive expression rates in CMML patients, but the expressions of them are not related to the prognosis.
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Objective: To analyze the clinical characteristics of patients with relapsed/refractory primary central nervous system lym-phoma (PCNSL) and to explore the factors that influence the prognosis, in order to provide evidence for the clinical diagnosis and treat-ment. Methods: Sixty-four patients with relapsed/refractory PCNSL diagnosed from October 2006 to August 2015 were selected. The clinical features, treatment plans, and laboratory examination data were retrospectively analyzed. Cox regression was used for multi-variate analysis. Results: Univariate and multivariate analyses showed that progression-free survival of first time (PFS1)≤1 year and Kar-nofsky performance status (KPS) score<70 points were independent prognostic factors in patients with first relapsed/refractory PCNSL. The median PFS2 and overall survival of second time (OS2) were 19 and 21 months, respectively, in patients with PFS1≥1 year, where-as the median progression free survival of second time (mPFS2) and OS2 were 10 and 14 months, respectively, in patients with PFS1<1 year. The median PFS2 (mPFS2) in patients with first relapse/refractory KPS score≥70 points and those with KPS score<70 points were 40 and 10 months, respectively, and the median OS2 were 43 and 12 months, respectively. The median PFS for the methotrexate (MTX) and non-MTX groups was 18 and 10 months, respectively. Multivariate analysis showed that the salvage therapy was a relevant factor influencing the patient's PFS. However, univariate analysis showed that the median OS2 in the MTX and non-MTX groups was 23 and 12 months, respectively, with significant difference but without any correlation with prognosis. Conclusions: progression-free sur-vival (PFS)≤1 year and KPS score<70 were independent prognostic factors in patients with first relapsed/refractory PCNSL. Patients with relapsed/refractory PCNSL who continuously received high-dose MTX-based treatment may have improved long-term treatment outcomes.
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Myeloproliferative neoplasm (MPN) is a kind of clone hematopoietic stem cell disease characterized by one or more myeloid cell lines hyperproliferation, including bcr-abl gene positive chronic myeloid leukemia (CML) and bcr-abl negative MPN, the later representing polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). A big stride has been made since the discovery of JAK2 and MPL gene mutations. However, the exact genetic basis of JAK2/MPL mutation double negative in MPN patients is still unclear. It has been reported recently that a new CALR mutation is discovered in the JAK2/MPL unmutated MPN patients who show unique clinical presentations, which provides a new diagnostic and prognosis-accessing criteria. The paper reviews CALR mutation and genetic mechanism mediating MPN.
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Objective Based on our previous established cohort of myelodysplastic syndrome (MDS), we investigated the potential effect of beta-tubulin (TUBB) gene in the transformation of MDS into acute leukemia Methods From our nested case-control study cohort of MDS patients, we chose 11 paired transformed and nontransformed MDS patients.TUBB gene expression was tested by quantitative real-time PCR.TUBB-siRNA transfection was used to down-regulate TUBB gene expression in SKM-1 cell line.The function of TUBB gene in SKM-1 cell line was evaluated by cell proliferation, soft agar clone formation and electron microscope.Results TUBB gene expression in MDS patients in transformed group were significantly higher than that in control group (2.91 ± 0.41 vs 0.90 ± 0.23, P ＜0.01).After TUBB-siRNA transfection, A450/630nm of SKM-1 cells at 24 h, 48 h and 72 h were 0.299 ± 0.045, 0.526 ± 0.034 and 0.652 ± 0.035, respectively, which were significantly decreased than those in negative-siRNA group (0.438 ±0.074, 0.858 ±0.064 and 0.974 ±0.044) (P ＜0.05).Soft agar clone formation in TUBB-siRNA group was (7.0 ±0.2)％, which was significantly reduced than that of negative-siRNA group (25.0 ± 0.2)％ (P ＜ 0.01).Electron microscope showed significant apoptotic signs in TUBB-siRNA group, including vacuoles in cytoplasm and karyorrhexis.Conclusion Our results indicate that TUBB gene may play a role in the transformation of MDS into acute leukemia by affecting the proliferation of malignant clones.
