Significance of Morphological Examination, Cytochemical Staining Combined with Bone Marrow Biopsy in Differential Diagnosis of Myelodysplastic Syndrome with Low Blasts and Hemolytic Anemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the value of morphological examination, cytochemical staining combined with bone marrow biopsy in the differential diagnosis between myelodysplastic syndrome (MDS) with low blasts and hemolytic anemia (HA).</p><p><b>METHODS</b>The clinical data of 85 cases of myelodysplastic syndrome with low blasts (< 5%) and 61 patients with hemolytic anemia in Chinese PLA's Gerneral hospital from September 2009 to March 2015 were retrospectively analysed. The clinical characteristics, cytogenetic and molecular features, bone marrow cell count and morphology features, cytochemical staining results and bone marrow biopsy features of above-methioned patients were compared.</p><p><b>RESULTS</b>There was no significant difference (P > 0.05) in clinical data between MDS group and HA group. Megakaryocytic dysplasia-positive rate, and ring sideroblasts positive rate, and PAS positive rate were significantly higher in MDS group than those that in HA group (P < 0.05). Abnormal localization of immature precursors (ALIP) and megakaryocytic dysplasia positive rate in bone marrow biopsy were significantly higher in MDS group than those that in HA group (P < 0.05), 90.6% of MDS with low blasts patients were identifiable by combined detections.</p><p><b>CONCLUSION</b>Combining detection of morphology, cytochemistry staining and bone marrow biopsy has been confirmed to be more useful for differential diagnosis between MDS with low blasts and HA.</p>

Clinical significance of bone marrow morphological examination and tumor marker detection for lymphoma diagnosis and prognosis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>This study was aimed to evaluate the significance of bone marrow(BM) morphological examination and many tumor marker(TM) detection, especially carcinoembryonic antigen (CEA), cancer antigen 125(CA125), cancer antigen 15-3 (CA15-3) and serum ferritin (SF) for lymphoma diagnosis and prognosis.</p><p><b>METHODS</b>A total of 47 confirmed patients with lymphoma in our hospital from January 2012 to October 2013 and 20 health peoplels as normal controls were performed with bone marrow morphological examination, at the same time, the electrochemistry luminescent technique was applied for detecting levels of TM (especially CEA, CA125, CA15-3 and SF) in serum samples of lymphoma patient and normal controls, then the BM immature lymphocyte counts of these people and clinical parameters were analyzed for diagnosis and prognosis.</p><p><b>RESULTS</b>There was significant differences in all the four TM levels between serum samples of lymphoma patients and normal control (P=0.029, P=0.000, P=0.005, P=0.000). These TM levels had no correlation with age, sex white blood cell, lymphocyte, platelet counts and anemia of lymphoma patients (P>0.05). It was also found that the patients with elevated TM levels had high BM immature lymphocytes (lymphoma cells) counts, B symptoms, advanced clinical stage and high IPI index (P<0.05). The CA15-3 and SF levels in serum samples of lymphoma patients with BM infiltration were higher than that in lymphoma patients without BM infiltration (P=0.002, P=0.000).</p><p><b>CONCLUSION</b>Combination of BM morphological examination with serum TM level detection plays an important role in diagnosis, clinical stage and prognosis evaluation of lymphoma patients. It is also very important for assessing BM infiltration status of lymphoma patients.</p>

