Establishment of Primary Adult MDS Nested Case-Control Study Cohort and Study of Risk Factors Associated with MDS Evolution to Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish a nested case-control study cohort in myelodysplastic syndrome (MDS) patients and investigate the clinical characteristics, WHO subtype and risk factors associated with MDS evolution to leukemia of this cohort.</p><p><b>METHODS</b>All patients, ≥18 years of age, provided by 24 Shanghai hospitals with initial clinical findings consistent with a hematopoietic abnormality between June 2003 and April 2007, were the candidates for inclusion in this study. The blood and bone marrow samples of every patient should be provided at baseline. Diagnosis was made by incorporating morphologic, immunophenotypic, cytogenetic and molecular features according to WHO classification criteria. Cytogenetic analysis was performed using conventional G-banding karyotyping and fluorescence in situ hybridization (FISH) techniques. Cumulative risk of evolution was estimated by Kaplan-Meier method. Prognostic factors were evaluated by univariate Log-rank method and multivariate Cox proportional hazard models.</p><p><b>RESULTS</b>A total of 435 patients were diagnosed as MDS. The median age of MDS onset was 58(18-90) years, with 248 male patients and 187 female patients (male: female 1.33: 1). The percentage of cases with refractory cytopenia with multilineage dysplasia (RCMD) was the highest (65.5%), while that of refraetory anemia (RA) (2.3%), refractory anenia with ring sideroblast (RARS) (1.1%) and 5q-syndrome (0.5%) was lower. Trisomy 8 (+8) was the most common chromosome abnormalities (71 cases, 12.7%). The mean follow-up time was 20.3 (4.2-57.1) months. Cases were patients with evolution by the end of follow-up, while controls were patients without evolution by that time. Case group included 41 patients and control group included 342 patients. Univariate analysis showed that the age, sex, WHO subtype, WBC count, absolute neutrophil count (ANC), IPSS cytogenetic subgroup, IPSS group and bone marrow blast percentage were significant risk factors for leukemia-free survival (LFS). Multivariate analysis of COX model showed that the age, sex, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast were independent risk factors for LFS.</p><p><b>CONCLUSION</b>A nested case-control study cohort of MDS patients is established. The clinical characteristics and WHO subtype of MDS patients in Chinese Shanghai are different from that in Western countries. The independent risk factors for MDS evolution are age, sex, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast percentage.</p>

Prognostic value of clinical characteristics and immunophenotypic biomarkers in 115 patients with primary central nervous system lymphoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Clinical outcome in patients with primary central nervous lymphoma (PCNSL) is variable and poorly predictable. This study investigated the association of clinical features and immune markers with prognosis of patients with PCNSL.</p><p><b>METHODS</b>One hundred and fifteen newly diagnosed PCNSL patients at the study institution were considered eligible for this study. Clinical characteristics and biochemical assay data were collected. Immunohistochemical staining of Cyclin D3, Cyclin E, Foxp1, and LMO2 were performed. All cases were followed-up regularly.</p><p><b>RESULTS</b>The common sites of involvement were frontal lobe (54.8%) and thalamus (16.5%). Diffuse large B-cell lymphoma composed of 96.5% of the cases. The median overall survival was 22 (4 - 41) months, and the 5-year survival rate was 22.8%. Age > 65 years, serum globulin > 40 g/L, large size of tumor, lymphocyte count ≥ 1 × 10(9)/L, and expression of Cyclin D3 and Cyclin E were associated with poor prognosis of PCNSL. Expressions of Foxp1, LMO2, and CD44 were not related to the survival. Expression of Cyclin E, large tumor size, and high serum globulin were independent prognostic factors for PCNSL.</p><p><b>CONCLUSIONS</b>PCNSL prognosis is relatively poor. Age, high tumor burden, higher lymphocyte count, expression of Cyclin D3, and Cyclin E are inferior prognostic factors for PCNSL.</p>

Establishment of human homoharringtonine-resistant SKM-1 cell line and its biological characteristics / 中华血液学杂志