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Objective To evaluate the sensitivity, repeatability and accuracy of microarray digital PCR system in detecting JAK2 V617F mutation, which was closely related to myeloproliferative neoplasms (MPN).Methods All of the 31 MPN patients with JAK2 V617F mutation, including 18 cases of polycythemia vera(PVs),11 primary thrombocythemias (ETs) and 2 primary myelofibrosis (PMFs), were collected from Huashan Hospital, Fudan University during 2014 -2015, while 10 normal controls and 6 cases with abnormal increased hemoglobin were involved.Human erythroleukemia cell line ( HEL ) and colorectal cancer cell SW480 were used as the mutant and the wild type control, respectively.The sensitivity of microarray digital PCR were verified by detecting the gradient diluted mutation standard harboring 30%, 10%, 1%, 0.1%and 0.01%mutant allele burden, respectively .Repeatability was evaluated by detecting 1%and 10% mutated samples for 5 times, respectively.MGB probe real time PCR was selected as the reference method to verify the accuracy of the digital PCR.Results With digital PCR, the accurate quantitation of JAK2 V617F mutation was achieved down to 0.1%, which is approximate to 0.16 copies per microliter.The results obtained from the two kinds of technique showed a high correlation by linear regression analysis (R2 =0.998 3).The results of repeated samples showed CVs as 17.18% for 1%mutant allele burden and 7.50%for 10%.Among all cases, the 31 patients known mutated were detected as positive and 10 controls as negative by both digital PCR and Real time PCR.In another 6 cases, 2 were found JAK2 V617F mutation of low allele burdens of 0.37% and 0.18% by digital PCR but detected as negative by real time PCR.Conclusions Microarray digital PCR offers a higher sensitivity and better repeatability than real time PCR which could help detect rare JAK2 V617F mutations in MPNs accurately.
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Objective To establish a novel glucocorticoid (GC)-resistant human diffuse large B lymphoma(DLBCL) cell line Toledo/dexamethasone (DEX) by the exposure to DEX,and observe the biological characteristics of resistant and parental cell line,investigate the mechanisms of glucocorticoid-resistance.Methods Toledo/DEX was established by the exposure to DEX,the dose of which was increased gradually and intermittently for long periods of time.Toledo,in the logarithmic growth phage,was incubated in the culture medium containing DEX at the concentration of 1×10-8 mol/L at first.The medium without DEX was replaced after for 96 hours until the cell line re-entered the logarithmic growth phase.Repeat the above steps to acquire the ultimate concentration of DEX in the medium as 1.024 ×10-5 mol/L.The biological characteristics of resistant and parental cell lines were evaluated.Results Toledo/DEX was more invasive in the aspects of ultrastructure,tumorigenicity and drug sensitivity.Meanwhile,Toledo/DEX achieved some stable biological characteristics such as morphology,karyotypes and immunophenotype.Furthermore,GC receptor (GR) α and GR β protein expression analysis showed that GR was involved in the mechanism of the GCresistance.Conclusions Toledo/DEX is a drug-resistant cell line with a stable biology backgroud.These results may help shed light on the knowledge of GC-resistance and lay the groundwork for searching new therapeutics to reverse drug-resistance.
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ObjectiveTo establish a simple and sensitive method to detect MPL515 mutations in peripheral blood of ET and PMF patients,and investigate the frequencies of the MPL515 and JAK2V617F mutations in Chinese patients.MethodsTotallv 261 patients of ET and 25 PMF cases were collected from Huashan Hospital of Fudan University and DNA samples were isolated from peripheral blood of these cases.SYBR GreenⅠreal-time PCR was used to detect JAK2V617F mutation.Taqman probe was designed to be specific for the three types of mutations ( MPl515wt,MPLW515L and MPIW515K).Real-time PCR was used to detect MPL515 mutations.Tbe results were confirmed by sequencing after T-A cloning.Results Among 261 ET patients,119 cases (45.6％ ) were identified as JAK2V617F mutation carriers and 7 cases (2.7％ ) were detected to be MPl515 mutation carriers,including 5 cases with MPLW515L,1 case with MPLW515K and 1 ease with MPLW515L + K.Additionally 10 cases with JAK2V617F(40.0％ ) and 3 cases with MPL515 ( 12.0％ ) were screened out in 25 PMF patients,including 1 case with MPLW515L and 2 cases with MPLW515L + K.One ET patient was found to harbor concurrent JAK2V617F and MPL515 mutations.ConclusionJAK2V617F mutation is the major molecular marker of ET and PMF,meanwhile MPL515 mutation is important and useful complement.