Clinical Significance of ID4 Gene Mehtylation in Demethylation-Treated MDS Cell Line and 2 MDS Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate significance of ID4 gene mehtylation in demethylating myelodysplastic syndrome(MDS) cell Line MUTZ1 and 2 patients with MDS.</p><p><b>METHODS</b>The methylation-specific PCR (MS-PCR) and reverse transcription-PCR (RT-PCR) were applied to identify the methylation status and gene expression of ID4 gene in MDS cell line MUTZ1, a patient with aplastic anemia(AA) and a donor with normal bone marrow (NBM). RT-PCR was applied to detect the ID4 gene expression status in MUTZ1 cell line treated with decitabine at 3 different concentrations. Then bisulfite sequencing PCR (BSP) was applied to detect ID4 gene methylation status in 2 MDS parients treated with decitabine.</p><p><b>RESULTS</b>The MDS cell line MUTZ-1 displayed a complete methylation of ID4 gene promoter with little mRNA expression. Inversely, bone marrow of an AA patient and NBM showed complete unmethylation of this gene with intensity mRNA expression. With the increase of decitabine concentration, ID4 gene mRNA expression was more and more increased. After decitabine treatment, ID4 gene methylation-positive frequencies of both the 2 MDS patients were much more decreased than that of the first treatment. So, ID4 gene mRNA expression inhibited by promoter hypemethylation could be recovered by using demethylation medicine.</p><p><b>CONCLUSION</b>ID4 as a new potential anti-oncogene suggests that its methylation may become a marker for selection and assessment of therapeutic schedules in patients with MDS.</p>

Quantitative Detection of ID4 Gene Aberrant Methylation in the Differentiation of Myelodysplastic Syndrome from Aplastic Anemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The diagnosis of myelodysplastic syndrome (MDS), especially hypoplastic MDS, and MDS with low blast counts or normal karyotype may be problematic. This study characterized ID4 gene methylation in patients with MDS and aplastic anemia (AA).</p><p><b>METHODS</b>The methylation status of ID4 was analyzed by bisulfite sequencing polymerase chain reaction (PCR) and quantitative real-time methylation-specific PCR (MethyLight PCR) in 100 patients with MDS and 31 patients with AA.</p><p><b>RESULTS</b>The MDS group had a higher ID4 gene methylation positivity rate (22.22%) and higher methylation levels (0.21 [0-3.79]) than the AA group (P < 0.05). Furthermore, there were significant differences between the hypoplastic MDS and AA groups, the MDS with low blast count and the AA groups, and the MDS with normal karyotype and the AA groups. The combination of genetic and epigenetic markers was used in much more patients with MDS (62.5% [35/56]) than the use of genetic markers only (51.79% [29/56]).</p><p><b>CONCLUSIONS</b>These results showed that the detection of ID4 methylation positivity rates and levels could be a useful biomarker for MDS diagnosis.</p>

Clinical Implications on ZO-1 Gene Methylation in Myelodysplastic Syndrome Progression / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical significance of ZO-1 gene methylation level in MDS progression in order to provide a theoretical basis for evaluating progrosis of MDS patients.</p><p><b>METHODS</b>The methylation specific PCR (MS-PCR) was performed to evaluate the ZO-1 gene methylation status in bone marrow samples of normal persons as control (NC). MDS and AML patients, the bisulfite sequencing PCR (BSP) was applied to detect the ZO-1 gene methylation status in serial bone marrow samples of MDS-RA, MDS-RAEB and AML stages of a MDS patients.</p><p><b>RESULTS</b>The possitive rate of ZO-1 gene methylation in samples of NC, MDS and AML patients displayed significant difference; in sample of NC group the positive of ZO-1 gene methylation was not observed, but the positive rate of ZO-1 gene methylation in samples of AML patients was highest (65.0%), the proportion of ZO-1 gene methylation in myeloid blast count of MDS/AML patients was higher (P=0.000). The serial samples in one MDS patient showed that along with progress of disease, the positive rate of ZO-1 gene methylation in MDS-RA, MDS-RAEB and AML stages was found to be obvious different (P=0.000), the positive rate of ZO-1 gene methylation in AML stage was highest (64.65%).</p><p><b>CONCLUSION</b>The high methylation in promoter region of ZO-1 gene has been found in MDS/AML patients, and along with clonal proliferation, the positive rate of ZO-1 methylation and positive froguency of methylation sites increase graduatly which suggests that the MDS progresses in a certain degree, and the ZO-1 gene methylation level may be used as an new indicator for monitoring desease progression from MDS to AML.</p>

