The Study Of Improved Technique Of Preparation Of Chromosome In Multiple Myeloma

Background and ObjectivesMultiple myeloma (MM) is a haematological disorder of clonal malignant plasma cells that accounts for about 10 percent of hematopoietic system tumors and approximately 2% of all human cancers.Despite the progress in therapy, the complete remission of the disease is still low, and the median survival is only 2-3 years. Pati-ents who achieved complete remission will develop drug resistance and recurrence inevitablely especially those with cytogenetic abnormalities (about 1/3) of the patients. Chromosome karyotypic analysis is important for diagnosis, classification, predicting prognosis and guiding treatment in hematologic malignancies. Cytogenetic studies performed in myeloma patients have been difficult and disappointing. The reasons for this are most likely manifold, and include a low mitotic index of the malignant cells and the presence of cytogenetically cryptic abnormalities as well as of complex karyotypes with poor chromosome morphology. Therefore, improving the culture enviroment is important in order to increase the yield of metaphases by promoting proliferation and inhibiting apoptosis of malignant plasma cell.Many cytokines are involved in the growth and survival of these tumour cells,such as interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1(IGF-1), HGF and so on.They affect these tumour cells via autocrine and/or paracrine regulation mechanisms. IL-6 has been recognized in the past few years as the key cytokines for human myeloma cells which can promoting proliferation and differentiation of the MM cells.GM-CSF increases IL-6 responsiveness of myeloma cells in vitro. In multiple myeloma, angiogenesis is related with disease progression and poor prognosis. VEGF can promote tumor angiogenesis, invasion and metastasis.VEGF can leads to a marked production of IL-6. IGF-1 is closely related with the occurrence of MM, which can induce the activation of membrane lipid rafts on the IGF-1Rwhich connect withβ1 integrin to mediated the migration and adhesion between MM cell and Fn. IL-6, VEGF can activate the JAK2/STAT3 to regulate survival,IL-6, IGF-1, and VEGF promotes MM cell proliferation through the Ras/ Raf/MEK/ERK pathway, IL-6, IGF-land VEGF lead to activate PI3-K/Akt anti-apoptotic pathway to inhibit apoptosis and induce drug resistance.Adding IL-6, GM-CSF, IGF-1 and VEGF to the culture enviornment to promote the proliferation of myeloma cells and inhibit apoptosis so that we can increase the yield of metaphases and thus of cytogenetic abnormalities in myeloma patients.Cell kinetics is different between Leukemia cells and normal cells in vivo and vitro. In vivo,the proliferation rate of leukemic cells are lower than normal cells.After short-term cultured, the proliferation rate of leukemic cells increases. Therefore, extending the incubation time can promote the proliferation of myeloma cells and inhibits their apoptosis so that we can increase the yield of metaphases.Some improvements were made at home and abroad in cytogenetic problems of multiple myeloma by the addition of GM-CSF (30 ng/mL), IL-6 (2000 IU/mL)+ GM-CSF (30 ng/mL), IL-6 (2000 IU/mL)+GM-CSF (30 ng/mL)+IL-3 (75 ng /mL) and extending incubation time. The yield of metaphases and the rate of abnorm karyotype increased. In this experiment, on the basis of previous studies, we add IL-6 (10 ng/mL)+GM-CSF (30 ng/mL) and IL-6 (10ng/ml)+VEGF (lOng/ ml)+IGF-1 (lOn g/ml) to create a new culture system to increase the yield of metaphases and the rate of abnorm karyotype.MethodsPart 1 The influences of cytokines on RPMI8226 and ARH-77RPMI8226 and ARH-77 were cultured for 6 days without any cytokines(A group), and 6 days in the presence of IL-6 (10 ng/mL)+GM-CSF (30 ng/mL) (B group), and 6 days in the presence of IL-6 (10 ng/mL)+VEGF (10 ng/mL)+ IGF-1 (10 ng/mL) (Cgroup),then counted on the third day and sixth day by trypan blue staining. Colony formation of RPMI8226 and ARH-77 clonyn in vitro treated with cytokines are tested by methyl cellulose semi-solid culture method. Mitotic indexs were tested and chromosome karyotype were analysed after cytokines acting on RPMI8226 and ARH-77 on sixth days.PartⅡThe influence on karyotype analysis of normal persons by different culture systems and different culture timeMononuclear cells were isolated from bone marrow aspirate of normal persons,and then cultured for 1 days and 6 days without any cytokines, and 6 days in the presence of IL-6 (10 ng/mL)+GM-CSF (30 ng/mL), and 6 days in the presence of IL-6 (10 ng/mL)+VEGF (10 ng/mL)+IGF-1 (10 ng/mL) before G-banding analysis. Leica CW4000 Karyo mining software is used to analyze chromosome karyotype. Whether the chromosome changes after adding cytokines and prolong incubation time would be dectected.Part III The iInfluence on karyotype analysis of multiple myeloma patients by different culture systems and different culture timeMononuclear cells were isolated from bone marrow aspirate of MM patients; and then cultured for 1 days and 6 days without any cytokines, and 6 days in the presence of IL-6 (10 ng/mL)+GM-CSF (30 ng/mL), and 6 days in the presence of IL-6 (10 ng/mL)+VEGF (10 ng/mL)+IGF-1 (10 ng/mL) before G-banding analysis. Leica CW4000 Karyo mining software is used to analyze chromosome karyotype. The rate of