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Backgrounds: The biologic characteristics of childhood acute lymphoblastic leukemia (ALL) is different from those of adult ALL. Tumor suppressor genes, p16, p14,and p15gene are inactivated either by promoter methylation, deletion or mutation, however, in leukemia, promoter methylation and deletion are the main mechanisms of inactivation.

Dysruption of cell cycle control genes (p16, p15 and p14) is known to be involved in the tumorigenesis of multiple myeloma (MM). We investigated the inactivation status of p16, p15 and p14 genes using promoter methylation study and fluorescent in situ hybridization (FISH) study in patients with MM and analyzed the association of inactivation of those genes and clinical prognosis.MM Promoter methylation study of p16, p14 and p15 gene was done with newly diagnosed 52 patients with by bisulfite modification and methylation specific PCR. Two sets of primers were used for p16 methylation study and one set of primer was used for p15, p14 methylation study. Deletion of p16, p14 and p15 gene was detected by dual color FISH (Vysis, Downers GroveIL, USA). Overall survival was analyzed by Kaplan Meier Method and Cox’s proportional hazard model. Methylation of p16, p15 and p14 promotor was detected in 36/52 (69.2%), 15/52(28.8%) and 7/52 (13.5%) patients with MM, respectively. Methylation of any of p16, p15 or p14 promotor was observed in 44/52 (84.6%) of MM patients Deletion of p16, p15 and p14 was detected in only one of the patients with MM. Of note, in the cases (15/52, 28.8%) showing that in cases with two positive methylation specific PCR of the p16 promotor with two sets of primers, significant lower overall survival rate (p<0.036) were observed compared with only one positive methylation specific PCR. Heavy methylation of p16 protomor was a independent predictive variable for overall survival [Hazard Ratio, 3.74; 95% CI, 1.44–9.68, P = 0.007]. Other adverse prognostic factors by univariate analysis were old age (p=0.014), β2-microglobuline (p=0.026), high serum creatinine levels (≥2.0 mg/dL, p=0.014). More than 80% of MM patients showed the methylation of any of p16, p15 or p14 promotor, which suggest the potential applicability of hypomethlyating agents to MM. The promoter methylation of p16, p14 or p15 was a major contributor to the disruption of cell cycle regulation in MM, whereas both the deletion and/or the promoter methylation of p16, p14, and p15 contributed to the disruption of cell cycle regulation in ALL. In our study, methylation of two positive methylation specific PCR of the p16 promotor was an independent adverse prognostic factor in MM. We infer that quantitative methylation study for p16 promotor is helpful for the evaluation of prognosis. Figure 1. Overall survival analysis of patients with according to p16 methylation amount (P = 0.036) Figure 1. Overall survival analysis of patients with according to p16 methylation amount (P = 0.036)

In our previous study, we have revealed that cases showing remission after treatment with G-CSF mostly had leukemia with AML1/ETOrearrangement and suggested that this finding might be explained by higher expression of G-CSF receptor (G-CSFR) in AML1/ETO+cells than in AML1/ETO−cells. To confirm the relationship between AML1/ETOfusion gene and G-CSFR expression, we applied small interfering RNAs (siRNAs) for efficient suppression of AML1/ETOgene expression and observed the change of G-CSFR expression. Moreover, we investigated the effect of most commonly used two forms of G-CSF (glycosylated lenograstim and nonglycosylated filgrastim) on AML1/ETOpositive leuekemic cells and other cell lines. Kasumi-1 cells (AML with AML1/ETO gene rearrangement) were transfected with AML1/ETOspecific siRNAs (siAM, siAGF1) and isolated for investigation of G-CSFR gene expression using real time PCR. The proliferation effect of two different forms of G-CSF on was evaluated using Cell-Titer 96® Non-Radioactive Cell Proliferation Assay (Promega Co., Madison, WI, USA). G-CSFR expression was decreased in AML1/ETOgene suppressed cells by both siRNAs. The levels of G-CSFR mRNA were 27% of untreated control with siAGF, 23% with siAM at 16 hours after trasnfection. Both forms of G-CSF significantly stimulated the proliferation of Kasumi-1 cells compared to control cells without G-CSF treatment (40–80% of control increased). The degree of proliferation was similar with different forms of G-CSF (filgrastim and lenograstim) at different concentrations (10, 50 and 100 ng/mL). In K562 cells(chronic myelogenous leukemia), proliferation was increased only at 50ng/ml filgrastim after 72 hours. The CTV-1 cells (AML without AML1/ETO gene rearrangement) showed slightly increased proliferation activities only after 72 hours of lenograstim treatment. In U266 cells (Multiple myeloma) showed no significant response on proliferation with G-CSF. In conclusion, our study revealed that AML1/ETOrearrangement increased the G-CSFR expression and G-CSF had proliferation effect on AML1/ETO-positive cells. Thus, the effect of G-CSF should be considered when we decide the remission or treatment plan, especially for AML1/ETO+AML with high G-CSFR expression.