Establishment of methylation-specific quantitative PCR system for ID4 gene in acute leukemia cells and its specificity and sensitivity / 中国实验血液学杂志

DNA methylation of ID4 gene promoter occurred frequently in patients with acute leukemia and was found to be highly related to the tumor progression. Due to lack of the appropriate methylation detection methods, the relation between the quantification of ID4 methylation and the states of acute leukemia is still unclear. This study purposed to set up a methylation-specific quantitative PCR system for ID4 and investigate the specificity and sensitivity of this methylation detection. The plasmids combined with target gene as well as with internal reference were constructed, and the standard curves were set up by using above mentioned plasmids. The specificity of this detection system in cell lines was verified through techniques of MSP and quantitative MSP. The sensitivity of this detection system was verified by mixing methylation-positive and negative cell lines in varying proportions and through amplification of qualitative MSP. The results showed that the standard curves were establish successfully. The results of quantitative MS-PCR in cell lines were consistent with those of MS-PCR, and as low as 1: 10(-5) of ID4 methylation positive cells could be detected by the new methylation detection assay. In newly diagnosed acute leukemia patients, the positive rate of quantitative MSP was higher. It is concluded that a complete quantitative MSP system for ID4 methylation detection has been established and this quantitative MSP method has good specificity and high sensitivity.

Clinical significance of ID4 methylation detection by quantitative methylation-specific PCR in acute leukemia / 中国实验血液学杂志

The advances of treatment improved the prognosis of the patients with acute leukemia (AL) in the last decade, but the lack of general biomarker for predicting relapse in AL, which is one of the most important factors influencing the survival and prognosis. DNA methylation of ID4 gene promoter occurred frequently in patients with AL and was found to be highly related to the tumor progression. Based on the previous work of the setup of methylation-specific quantitative PCR system for ID4 gene, this study was designed to investigate the relation between the quantitative indicator of methylation density, percentage of methylation reference(PMR) value, and different disease status of AL. PMR of ID4 was detected by MS-PCR in bone marrow (BM) samples of 17 healthy persons and 54 AL patients in the status of newly diagnosis, complete remission and disease relapse. The results showed that at different disease status, PMR value in newly diagnosed group was significantly lower than that in complete remission group (P = 0.031). Among serial samples, PMR value remained very low at the status of patients with continuous complete remission (<1.5‰), and increased along with the accumulation of tumor cells at relapse. In 1 relapse case, the abnormal rise of PMR value occurred prior to morphological relapse. PMR value seemed to be related to body tumor cell load. It is concluded that the quantitative indicator of methylation density and PMR value may reflect the change of tumor cell load in acute leukemia patients. Dynamic monitoring of PMR maybe predict leukemia relapse.

Perioperative aortic dissection rupture after endovascular stent graft placement for treatment of type B dissection / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The perioperative aortic dissection (AD) rupture is a severe event after endovascular stent graft placement for treatment of type B AD. However, this life-threatening complication has not undergone systematic investigation. The aim of the study is to discuss the reasons of AD rupture after the procedure.</p><p><b>METHODS</b>The medical record data of 563 Stanford type B AD patients who received thoracic endovascular repair from 2004 to December 2011 at our institution were collected and analyzed. Double entry and consistency checking were performed with Epidata software.</p><p><b>RESULTS</b>Twelve patients died during the perioperation after thoracic endovascular repair, with an incidence of 2.1%, 66.6% were caused by aortic rupture and half of the aortic rupture deaths were caused by retrograde type A AD. In our study, 74% of the non-rupture surviving patients had the free-flow bare spring proximal stent implanted, compared with 100% of the aortic rupture patients (74% vs. 100%, P = 0.213). The aortic rupture patients are more likely to have ascending aortic diameters = 4 cm (62.5% vs. 9.0%, P = 0.032), involvement the aortic arch concavity (62% vs. 27%, P = 0.041) and have had multiple stents placed (P = 0.039).</p><p><b>CONCLUSIONS</b>Thoracic AD endovascular repair is a safe and effective treatment option for AD with relative low in-hospital mortality. AD rupture may be more common in arch stent-graft patients with an ascending aortic diameter = 4 cm and with severe dissection that needs multi-stent placement. Attention should be paid to a proximal bare spring stent that has a higher probability of inducing an AD rupture. Post balloon dilation should be performed with serious caution, particularly for the migration during dilation.</p>

