Detection of BCR/ABL Fusion Gene and ASS1 Gene Deletion by Using Tricolor Dual-fusion Probe / 中国实验血液学杂志

OBJECTIVE@#To analyze the significance of various abnormal signal patterns appreared in CML and B-ALL patients by using BCR/ABL/ASS1 tricolor dual-fusion probe, and to explore its application value in detecting BCR/ABL fusion gene and ASS1 gene deletion.@*METHODS@#50 newly diagnosed CML patients and 50 newly diagnosed B-ALL patients were detected by fluorescence in situ hybridization (FISH) with BCR/ABL/ASS1 tricolor dual-fusion probe. Meanwhile, karyotype analysis was performed on all the patients using the 24 hours short-term culture and R-banding.@*RESULTS@#Among the 50 CML patients, Ph was found in 49 cases, 5 normal interphase karyotype was observed in 1 case. FISH detection showed that BCR/ABL fusion gene existed in all patients (100%), while the positive signal pathway showed that 1R1G2B2F was observed in 39 cases (78%), 2R1G2B1F in 2 cases (4%) and 1R1G2B1F in 6 cases (12%), simultaneous existence of 1R1G1B1F and 1R1G2B3F in 1 case (2%), 2R1G1B1F in 1 case (2%) 1R1G3B3F in 1 case (2%). FISH detection also showed that the karyotype of 6 case at ASS1 gene deletion (1R1G1B1F) all were simple t (9; 22) translocation, and other abnormalities not were observed. Among 50 cases of B-ALL, Ph was found in 13 cases, the numerical aberration and structural aberration of non t (9; 22) in 16 cases, normal karyotype in 20 cases, absence of mitotic phase in 1 case. FISH detection showed that 16 cases (32%) had BCR/ABL fusion gene including 13 cases (26%) of 1R1G2B2F, 1 case (2%) of stimultaneous exitance of 1R1G2B2F and 1R1G3B3F 1 case (2%) of 2R1G1B1F, 1 case (2%) of 1R1G3B2F. FISH detection also showed that 3 cases had BCR/ABL fusion gene, including 1 case with ASS1 gene deletion (2R1G1B1F), 1 case with classical t (9; 22) translocation (1R1G2B2F) and 1 case with BCR/ABL fusion gene and increase of ASS1 gene copy (1R1G3B3F).@*CONCLUSION@#Tricolor dual-fusion FISH probe for detecting BCR/ABL fusion gene and ASS1 gene deletion is simple, rapid, sensitive and stable. It can detect various forms of molecular fusion and avoid the false positive results due to coincidental overlap of signals generated by D-FISH probe and ES-FISH probe. In addition, this detection method not only can directly observe the presence or absence of ASS1 gene deletion, but also improve the reliability of the positive results of newly diagnosed BCR/ABL fusion gene and accuracy of monitoring results of minimal residual disease for the subsequent visit.

Clinical Efficacy of Allo-HSCT on FLT3-ITD Positive AML Patients / 中国实验血液学杂志

OBJECTIVE@#To detect the expression levels of FAM19A5 in patients with mantle cell lymphoma (MCL), and to determine the relationship between FAM19A5 and the prognosis of MCL patients.@*METHODS@#Twenty-five MCL patients were choosen in the study, cytometric bead assay was used to detected the concentration of FAM19A5 in serum and immunohistochemical analysis were used to detect the expression levels of FAM19A5 in lymph nodes. The relationship of the FAM19A5 expression in serum and tissue were analyzed, the relationship of FAM19A5 and clinical characteristics of MCL patients, treatment and prognosis of MCL patients was analyzed.@*RESULTS@#The average serum concentration of FAM19A5 in MCL patients was 90.55±38.24 (ng/ml), which was significantly higher than that in control (P=0.0461). The proportion of high, medium, and low expression of FAM19A5 in lymph nodes was 32%, 36% and 32%, respectively, which showed significant difference from that in control group (P=0.001). The expression of FAM19A5 in serum and lymph nodes showed significant correlation (r=0.8683，P=0.001). The serum concentration of FAM19A5 showed positive correlation with the proportion of Ki67 (P=0.0222, r=0.4554). The mean survival time without relapse/death of MCL patients with high, middle and low expression of FAM19A5 was 17, 27 and 37.5 months, respectively，which showed significant statistical difference (P=0.0360). ROC curve analysis showed that serum concentration of FAM19A5 could predict the therapeutic effect in MCL patients, the cut-off value was 91.49 ng/ml. The proportion of recurrent/death in AML patients with FAM19A5 ＞91.49 ng/ml was significantly higher than that in patients with FAM19A5＜91.49 ng/ml (P=0.0156).@*CONCLUSION@#The expression level of FAM19A5 is increased in MCL patient, and patients with high expression of FAM19A5 are more likely to relapse or die. FAM19A5 may be a new prognostic marker and potential therapeutic target for MCL.