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Cancer stem cells are considered to be the origin of tumor relapse.Isolating and identifying them by special immune-phenotypes is the most commonly used method.The technique of isolating SP cells is very mature.More attentions are being paid to the isolation of cancer stem cells using differential expression activities of Wnt pathway on surface of tumor cells.Establishing stem cells niches to capture cancer tumor cells remains to be further evaluated.
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Objective To investigate the role of endothelial bioreacter device in sepsis porcine model.Method Sepsis porcine model was induced gy established endotoxin (LPS,0.25 mg·kg~(-1)) in healthy hybrid swines. The animals were randomly divided(random number) into endothelial bioreactor device group(EBR group) and sham circulation group( Sham group)( n = 6, respectively). After the infusion of endotoxin, extracorporeal circulation was started with the blood flow of 30 mL/min. The blood went through the endothelial bioreactor, then went back to the body via internal jugular vein in the EBR group. The bioreactor with the same size and without endothelial cells(ECs) was used in the sham group. Hemodynamic variables, blood biochemistry, inflammatory markers, Endothelin-1(ET-11) and yon Willebrand Factor(vWF) were examined just before and every hour after the injection. When the survival time of the animals was recorded,the animals were sacrificed to calculate the lung injury score. The time-dependent hemodynamics and cytokine data were compared between groups by repeated measurement ANOVA .Student's t -test was used to analyze the survival time. Results The mean artetial blood pressure (MAP) remarkably decreased in both groups after LPS injection, while the decreasing rate in EBR group was significantly lower than that in control group after 2 hours( P < 0.05). The ET- 1 level in EBR group increased after a slight decrease at the beginning, while that in the sham group went on increasing(P<0.01). The vWF levels increased first, then returned to the baseline in the sixth hour in both groups, while the change in EBR group was significantly less than that in the sham group(P<0.05). The Lung Injury Score in EBR-treated group was significantly lower than that in the sham group(6.1 ± 0.9 vs. 8.2 ± 1. 0, P < 0.05). These physiologic and biochemical alterations were associated with a significant advantage to the survivals in the EBR group when compared with the control sham group(6.7 ± 1.32 vs. 5.2 ± 0.61 h, P < 0.01 ). Conclusions Timely intervention in endotoxin shock with EC therapy by using tissue-engineered bioreactor may improve cardiovascular performance and alter the natural course of this disease process, probably via modulating ioflammation and coagulation cascades.
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Objective To investigate the characteristics of dysplasia in myeledysplastic syndrome (MDS). Methods 716 samples of adult patients with abnormal blood routine and unelear cause were collected between July 04, 2003 and March 14, 2007. Based on the gold diagnostic standard of WHO MDS classification, all eases were detected on cytomorphology, cytochemical stain, bone marrow pathological assay,cytogenetics, flow eytometry et al. The cytological study of bone marrow on some abnormal hematopaietie cells has a diagnostic value to determine clonal or non-clonal diseases and assess sensitivity and specificity. Results in the complicated various dysplasia of hematopeiefic ceils, the following characteristics can be the main basis of cytomorphological diagnosis: one of granular Auer bodies, micronuclens (MN), or nuclear budding, erythroid nuclear budding, megakaryocytes presented in peripheral blood, myeloblast or prorubricyte exhibited in peripheral blood, ringed sideroblasts>1%. The subordinate basis of cytomorphological diagnosis development of nuclei, ring-shaped nuclei, and aggregation of nuclear chromafin, erythroid multi-nuclei, odd nucleus, mother-daughter nucleus, nuclear fragmentation, vacuole, anisoeytosis and mieromegakaryocytes.Conclusion Cytomorphologic assay is the base for the diagnosis of MDS, however, it presents certain limit,especially when eytomnrphoiogical change does not possess specificity for early MDS. Hereby, it requires to combine other deteetion methods.