Significance of methylation status of zo-1 gene in differential diagnosis for myelodysplastic syndrome / 中国实验血液学杂志

It is hard to discriminate myelodysplastic syndrome(MDS) from many benign hematological diseases. To identify the methylation status of zo-1 gene in MDS, the methylation specific PCR (MS-PCR) and reverse transcription-PCR (RT-PCR) were applied to detect the MDS cell line MUTZ-1, bone marrow of a healthy donor and an aplastic anemia patient. MS-PCR was also employed to detect the bone marrow of 72 patients with benign hematological diseases, 35 MDS-RA patients, and 20 MDS-like patients. The results showed that MDS cell line MUTZ-1 displayed complete methylation of zo-1 promoter without mRNA expression. Inversely, a patient with benign hematological disease and a donor with normal bone marrow showed complete unmethylation of this gene with unaffected mRNA expression. No zo-1 promoter methylation was detected in patients with benign hematological diseases, while aberrant hypermethylation of zo-1 gene promoter were found in 48.6% (18/37) of MDS-RA patients. The positive rate of zo-1 methylation in MDS-RA patients was higher than that in patients with benign hematological diseases (p < 0.05). Seven suspected MDS patients manifested hypermethylation status of zo-1 gene (7/20), 2 were followed up for 1 year and transformed into MDS. It is concluded that relatively high hypermethylation rate of zo-1 promoter is observed in MDS-RA, and no methylation in patients with benign hematological diseases. Therefore, zo-1 gene hypermethylation may be served as a useful epigenetic marker in the differential diagnosis for MDS.

Poliovirus receptor on surface of acute B lymphoid leukemia cells modulated by epigenetic mechanism / 中国实验血液学杂志

The aim of this study was to identify if the expression of poliovirus receptor (PVR) on the surface of acute B lymphoid leukemia (B-ALL) cells RS4:11 and SUP-B15 is modulated by epigenetic mechanism. B-ALL cell lines RS4:11 and SUP-B15 were treated with demethylation agent. Bisulfite PCR was performed to detect percentage change of the methylated CpG islands in the promoter region of PVR. In the meantime, the expression levels of PVR at the translation and transcription levels were detected by flow cytometry and RT-PCR respectively. The B-ALL cell lines were also treated with histone deacetylase (HDAC) inhibitor. The expression level of the gene mRNA and protein was detected too. The results indicated that after treated with 5-azacytidine, the hypermethylated status of PVR promoter region was partly reversed, and the expression of PVR at both mRNA and protein levels was restored in the meanwhile. HDAC inhibitor suberoylanilide hydroxamic acid could also increase the PVR expression. But there was no synergistic function between hypermethylation and HDAC as for repressing PVR transcription in B-ALL cell lines. It is concluded that the expression of PVR in B-ALL cells is modulated by epigenetic mechanisms. Treatment with corresponding inhibitors can partly restore the gene's expression in both mRNA and protein levels.

Methylation status of zo-1 gene promoter in acute leukemia / 中国实验血液学杂志

This study was purposed to investigate the difference of zo-1 gene promoter methylation between healthy individuals and acute leukemia patients. BS-PCR method was used to detect the status of zo-1 gene methylation in healthy individuals, acute leukemia patients and leukemic cell line NB4 cells. The results showed that zo-1 gene was hypomethylated in bone marrow samples from healthy individuals (1.9%). In newly diagnosed AL and relapsed patients, the rate of zo-1 gene methylation was 93.2% and 66.9% respectively, while it was 16.4% in AL patients in complete remission, which was much higher than that in healthy individuals. There was significant difference between them. It is concluded that as compared with healthy individuals, zo-1 gene in acute leukemia patients is hypermethylated and with different degrees in various phases of leukemia. Analysis of zo-1 gene methylation status may be useful to monitor the development of acute leukemia.

