Objective To investigate the pathogenesis of Philadelphia (Ph)-positive acute lymphocytic leukemia (ALL), we established a lymphoblastoid cell line. Methods Bone marrow cells from a patient with Ph-positive ALL were enriched by Ficoll-Hypaque centrifugation and cultured in medium with fetal calf serum. Materials The mononuclear cells of bone marrow aspirate were obtained from an adult man with ALL after he experienced relapse following induction therapy including imatinib mesylate. Results The cell line termed TNA-M was established, carrying a three-way Ph translocation involving two chromosome 9s and one chromosome 22 as a sole karyotypic abnormality. Furthermore, the cells were positive for CD13 and CD33 in addition to CD19, CD22 and CD79a antigens. Conclusion This unique cell line is expected to be a valuable tool for understanding the pathogenesis of Ph-positive ALL.

Deletion of the long arm of chromosome 20 (del(20q)) is observed in 5-10% of patients with myelodysplastic syndromes (MDS). We examined the expression of 28 genes within the common deleted region (CDR) of del(20q), which we previously determined by a CGH array using clinical samples, in 48 MDS patients with (n = 28) or without (n = 20) chromosome 20 abnormalities and control subjects (n = 10). The expression level of 8 of 28 genes was significantly reduced in MDS patients with chromosome 20 abnormalities compared to that of control subjects. In addition, the expression of BCAS4, ADA, and YWHAB genes was significantly reduced in MDS patients without chromosome 20 abnormalities, which suggests that these three genes were commonly involved in the molecular pathogenesis of MDS. To evaluate the clinical significance, we analyzed the impact of the expression level of each gene on overall survival (OS). According to the Cox proportional hazard model, multivariate analysis indicated that reduced BCAS4 expression was associated with inferior OS, but the difference was not significant (HR, 3.77; 95% CI, 0.995-17.17; P = 0.0509). Functional analyses are needed to understand the biological significance of reduced expression of these genes in the pathogenesis of MDS.

Chromosome translocations involving the immunoglobulin heavy chain (IGH) gene locus at chromosome region 14q32 are often observed in B-cell lymphoid neoplasms. Of these, t(14;18)(q32;q21) results in juxtaposition of the IGH gene on chromosome 14 and the BCL2 gene on chromosome 18, leading to the overexpression of BCL2 anti-apoptotic protein, which plays a critical role in the development of follicular lymphoma (FL). However, BCL2 overexpression is not observed in approximately 10 % of FL, and the molecular pathogenesis of BCL2-negative FL has not been elucidated. Here, we identify the SRY-related high-morbidity-group (HMG) box 5 (SOX5) gene on chromosome 12p12 as a novel IGH-involved translocation partner in the case of BCL2-negative follicular lymphoma (FL) with a complex karyotype including t(12;14)(p12.2;q32) by long-distance inverse PCR. As a result of this translocation, the SOX5 gene is juxtaposed to the enhancer of the IGH gene; SOX5 overexpression in neoplastic cells was demonstrated by immunohistochemistry. The results of the present study suggest a role for SOX5 in the molecular pathogenesis of FL.

L265P mutation in the MYD88 gene has recently been reported in Waldenström's macroglobulinemia; however the incidence has been different according to the methods used. To determine the relevance and compare the incidence by different methods, we analyzed the L265P mutation in bone marrow mononuclear cells from lymphoid neoplasms. We first performed cloning and sequencing in 10 patients: 8 Waldenström's macroglobulinemia; 1 non-IgM-secreting lymphoplasmacytic lymphoma; and 1 low grade B-cell lymphoma with monoclonal IgG protein. The L265P mutation was detected in only 1/8 Waldenström's macroglobulinemia patients (2 of 9 clones). To confirm these results, direct sequencing was performed in the 10 patients and an additional 17 Waldenström's macroglobulinemia patients and 1 lymphoplasmacytic lymphoma patient. Nine of 28 patients (7/25 Waldenström's macroglobulinemia, 1/2 lymphoplasmacytic lymphoma, and B-cell lymphoma) harbored the mutation. We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma. Aberrant bands corresponding to the mutation were detected by BSiE1 digestion in 19/25 patients with Waldenström's macroglobulinemia (76%), 1/2 lymphoplasmacytic lymphoma and B-cell lymphoma, but not in the 38 myeloma patients. The L265P mutation was more frequent in patients with Waldenström's macroglobulinemia than in those with myeloma (p=1.3x10(-10)). The mutation was detected by allele-specific polymerase chain reaction in 18/25 Waldenström's macroglobulinemia patients (72%). In the 25 Waldenström's macroglobulinemia patients, the L265P was more frequently detected by BSiE1 digestion than by direct sequencing (p=5.3x10(-4)), and in males (15/16, 94%) than in females (4/9, 44%) (p=1.2x10(-2)). No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32). These results suggest that the L265P mutation is involved in the majority of Waldenström's macroglobulinemia. BSiE1 digestion and allele-specific polymerase chain reaction may detect a small fraction of mutated cells in some cases.