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The patient was a 38-year woman.Six years prior to presentation,she developed erythema and papules with occasional pruritus in both labium majora,which gradually confluenced into plaques with the formation of superficial erosion and ulcer;extension into the vulva and crissum occurred later.Half a year prior to presentation,a subcutaneous firm nodule measuring about 1.5cm in diameter with intact epidermis,developed on the right submaxilla,associated with mild swelling in the area;the nodule enlarged gradually.A subcutaneous induration measuring 0.5 cm in diameter was observed in the left chin 2 months later.She also reported a 20-year history of dry eye and dry mouth.The patient tested postive for antinuclear antibody cally,there was a focal infiltration of lymphocytes in tissue of minor salivary glands.The pathology of lesions on the right labium majus showed a dense dermal infiltration with S100-and CD1a-positive,irregulady-shaped histiocyte-like cells with abundant eosinophilic cytoplasm and few mitotic figures.Needle biopsy of the right submaxilla area showed tumor cells.A diagnosis of Langerhans cell histiocytosis and Sj(o)gren syndrome was made based on clinical manifestation,laboratory findings and histopathological features.Combination chemotherapy with cyclophosphamide,vincristine,corticosteroids and etoposide resulted in clinical improvement.
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Objective To evaluate the DLC-l gene promoter methylation in acute lymphoblastic leukemia(ALL)in children by methylation specific-PCR(MSP),explore the its prognostic value.Methods Both pyrosequecing and MSP was used to detect DLC-1 gene methylation in bone marrow samples from 34 children with acute lymphoblastic leukemia,5 normal bone marrow samples and acute leukemia T cell line 6T-CET.DLC-1 mRNA expression in cells with、or without treatment with 5-aza-2'-deoxycytidine wag investigated by real time RT-PCR Results MSP analysis showed that DLC-1 promoter methylation Was identified in 21 of bone marrow samples from ALL patients,but was absent in 5 normal bone marrow specimens.These results were completely agreement with pyrosequencing analysis.In additional,the latter can give the quantitative analysis of methylation status in specific CpG sites.No association Was observed between DLC-1 methylation and patient’S clinical-biologic characteristics including age,gender,WBC count,FAB classification,immunology typing and clinical typing(P>0.05)In 18 ALL patients achieving complete remission(CR)witIl DLC-1 methylation,14 relapsed in 12 monthhs,while only 4 relapsed in 1 patients without DLC-1 gene metllylation.Treatment of the cell line 6T-CEM harboring methylation with 5-aza-2'-deoxycytidine increased DLC-1 expression in dose dependent manner. Conclusions This is the first report of high frequency of promoter methylation of DLC-1 in ALL It shows that DLC-1 methylation iS useful for predicting relapse of ALL.Pyrosequencing assay Call provide a sensitive,easy-to-use method for quantitative assessment of DLC-1 methylation.
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PurposeTo explore the relationship between expression of apoptosis-modulating proteins and chemotherapeutic efficacy in acute leukemia. MethodsImmunocytochemical method was used to detect the expression of Bcl-2、Bcl-XL、Bax and Bak in 36 cases of acute leukemia including previously untreated/ drug-sensitive group and refractory/relapse group. ResultsThe average positive cell rates of Bcl-2 and Bcl-XL in refractory/relapse group were (41.68 ± 14.39) % and (35.96 ± 9.95 ) %, while the rates in previously untreated/drug-sensitive group were (15.64 ± 8.51 )% and (12.91 ± 8.63 )%. Statistical analysis showed the average positive cell rates of Bcl-2 and Bcl-XL in refractory/relapse group were higher than those in previously untreated/drug-sensitive group (P ＜ 0.01 ). There was no significant difference in average positive cell rates of Bax and Bak between refractory/relapse group (25.28 ± 15.49) %, (15.53 ± 10.64) % and previously untreated/drug-sensitive group (21.55 ± 12.58)%, (13.23 ± 8.36)%. The Logistic regression of expression of Bd-2 、Bcl-XL、Bax and Bak to complete remission rate (CR) of 36 cases of acute leukemia showed that Bcl-XL was the most risk factor in reducing the CR.ConclusionsBcl-2 and Bcl-XL might play important roles in multi-drug resistance of acute leukemia and Bcl-XL was more important than Bcl-2.