Clinical significance of zo-1 and id4 gene abnormal methylation in multiple myeloma / 中国实验血液学杂志

Multiple myeloma (MM) is an incurable heterogeneous disease derived from malignant clonal expansion of plasma cells. The evaluation of prognosis, detection of minimal residual disease (MRD) and treatment of MM are unclear for decades. Recently, Velcade and autotransplantation have been broadly applied to MM patients and achieved better outcomes, but there is yet no effective and universal marker for MRD detection in MM. Both genetic and epigene-tic aberrations play important roles in the pathogenesis and development of cancer. Our preliminary data showed that aberrant promoter methylation of zo-1 and id4 genes was correlated with their gene silencing in several types of hematological malignancies. Therefore, this study was aimed to identify the promoter methylation status of zo-1 and id4 genes in MM and their relationship with the prognosis, MRD and treatment of MM. The methylation status of zo-1 and id4 genes of MM cell lines U266, H929 and IM9 was tested by using MS-PCR; the methylation status of zo-1, id4 gene promoters in bone marrow samples of 20 MM patients and 6 healthy persons was detected by MS-PCR. The results showed that the zo-1, id4 gene in MM cell lines all were methylation positive (complete or partial methylation), the zo-1, id4 gene in samples of 5 healthy persons all were completely unmethylated. The methylation positive rate of zo-1 and id4 genes were 50% and 85% respectively, which were significantly higher than that in normal bone marrow (0%). The coverage rate of zo-1 and id4 gene methylation was 95%. There were no significant differences in the methylation status of both genes among the patients with different heavy chains, different light chains and symptoms. It is concluded that the change of zo-1, id4 genes methylation status occurs in MM patients and has specificity, which may be a new gene marker of MM, methylation analysis of zo-1 and id4 genes may be important for MRD monitoring in patients with MM.

Methylation status of id4 gene promoter in patients with chronic myeloid leukemia / 中国实验血液学杂志

This study was purposed to investigate the methylation status of id4 gene promoter in patients with chronic myeloid leukemia (CML) and explore the relationship between methylation of the id4 gene and progress of CML. The methylation status of id4 gene in 48 chronic myeloid leukemia patients and 10 healthy individuals was detected by using methylation-specific polymerase chain reaction (MS-PCR). The results showed that id4 gene was unmethylated in bone marrow samples from both healthy individuals and CML patients in chronic phase (CP). The rate of id4 gene methylation in both CML patients in accelerated phase (AP) and blast crisis (BC) was 66%, and was higher than those of CML patients in CP phase. There was significant difference between them (p < 0.05). In one CML patient who received a serial observations, the status of id4 was unmethylated in CP, but it was methylated in AP and BC phase. It is concluded that the id4 gene in CML patients is unmethylated in CP, while it is methylated in AP or BC. The detection of id4 gene methylation status may be useful for monitoring disease advance in CML and may be used as a marker of disease progression in CML.

GFP used as a reporter system for optimization of K562 cell transfection / 中国实验血液学杂志

This study was aimed to obtain higher efficiency in gene transfection into K562 cells and to study the role of green fluorescence protein (GFP) as a reporter system. Transfection efficiencies with different methods including electroporation and lipofectamine 2000 transfection, were observed and calculated under fluorescent microscopy by using GFP as a reporter system. The results showed that the transfection efficiency with electroporation (10%) was higher than that with lipofectamine 2000 (1%). In conclusion, the electroporation is a more ideal method for introduction of foreign gene into K562 cells. GFP can be used as a reporter system for optimizing transfection of K562 cells.