Dysruption of cell cycle control genes (p16, p15and p14) is known to be involved in the tumorigenesis of multiple myeloma (MM). We investigated the inactivation status of p16, p15and p14genes using promoter methylation study and fluorescent in situ hybridization (FISH) study in patients with MM and analyzed the association of inactivation of those genes and clinical prognosis.MM Promoter methylation study of p16, p14and p15gene was done with newly diagnosed 52 patients with by bisulfite modification and methylation specific PCR. Two sets of primers were used for p16 methylation study and one set of primer was used for p15, p14 methylation study. Deletion of p16, p14and p15gene was detected by dual color FISH (Vysis, Downers GroveIL, USA). Overall survival was analyzed by Kaplan Meier Method and Cox's proportional hazard model. Methylation of p16, p15and p14promotor was detected in 36/52 (69.2%), 15/52(28.8%) and 7/52 (13.5%) patients with MM, respectively. Methylation of any of p16, p15or p14promotor was observed in 44/52 (84.6%) of MM patients Deletion of p16, p15and p14was detected in only one of the patients with MM. Of note, in the cases (15/52, 28.8%) showing that in cases with two positive methylation specific PCR of the p16promotor with two sets of primers, significant lower overall survival rate (p<0.036) were observed compared with only one positive methylation specific PCR. Heavy methylation of p16protomor was a independent predictive variable for overall survival [Hazard Ratio, 3.74; 95% CI, 1.44–9.68, P = 0.007]. Other adverse prognostic factors by univariate analysis were old age (p=0.014), β2-microglobuline (p=0.026), high serum creatinine levels (≥2.0 mg/dL, p=0.014). More than 80% of MM patients showed the methylation of any of p16, p15or p14promotor, which suggest the potential applicability of hypomethlyating agents to MM. The promoter methylation of p16, p14or p15was a major contributor to the disruption of cell cycle regulation in MM, whereas both the deletion and/or the promoter methylation of p16, p14,and p15contributed to the disruption of cell cycle regulation in ALL. In our study, methylation of two positive methylation specific PCR of the p16promotor was an independent adverse prognostic factor in MM. We infer that quantitative methylation study for p16promotor is helpful for the evaluation of prognosis.

Recent studies proposed the classification of multiple myeloma (MM) by the pathways involved in the early pathogenesis; nonhyperdiploid variants with a high incidence of IgHtranslocations and hyperdiploid variants associated with no IgHtranslocation. Most studies applied cytogenetic study or flow cytometry to define the ploidy. In this study, we combined the cytogenetic results and fluorescent in situ hybridization results to define the ploidy and investigated IgHtranlocation and 13q deletion in relation to the ploidy level on Korean patients with MM. A total of 135 cases diagnosed as MM between 1997 and 2003 from Seoul National University Hospital and the Asan Medical center were enrolled in this study. Conventional cytogenetic studies and FISH studies with different probes specific for the regions containing the genes or chromosomes (RB1, D13S319, D13S25, IgH/FGFR3, IgH/BCL2, IGHdual color, break apart rearrangement probe, IgH/CCND1, 1q, p53, p16, MLL,CEP 7, 11, 12) were performed. Of 135 patients with MM, 62 (45.9%) patients had hyperdiploid karyotype by cytogenetics and FISH. IgHtranslocations were observed in 37.4% of Korean patients with MM and were more frequent (54.7%) in hyperdiploid variants than in nonhyperdiploid variants (17.4%). Incidence of deletion 13q was 34.7% and also more frequent in hyperdiploid variants (54.2%) than in nonhyperdiploid variants (16.1%). In conclusion, IgHtranslocations and 13q deletions were not associated with nonhyperdiploid MM and appeared more frequently in hyperdiploid variant in Korean patients with MM.