Abstract 3834 Del(20q), one of the common chromosome abnormalities in myeloid neoplasms, is observed in 5 to 10% of patients with myelodysplastic syndromes (MDS). However, the clinical and molecular biological significance of del(20q) has not been fully elucidated. We hypothesized that the genes involved in the molecular pathogenesis of MDS as tumor suppressor genes, are present within a common deleted region (CDR) of del(20q). Therefore, we attempted to determine CDR of del(20q), and, then, analyze the genes located in CDR. Microarray comparative genomic hybridization (CGH) analysis was performed using genomic DNA derived from bone marrow samples of five MDS patients (2 RA and 3 RCMD) with del(20q). In addition, five cases of acute myeloid leukemia with del(20q) were also included in the analysis. The results from microarray-CGH demonstrated that the size of CDR was 11.2 Mb. There are approximately 150 genes in CDR. Next, we performed mutation analysis of the genes located within the CDR using the next generation sequencing method, based on the “two-hit theory”, to identify TSGs which are involved in the molecular pathogenesis of MDS. We applied the SOLiD system to determine the sequences of genes located within CDR in eight patients of MDS with del(20q). At first, we selected and analyzed 32 genes located within or around CDR, which include candidate TSGs, or genes possibly involved in normal and/or malignant hematopoiesis. A total of 16 single nucleotide changes, which have not been reported as single nucleotide polymorphisms (SNPs), were found in the coding regions of the 32 genes. Of the 16 single nucleotide changes, two nonsynonymous nucleotide changes of the STK4 (R117Q) and NCOA3 (P467Q) genes were identified. After confirmation of the results by the Sanger sequencing method, we analyzed mutations for whole coding exons of the NCOA3 and STK4 genes in an additional 30 cases of MDS with del(20q) or monosomy 20. Two additional nonsynonymous single nucleotide changes of the NCOA3 gene, R353L and R1163W, which also have not been reported as SNPs, were found, while no additional mutations were found in the STK4 genes. Therefore, the nonsynonymous and non-SNPs single nucleotide changes in the NCOA3 gene were recurrently found in 3 (7.9%) of 38 cases of MDS with del(20q) or monosomy 20. The NCOA3 gene encodes a nuclear receptor coactivator that form coactivator complex with various molecules including nuclear receptors, and stimulates the transcriptional activities in multiple cellular pathways. Haploinsufficiency of TSGs may be another molecular mechanism in the pathogenesis of MDS. If target genes located within CDR of del(20q) exhibit haploinsufficiency, the loss of one allele as a result of del(20q) may be sufficient, and mutations of the remaining allele are not necessary. Therefore, we then examined the expression of 32 genes by quantitative RT-PCR, in 20 patients with MDS with del(20q) or monosomy 20, and compared it to those in 18 control subjects. In MDS patients with del(20q) or monosomy 20, expression of 8 out of the 32 genes was significantly reduced, compared to control subjects. We also examined expression of the 32 genes in 20 patients with MDS without chromosome 20 abnormalities. Interestingly, among the 32 genes, expression of 5 genes was significantly reduced in MDS patients without chromosome 20 abnormalities compared to control subjects. Expression of three genes (BCAS4, ADA, and ZNF335) was reduced in both MDS patients with del(20q) or monosomy 20 and those without chromosome 20 abnormalities, suggesting the significance of a decreased expression of those three genesin molecular pathogenesis of MDS. Molecular mechanisms other than chromosome deletion, including methylation of promoter regions may result in decreased expression of those genes. In the present study, we determined CDR of del(20q) and analyzed the genes located within or around CDR. Our present results showed recurrent mutations in the NCOA3 gene in MDS patients with del(20q), and decreased expression of genes within CDR in not only MDS patients with del(20q) or monosomy 20, but also in those without chromosome 20 abnormalities. The clinical and molecular biological significance of mutations of the NCOA3 gene and that of the decreased expression of the genes within CDR is also unclear. Further study is on going. Disclosures: No relevant conflicts of interest to declare.