Construction of monoclonal cells derived from the human glioblastoma U87 cell line and study of their heterogeneity / 医学研究生学报

Objective The human glioblastoma (GBM) U87 cell line is employed as a model for studying the heterogeneity of GBM. This study was to examine the phenotypic profiles and genetic backgrounds of different monoclonal cells derived from the human GBM U87 cell line and explore the molecular mechanisms underlying the phenotypic difference. Methods Using the finite dilution method labeled with 5(6)-carboxyfluorescein diacetate N-hydroxy succinimidyl ester (CFSE), we constructed the monoclonal cell lines CF5 and G11 with typical morphological characteristics derived from the human GBM U87 cell line and identified them by short tandem repeat (STR). We detected the proliferation of the cells by CCK8 assay, EdU incorporation and colony-formation assay, their self-renewal capability by tumor sphere formation assay, their adhesion ability by immunofluorescence and CCK8 adhesion assay, their invasion ability with a 3D culture model, and their sensitivity to chemotherapeutic agents by Annexin V/PI double-staining flow cytometry. We performed transcriptome sequencing and bioinformatics analysis on the genetic profiles and determined the mRNA expressions of the representative differential genes in the enriched pathway by real-time quantitative PCR (qRT-PCR). Results The CF5 and G11 monoclonal cell lines morphologically typical of U87 were successfully constructed, the former small, short and thick, while the latter big, long and thin. Compared with the U87 and G11 cell lines, the CF5 cells showed a significantly higher proliferation ability (P < 0.01), though higher in the U87 than in the G11 cell line, a higher proportion of EdU-positive cells (0.35 ± 0.03 and 0.44 ± 0.03 vs 0.54 ± 0.05, P < 0.01), though higher in the U87 than in the G11 cell line, and a higher tumor-sphere formation ability (P < 0.01), though higher in the U87 than in the G11 cell line. In comparison with the U87 and CF5 cell lines, the G11 cells exhibited remarkably higher abilities of adhesion (P < 0.01) and invasion (P < 0.05), though both higher in the U87 than in the CF5 group. Totally, 159 genes were down-regulated and 303 up-regulated in the CF5 cells compared with those in the U87 and G11 cells, while 281 were down-regulated and 116 up-regulated in the G11 cells compared with those in the CF5 and U87 cells. The CF5 and G11 cells manifested the highest enrichment in the extracellular matrix-associated pathways, which were shown to be closely associated with the invasiveness and drug-resistance of the tumor. Conclusion We successfully constructed human GBM U87-derived monoclonal cell lines CF5 and G11 with different morphological features, phenotypic profiles and genetic backgrounds, which has paved the ground for further studies of the heterogeneity of GBM.

Detection of the Cytogenetic Aberrations in Multiple Myeloma by Using Microrray Comparative Genomic Hybridization / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the molecular cytogenetic abnormalities of multiple myeloma (MM) by using microrray-based comparative genomic hybridization (array-CGH) technology and to investigate its value of application in MM.</p><p><b>METHODS</b>The whole-genoine copy number variants (CNV) of bone marrow samples acquired from 20 cases of newly diagnosed MM patients were detected by genome-wide hybridization and scanning by CytoScan 750K Array (Affymetrix). At the same time, the chromosome abnormalities of bone marrow cells were detected by karyotype analysis and FISH using 9 specific probes: D13S319, RB1, p53, 1q21, IgH, IgH/CCND1, IgH/FGFR3, IgH/MAF, IgH/MAFB.</p><p><b>RESULTS</b>Among the 20 MM patients, the incidence of chromosome abnormalities detected by karyotype analysis, FISH and array-CGH were 15%, 65% and 90%, respectively. The types of CNV detected by array-CGH included the gain (106), loss (156) or UPD (23). There were many different CNVs in every chromosomes except chromosome 5, 9, 18, 21 and Y. Comparison of chromosome abnormalities detected by FISH and array-CGH showed that, the positive ratio of del (13q) was 35% and 40% respectively; the positive ratio of amp (1q) was 40% and 50% respectively; the positive ratio of del (17p) was both 15%. FISH detection showed 8 cases with IgH rearrangement, meansahile the array-CGH detection showed that 4 cases had amp (11q13) (CCND1 gene), 3 cases had amp (16q23) (MAF gene), 1 case had amp (4p16) (FGFR3 gene) and 2 cases had amp (20q12) (MAFB gene). Besides, many other new chromosome abnormalities were found.</p><p><b>CONCLUSION</b>More than half of MM patients have cytogenetic changes, and most of them are complex chromosomal abnormalities. By using array-CGH, more chromosome abnormalities can be detected and more cytogenetic information can be provided for clinician.</p>