<p><b>OBJECTIVE</b>To establish a homoharringtonine (HHT)-resistant SKM-1 cell line and explore its biologic characteristics and mechanisms for drug resistance.</p><p><b>METHODS</b>The HHT-resistant SKM-1 cell line was established by repeatedly exposing the cells to comparatively large doses of HHT with a short-time duration, and gradually elevating the drug concentration to an endurable level. The morphology of the resistant and parental cell lines was observed through optical microscope. The MTT assay was used to determine the doubling time and the resistance index to draw growth curve. The immunophenotype, cell cycle distribution and DNR accumulation between SKM-1 and SKM-1/HHT were analyzed by flow cytometry, and the karyotypes by R-banding. Semi-quantitative real-time PCR was performed to evaluate the expression levels of mdr1, MRP and topo-IIa.</p><p><b>RESULTS</b>The HHT-resistant cell line SKM-1/HHT was eventually established following 7-month drug induction. Both the resistant and the parental cell lines were similar with regard to morphology and immunophenotype. The karyotypes of the former was more complicated with differences located in chromosome 20, X, 4, 5, 9 and 11. The resistant cell line had more G(1) phase cells (64.04% vs 41.91%), less S phase cells (34.92% vs 53.53%), and less G(2) phase cells (1.04% vs 4.56%) compared with the parental cell line. The SKM-1/HHT cell line showed significant drug resistance to HHT, VCR, DNR and etoposide, the resistance indices of HHT, VCR, DNR and etoposide were 17.94, 8.75, 5.99 and 13.76 respectively. DNR accumulation was impaired in SKM-1/HHT cell line as less fluorescence of DNR (698 ± 36 vs 858 ± 54). The expression of mdr1 increased dramatically in the resistant cell line, its 2(-ΔCt) value was 20.1 higher than that of the parental cell line \[(3.42 ± 0.46)×10(-2) vs (0.17 ± 0.01)×10(-2), P < 0.05\], while MRP also increased in the resistant by 3.56 folds \[(4.77 ± 0.87)×10(-3) vs (1.34 ± 0.56)×10(-3), P < 0.05\]; However there was a slightly decrease of topo-IIa, the ratio of the resistant to the parental calculated by their 2(-ΔCt) values was 0.619:1 \[(1.91 ± 0.30)×10(-4) vs (3.08 ± 0.21)×10(-4), P < 0.05\].</p><p><b>CONCLUSION</b>A HHT-resistant cell line SKM-1/HHT was established. The prominent overexpression of mdr1 may be the main cause for multidrug resistance.</p>

Expression of FOXP1 and cyclinE in primary central nervous system lymphoma and its significance / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the value of FOXP1 and Cyclin E gene in primary central nervous system lymphoma(PCNSL) of immunocompetent patients on prognostic significance.</p><p><b>METHODS</b>Clinical data of 71 patients with newly diagnosed PCNSL from 2002 to 2007 was analyzed retrospectively. Immunohistochemistry method (HRP-EnVision(TM)) was performed to observe the expression of FOXP1 and Cyclin E gene in tumor tissue samples. The survival was analyzed by Kaplan-Meier survival curve, survival factors analysis by the Log-rank test and COX proportional hazards regression model.</p><p><b>RESULTS</b>FOXP1 positive was observed in 35 of 51 patients (68.63%) and Cyclin E staining was present in 29 of 50 cases (58.00%). FOXP1(+) patients had a shorter overall survival (OS) than FOXP1(-) ones. 2-year OS rate in FOXP1(+) and FOXP1(-) patients were 23.33% and 73.56%, respectively(P = 0.0015). Cyclin E(+) patients had a shorter overall survival(OS) than cyclinE(-) ones. 2-year OS rate in Cyclin E(+) and Cyclin E(-) patients were 17.56% and 69.76%, respectively (P = 0.0017). Multivariate analysis showed that Cyclin E expression was an independent prognostic factor for shorter OS (P = 0.048). FOXP1 expression might be an important prognostic factor for shorter OS (P = 0.065).</p><p><b>CONCLUSION</b>Cyclin E expression is an independent prognostic factor and FOXP1 expression is a possible prognostic factor for poor clinical outcome in patients with PCNSL.</p>