Methylation status of ZO-1 gene in patients with myelodysplastic syndrome / 中国实验血液学杂志

The objective of this study was to investigate the methylation status of zonula occluden protein-1 (ZO-1) gene in patients with myelodysplastic syndrome (MDS) and to identify its roles in pathogenesis, development and classification of MDS. 85 patients with MDS and 30 healthy individuals were tested by methylation specific polymerase chain reaction (MS-PCR). The results indicated that no ZO-1 promoter methylation could be detected in healthy controls. methylation of ZO-1 gene promoter of bone marrow was found in 56.5% (48/85) MDS patients. The difference between these two kinds of subjects was statistically significant (p<0.05). The methylation status of ZO-1 gene promoter region in the subtypes of MDS was as following: RA (18/37, 48.6%), RAS (4/6, 67%), RCMD (19/30, 63%), RAEB (7/12, 58%). Every subtype of MDS patients had statistical difference from healthy people (p<0.05), but between the subtypes of MDS there were no significant statistical differences in the methylation status of ZO-1 gene, while the level of ZO-1 promoter methylation in group of RA was lower than that in other groups. It is concluded that the ZO-1 promoter region in bone marrow of MDS patient shows a hypermethylation status, which is specific for MDS. MDS is a common hematologic malignancy with clonal proliferation, it is difficult to differentiate from many other hematologic malignancies in clinical diagnosis. However, the change of ZO-1 gene methylation status is closely related to pathogenesis of MDS, therefore the ZO-1 gene as valuable diagnostic marker has important clinical significance. The ZO-1 gene may be a potential gene related to hematologic malignancies.

Construction and identification of Kir2ds4 RNAi lentiviral vector / 中国实验血液学杂志

This study was aimed to construct a lentiviral vector of RNA interfered (RNAi)-kir2ds4 gene. In accordance with study-confirmed effective sequence of siRNA targeting kir2ds4 gene, the complementary DNA containing both sense and antisense oligonuctide of the targeting sequence was designed, synthesized and inserted into pSicoR-GFP vector containing U6 promoter and GFP sequence. The resulting lentiviral vector containing kir2ds4 shRNA was named as LV-sh kir2ds4, and confirmed by PCR and sequencing. 293T cells were co-transfected with lentiviral vector LV-sh kir2ds4 and packaging system. All virus stocks were produced by Lipofectamine 2000-mediated transfection. The titer of virus was tested according to the expression level of GFP. As a result, PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of kir2ds4 was constructed successfully. The titer of virus tested by expression level of GFP was 6 x 10(8) TU/ml. It is concluded that the lentivirus RNAi vector of kir2ds4 has been successfully constructed.

Methylation pattern of LRP15 gene in leukemia / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the methylation status of LRP15 gene in acute leukemia (AL) patients and its role in the tumorigenesis.</p><p><b>METHODS</b>The methylation of LRP15 promoter and first exon of bone marrow mononuclear cells in 73 patients with AL, 10 with chronic leukemia (CL), 9 with hematological benign diseases, and 20 healthy transplantation donors was analyzed by using methylation specific polymerase chain reaction. The methylation of LRP15 gene promoter and first exon in COS7, K562, and HL60 cell lines was also assayed.</p><p><b>RESULTS</b>No LRP15 gene promoter methylation was detected in COS7 cell line. LRP15 gene promoter was methylated in K562 and HL60 cell lines. No deletion of LRP15 gene was detected in all samples. In nearly all French-American-British leukemia subtypes, we found that frequency of LRP15 methylation in adult patients with AL was 71.23% (52/73). There was no detectable methylation in any of the 20 healthy donors and 8 chronic myeloid leukemia patients. The difference in frequency of LRP15 methylation between AL patients and healthy donors or CL patients (10.00%, 1/10) was significant (P < 0.01). Hypermethylation of LRP15 gene was found in 57.14% (16/28) of newly diagnosed AL patients, 83.33% of relapsed AL patients respectively, which was significantly different (P < 0.05). We also demonstrated LRP15 methylation in 55.56% (5/9) adults with benign hematological diseases.</p><p><b>CONCLUSIONS</b>LRP15 methylation changes are common abnormalities in leukemia. LRP15 is postulated to be a tumor suppressor gene.</p>