The chromosomal abnormality del(20q) is mostly found in various myeloid disorders, including myelodysplastic syndromes, myeloproliferative neoplasms, and acute myeloid leukemia. Here, microarray comparative genomic hybridization (aCGH) analyses of 14 patients cytogenetically confirmed to carry the del(20q) aberration in their bone marrow demonstrated that all deletions were interstitial and both the proximal and distal breakpoints varied among individuals. The centromeric breakpoints were located in the 20q11.21-12 region, and the telomeric breakpoints, in the 20q13.13-13.33 region. The extent of the deletion ranged from 11.2 to 27.3 Mb, and the commonly deleted region (CDR) was estimated to be 7.2 Mb in size. Two commonly retained regions were present, the proximal region adjacent to the centromere (20q11.1-11.21) and a subtelomeric one (20q13.33). The CDR of our study was more distal than reported previously. Furthermore, in three patients fluorescence in situ hybridization (FISH) demonstrated that del(20q) cells were detected at a higher frequency in the karyotype analyses than by interphase FISH and aCGH analyses. As the size and breakpoints of del(20q) have been reported to vary among patients, the presence of one or more tumor suppressor genes in the CDR has been suggested. Our study will contribute to the identification of candidate tumor suppressor genes on 20q.

Retinoblastoma protein-interacting zinc finger, RIZ1, is a tumor suppressor gene that is inactivated in various solid tumors. However, the role of the RIZ1 gene has not been well examined in adult acute lymphoblastic leukemia (ALL). We analyzed the expression and promoter methylation status of the RIZ1 gene in patients with newly diagnosed ALL by quantitative real-time reverse transcription polymerase chain reaction (PCR) and methylation-specific PCR, respectively. RIZ1 expression in 67 cases of ALL (mean 1.043) was decreased compared with that in normal bone marrow (mean 1.471) (p = 0.030). Methylation was detected in 11 of 71 patients (15.5%) but not in healthy controls. Methylation was associated with decreased RIZ1 expression in many ALL cases examined, but this was not statistically significant. In T-ALL, RIZ1 methylation was more frequent (63.6%) than in B-ALL (6.7%) (p < 0.0001) and the decrease of RIZ1 expression was more significant than in B-ALL (p = 0.045). 5-Aza-2'-deoxycytidine treatment of MOLT-4 cells with RIZ1 methylation induced demethylation of RIZ1 and restoration of expression. Forced RIZ1 expression in T-ALL cell lines suppressed cell growth accompanied by G2/M arrest and apoptosis. No mutations were found by PCR-single strand conformation polymorphism analysis in hotspots of the gene. These results suggest that RIZ1 is inactivated in adult ALL, and this inactivation is associated with methylation in T-ALL.

We performed methylation specific PCR analysis on the RIZ1 promoter in MDS and AML. Methylation was detected in 17 of 34 MDS (50%) and 22 of 72 AML (31%) (p=0.053). Methylation was detected in eleven of 17 secondary AML from MDS (65%), and eleven of 55 de novo AML (20%) (p=0.0005). Bisulfite sequence revealed methylation at many CpG sites in the promoter. Decreased RIZ1 expression was accompanied by methylation in six of nine samples examined, while it was also observed in seven of 13 without methylation. Treatment of AML cells, that have RIZ1 methylation, with 5-Aza-dC, induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene is inactivated in MDS and AML in part by methylation, whereas another mechanism should be involved in others.

Background: The relation with SNF5 mutation and chromosome 22 abnormalities is not clear in hematological neoplasms. Methods: To elucidate the relevance of the SNF5 gene on 22q11.2, karyotypes were reviewed in 283 hematological neoplasms. Loss of heterozygosity (LOH) on 22q was analyzed in 21 plasma cell myelomas without chromosome 22 abnormalities. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) on the SNF5 gene was analyzed in 8 hematological neoplasms with 22q- or -22, and 8 chronic myelogenous leukemias (CMLs) in blast crisis. Fluorescence in situ hybridization (FISH) was performed in 1 myelodysplastic syndrome (MDS) case with -22,del(22)(q11.2 q13). Results: 22q- or -22 was observed in 36 patients. LOH on 22q was detected in 1 of the 21 myelomas. Mobility shifts were found by PCR-SSCP analysis in 2 CMLs, whereas sequence analysis showed polymorphisms. FISH analysis revealed the SNF5 gene was not deleted in the MDS case. Conclusion: These results suggest that alterations of the SNF5 gene are rare in hematological neoplasms with chromosome 22 abnormalities. Haploinsufficiency may contribute to the development of these neoplasms.