Functional study of mdr1 and GSTpi expression reversed by hairpin siRNA in K562/A02 cell line / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of hairpin small interference RNA (shRNA) on mdr1 and GSTpi protein expression in multidrug resistance human leukemia cell line K562/A02.</p><p><b>METHOD</b>The shRNAs were synthesized targeting the coding region sequences of mdr1 (79 - 99 nt) and GSTpi (308 - 327 nt) respectively, and cloned to plasmid pSilencer2.1-U6 neo. The cloned products pSilence mdr1 and pSilence GSTpi were transfected into K562/A02 cells. Western blot and immunofluorescence analysis were used to detect the effectiveness and the specificity of the gene silence. 50% inhibition concentration (IC(50)) of doxorubicin (ADM) on K562/A02 cells was determined by MTT method.</p><p><b>RESULT</b>pSilence mdr1 and pSilence GSTpi reduced the expression of P-gp and GSTpi protein from 0.75 +/- 0.02 and 0.54 +/- 0.02 to 0.48 +/- 0.05 and 0.39 +/- 0.02 (P < 0.01) respectively, with no effect on alpha-tubulin expression in comparison with the mock treatment. Transfection of pSilence lamin A/C into K562/A02 decreased lamin A/C expression but had no effect on the expression of P-gp and GSTpi. Immunofluorescence assay also showed that shRNAs significantly reduced the P-gp and GSTpi positive cells from (71.25 +/- 9.65)% and (81.25 +/- 6.49)% to (35.25 +/- 5.97)% and (41.25 +/- 4.43)% (P < 0.01), respectively, compared with the mock treatment. The resistance indexes after transfection were decreased to 8 (pSilence mdr1) and 10 (pSilence GSTpi) respectively from 23 (mock transfection) (P < 0.01).</p><p><b>CONCLUSION</b>The shRNA could effectively and specifically reverse the multidrug resistance on K562/A02 cell line.</p>

Study on the targeting effects of M1-GS RNA on K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To determine the effects of M1-GS RNA (M1 RNA) on bcr-abl mRNA and oncoprotein after M1 RNA with guide sequence (M1-GS RNA) targeting the oncogene was transfected into K562 cells.</p><p><b>METHODS</b>pAVGS4 (an eukaryocyte expression vector containing M1-GS RNA sequence) and pNAV-1 (as the control) were transfected into K562 cells by X-tremeGENE Q2. Total RNA was extracted at 24, 48, 72 and 96 hours after transfection. Then RT-PCR was done to compare the products at different time point. After collecting pAVGS4-transfected cells and the control cells at 48 and 96 hours after transfection, total protein was extracted and quantified. Change of P210 was determined by Western blot. Colony formation was analyzed at 96 hours after transfection.</p><p><b>RESULTS</b>RT-PCR based on transfected cells at different time point showed that the amount of bcr-abl mRNA began to decrease at 24 hours and reduced to 9.2% and 2.5% respectively at 48 and 72 hours after transfection. Western blot showed that the expression of P210 in the pAVGS4 group reduced to 10.4% of the control at 48 hours and 6.7% of the control at 96 hours after transfection. The inhibition rate of colony formation was 81.3% after K562 cells were transfected by pAVGS4.</p><p><b>CONCLUSION</b>pAVGS4 can efficiently destroy bcr-abl mRNA in K562 cells. The transcript level of bcr-abl mRNA was reduced with the time after transfection. The expression of P210 was decreased significantly at 48 and 96 hours after transfection. K562 cell colony formation was prominently inhibited.</p>

Experimental study of K562 cell apoptosis induced by siRNA / 中华血液学杂志

<p><b>OBJECTIVES</b>To construct a siRNA expression vector pBCR6 that produces siRNA against bcr/abl mRNA and detect apoptosis rate of K562 cells after pBCR6 transfection.</p><p><b>METHODS</b>Template sequence for siRNA was designed, synthesized and inserted into an expression vector pSilencer1.0-U6. Restriction analysis and sequencing were performed to verify the pBCR6 vector. Then pBCR6 was transfected into K562 cells by X-tremeGene Q2. pSilencer1.0-U6 was used as the control. At different time point after transfection, apoptosis rate was determined by Tunel and Annexin V+ PI with FCM.</p><p><b>RESULT</b>pBCR6 was verified by restriction analysis and sequencing. The apoptosis rate of K562 cells markedly increased at 48 and 72 hour after transfected with pBCR6, and increased in a time-dependent manner [the apoptosis rate of transfected K562 cells was (47.80 +/- 1.63)% at 72 hrs, whereas the control group was (6.67 +/- 0.37)%, P < 0.0001] No prominent change in apoptosis rate was found in the control.</p><p><b>CONCLUSION</b>The siRNA expression vector against bcr/abl mRNA was successfully constructed. The pilot study showed that pBCR6 could effectively induce K562 cells apoptosis. siRNA may be a new tool for molecular target therapy for chronic myelogenous leukemia.</p>