abnormal karyotype after adding cytokines and prolong incubation time would be dectected. whether the stage of the disease and the proportion of plasma cells affect the the rate of abnorm karotype would be dectected.Statistical analysisThe analysis was performed using SPSS 13.0 software package.The data was represented as the mean±standard deviation. Comparisons of means among groups were performed using one-way analysis of variance(ANOVA) andχ2 test. If variance were homogenous among groups, the bonferroni method was used for multiple comparisons, otherwise Dunnett'S T3 method was used, P<0.05 were considered to be statistically significant.ResultsPart I The influences of cytokines on RPMI8226 and ARH-771.1 cytokines promote the proliferation of myeloma cellsThere were different responses for different cells treated by cytokines. Cytokines promoted the proliferation of ARH-77,(the third day F= 57.488, P 0.001;the sixth day, F=151.817, P<0.001,there is statistical significance). The third day, inter-group comparision,PAB=0.001; PAC<0.001, there is statistically significance; PBC= 0.334> 0.05, there is not statistically significance. The sixth day, inter-group comparision,PAB< 0.001, statistically significant; PAC<0.001, statistically significant; PBC= 0.539>0.05, there is not statistically significance.. Cytokines did not play a role in RPMI8226, (the third day F=2.616, P=0.152> 0.05, the sixth day, F=4.659, P=0.060> 0.05), there is not statistically significance.1.2 Colony formation of RPMI8226 and ARH-77 cells treated with cytokinesIn vitro colony assays indicated, cytokines played a role in colony formation of RPMI8226 and ARH-77 cells.For RPMI8226,the number of clones were 91.00±4.58,205.67±6.03,175.67±6.03 for ABCgroups,respectively. Compared between group adding cytokines and the group without adding cytokines, F=339.77, P<0.001, The numbers of Cloning was statistically significant. Pairwise comparisons among the three groups, P<0.05, statistically significant. For ARH-77, the number of clones were 17.00±3.08,26.60±2.70,24.00±3.81 for ABCgroups,respectively.Compared between group adding cytokines and the group without adding cytokines, F=31.394,P=0.001, The numbers of Cloning is statistically significant. Pairwise comparisons among the three groups, PAB=0.004<0.05, PAC=0.001<0.05, The numbers of Cloning was statistically significant. PBC=0.231>0.05, The numbers of Cloning was not statistically significant. Cytokines changed not only the number of clones in vitro, and the size of the clone.1.3 Mitotic indexs of RPMI8226 and ARH-77 cells treated with cytokinesCytokine played a role on the mitotic indexs of RPMI8226 and ARH-77 cells. For RPMI8226, the mitotic indexs of ABCgroups were 1.96±0.40%,3.16±0.42%, 2.84±0.37% respectively. Compared between group adding cytokines and the group without adding cytokines, F=12.350,P=0.001<0.05,the mitotic indexs were statistically significant. Pairwise comparisons among the three groups, PAB=0.001<0.05, PAC=0.013<0.05,the mitotic indexs were statistically significant. PBC=0.675>0.05,the mitotic indexs were not statistically significant. For ARH-77, the mitotic indexs of ABCgroups were 1.70±0.31%,2.67±0.27%,2.40±0.31% respectively. Compared between group adding cytokines and the group without adding cytokines, F=11.815,P=0.001,The mitotic indexs were statistically significant. Pairwise comparisons among the three groups, PAB=0.002<0.05, PAC=0.015<0.05, mitotic indexs were statistically significant. PBC=0.682>0.05, the mitotic indexs were not statistically significant.1.4 karyotype of RPMI8226 and ARH-77 treated with cytokinesAnalysed by cytogenetics,RPMI8226 was hyperdiploid, and the number of chromosome was 62-64, and complex structural changes appeared, such as 1p-,6q-,15q-,+mar and so on.The number of ARH-77 is 42-46, and complex changes appeared, such as-1,+2,-3,+4,6q-,-9,13q-,14q+,+mar and so on.There was no difference between adding cytokines and no cytokines.PartⅡThe influence on karyotype analysis of normal persons by different culture systems and different culture time 7 karyotypes of normal persons were analysed, in which 5 were males, the karyotype were 46, XY and 2 were females,the karyotype were 46, XX.There were no changes in different culture systems.Adding cytokines and extending cultured time will not change the karyotype.Part III Karyotype analysis of patients with multiple myeloma in different culture systemsThe experiment was failure in 3 cases because there is no metaphases,and another 2 cases were failure in 1days culture. The karyotype abnormalities were found from 3 cases of 21 available patients in 1 day culture,4cases in 6 days culture,7cases in the presence of IL-6 (10 ng/mL)+GM-CSF (30 ng/mL),and 5 cases in the presence of IL-6 (10 ng/mL)+VEGF (10 ng/mL)+IGF-1 (10 ng/mL). Most of them had complex karyotypes. The rate of abnormal karyotype of Phase III MM patients and patients with high plasma cell percentage was high.Conclusion1 Cytokines promote proliferation of ARH-77cells,but them is no role on RPMI8226.2 Cytokines promote colony formation of the ARH-77, RPMI8226, and promote their division.3 Adding cytokines and extend ing cultured time does not cause chromosomal changes.4 Extended culture in the presence of cytokines and extended culture without cytokines could improve the efficiency of cytogenetic analysis of MM.