Abstract 5112 Inactivation of a tumor suppressor gene is often caused by a mutation, small deletion of one allele accompanied by loss of the second allele. Methylation in a promoter CpG of several tumor suppressor genes has recently been reported and has been associated with loss or decreased expression in many tumors. We previously reported frequent loss of heterozygosity on the short arm of chromosome 1 (1p) in the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML). The retinoblastoma protein-interacting zinc finger gene RIZ maps to 1p36. Mouse gene knockout models show that RIZ1 inactivation can cause tumor susceptibility. Inactivation of the RIZ1 gene by promoter hypermethylation has been reported in breast, liver, and gastric carcinoma. Previous study showed altered expression of the RIZ1 gene in human leukemia. However, methylation status of the RIZ1 gene has not been well studied in hematological neoplasms. To determine the relevance of the RIZ1 methylation, we performed methylation specific-polymerase chain reaction (PCR) analysis on the RIZ1 gene in 34 patients with MDS and 17 with AML evolved from MDS (secondary AML) as well as 55 patients with de novo AML. The 34 MDS samples consisted of 13 refractory anemia (RA), 1 RA with ringed sideroblasts (RARS), 10 RA with excess of blasts (RAEB), 6 RAEB in transformation (RAEB-t), and 4 chronic myelomonocytic leukemia. The 55 de novo AML consisted of 1 M0, 12 M1, 17 M2, 7 M3, 8 M4, 7 M5, 1 M6, and 2 M7. Written informed consent was obtained from the patients. Methylation of the RIZ1 gene was detected in 17 of the 34 MDS (50%) and 22 of 72 de novo and secondary AML (31%) (p=0.053). Methylation was detected in 7 of 14 low risk MDS (50%) and 10 of 20 high risk MDS (50%). Patients with MDS were classified using the IPSS score. Frequency of methylation was not statistically different among IPSS subgroups (p=0.419). No statistical differences were observed between methylation and overall survival (3 years) or progression to AML. In AML patients, methylation was more frequent in secondary AML (11/17, 65%) than in de novo AML (11/55, 20%) (p=0.0005). To define the methylation status of the CpG in the RIZ1 promoter region, we performed bisulfite sequence in several samples with methylation. Bisulfite sequence analysis revealed methylation at many CpG sites in the promoter region. Expression of the RIZ1 gene was examined by quantitative real time reverse transcriptase-PCR analysis in 22 samples of MDS and AML. RIZ1 expression (mean) was not statistically different in secondary AML and de novo AML (2.026 vs. 1.900, p=0.815). RIZ1 expression (mean) was not statistically different in methylation-positive group and methylation-negative group (1.996 vs. 1.810, p=0.728). In comparison with expression of normal bone marrow cells, decreased RIZ1 expression was accompanied by methylation in 6 of 9 samples examined, while it was also observed in 7 of 13 without methylation. HL-60 myeloid leukemia cells with RIZ1 methylation were cultivated for 3 days in the presence of various concentrations of 5-Aza-dC. Treatment of the leukemia cells with 5 Aza-dC induced growth suppression with RIZ1 restoration. Our results suggest that the RIZ1 gene was inactivated in MDS and AML in part by methylation, whereas another mechanism of inactivation should be involved in others. Disclosures: No relevant conflicts of interest to declare.

発芽玄米の糖代謝・脂質代謝に対する影響を知ることを目的に,糖尿病患者に試験食 (発芽玄米: 白米を1:1に調整) を3か月間摂食させて,その前後で糖・脂質のパラメーターを比較検討した。3か月間の試験食摂取によって,グリコヘモグロビンは,摂取前の6.40±0.23%から6.23±0.19%へと有意な低下が認められた。空腹時血糖値に有意な変化はなかったが,インスリン値とHOMA-IRは低下傾向を示した。T-CHO値,TG値は試験食の摂取によって変化はみられなかったが,LDL-c値は低下傾向,HDL-c値は増加傾向を示し,LDL/HDL比は摂取前の2.03±0.13から,摂取3か月後には1.83±0.12へと有意に低下した。試験食摂取量を多い群と少ない群に分けて検討すると,試験食の摂取量が多い群で,LDL-c値は有意に低下,HDL-c値は有意に増加した。今回,糖尿病患者において,3か月間の発芽玄米摂取によって糖代謝と脂質代謝がともに改善する結果が得られた。これらは,糖尿病患者の食事療法として発芽玄米が有効であることを示すものであるとともに,高コレステロール血症の治療にも有効である可能性を示すものと考えられた。

Nucleophosmin (NPM1) gene exon 12 mutations are frequently present in patients with acute myeloid leukemia (AML) with normal karyotype. The NPM1 gene is located on chromosome 5q35, which is often affected in myeloid malignancies including myelodysplastic syndrome (MDS). This suggests that the NPM1 gene is a one of the target genes affected by chromosome 5 abnormalities and play a role in the development of MDS. It has not been clarified whether MPM1 mutations are present in patients with MDS and AML with chromosome 5 abnormalities. Therefore, we carried out a mutational analysis on the NPM1 gene exon 12. NPM1 mutations were not detected in the 28 patients with MDS and AML with chromosome 5 abnormalities.