ALBERT

Description: Our previous studies showed that DAZAP2 (deleted in azoospermia associated protein 2) was the most profoundly downregulated gene in bone marrow mononuclear cells from multiple meyloma (MM) patients. The results implicated a role for DAZAP2 as a potential tumor suppressor involved in the origin and development of MM. To further understand the molecular mechanism underlying this deregulation, we analyzed epigenetic changes (i.e., DNA methylation) associated with DAZAP2 in MM, given that aberrant promoter methylation contributes to tumorigenesis through inducing transcriptional suppression and tumor suppressor inactivation. Bioinformatics analysis showed that there are three CpG islands in the DAZAP2 promoter region. The fragments of DAZAP2 promoter region with different length were separately PCR-amplified and inserted into the luciferase reporter, pGL2-Basic vector. The reporter constructs were transfected into COS-7 cells and their luciferase activities measured. The results showed that the fragment containing the third CpG island was the most active among the constructs tested. When this fragment was treated with M. Sss I methylase, its luciferase activity was almost lost. On the other hand, after 5-aza-2′-deoxycytidine treatment, the expression of DAZAP2 was restored in KM3 cells, an MM cell line associated with DAZAP2 downregulation. To extend these observations, bone marrow samples from MM patients and normal controls were collected and genomic DNA, RNA and proteins were extracted from each sample for RT-PCR, MSP (methylation-specific PCR) and Western blot analyses. The results were in general agreement with that reported previously, demonstrating that the protein and mRNA of DAZAP2, while detectable in 100% normal controls, were undetectable in 62.5% MM patients. The MSP data further corroborated with RT-PCR and Western blot analysis (94%), which is characterized by a negative correlation between promoter methylation and downregulation of DAZAP2 in multiple myeloma. These results indicate that promoter hypermethylation may play an important role in downregulation of DAZAP2 gene expression in multiple myeloma.

Description: Abstract 4421 Most malignant features of cancer cells are triggered by activated oncogenes and the loss of tumor suppressors due to mutation or epigenetic inactivation. We previously reported that DAZAP2 was down-regulated in newly diagnosed myeloma (MM) and this may influence MM cell growth and survival. This study was undertaken to evaluate the mechanism of down-regulation DAZAP2 and determine the methylation status and loci of its promoter by using bisulphite genomic-sequencing (BGS) method in KM3 myeloma cell line. Two islands with rich GC box in the promoter of DAZAP2 gene were identified and amplified by using bisulfite-sequencing PCR (BSP). Island 1 spans -472 to -247 including 9 CpG sites (CpGs) and island 2 covers -226 to -13 including 23 CpG sites. The ratio of methylated CpGs in two CpG islands was 46.25%. The above sequences were demethylated and inserted into a pGL2-basic vector. COS-7 cells were transfected with recombinant plasmids and the activity of luciferase was evaluated. The results showed that the CpG island 1 had a weakly transcriptional activity, whereas the CpG island 2 had a strong transcriptional activity (2.38 folds compared with the positive control). The other sequence which covered CpG island 1 and 2 showed a remarkable activity (15.1 folds compared with the positive control). These data indicated that CpG island 2 of DAZAP2 gene may be hypermethylated and suppressed its expression in MM cells. We further correlated DAZAP2 expression with normal plasma cells and malignant myeloma cells, as well as the molecular subtypes which the dataset includes 8 genetic subtypes (MY, PR, LB, MS, HP, CD-1, CD-2, and MF) from 351 newly diagnosed myeloma cases (Zhan, 2006). Interestingly, we extended and confirmed our previous discovery that DAZAP2 was significantly down-regulated in MM cells by using a large uniform dataset (P = 0.004). The low expression of DAZAP2 was especially significant in the subgroups of MY, PR, LB, HP, and CD-1. This study warrants further investigation of DAZAP2 and its potential role in myeloma. Disclosures: No relevant conflicts of interest to declare.

Description: Objective: To study the expression of MUC1 in acute leukemia and its clinical significance. Methods: Expression of MUC1 mRNA was detected in 73 newly diagnosed and relapsed patients of acute leukemia by reverse transcriptase polymerase reaction(RT-PCR). The MUC1 positive PCR products were analyzed by digestion with pst I. With clinical observation, the relationship of expression of MUC1 gene and treatment results were done. Results: The expression of MUC1 was positive in 36 of 73 AL patients (49.3%). The MUC1 expression rate was 39.1% in ALL and 52.2% in AML and they show no significance difference. MUC1 gene was undetectable in 23 healthy subjects. Nineteen of 21 (90.5%) MUC1-negative non-M3 acute leukemia patients got CR while 14 of 22 (63.6%) MUC1-positive non-M3 acute leukemia patients got CR which showed significance difference (p

Description: Abstract 4295 Syngeneic blood and marrow transplantation (BMT) has been applied in the treatment of many malignant or nonmalignant hematologic disorders with no or minimal and transient graft-versus-host disease (GVHD), much less transplant-related mortality (TRM) in contrast to allogeneic BMT, and lower relapse rate compared with autologous BMT. However, limited data in a single BMT center is not sufficient for statistical analysis. To evaluate the clinical outcomes of syngeneic BMT, CSBMT has performed a cooperative survey among BMT centers in mainland, Taiwan, and Hong Kong. From January 1964 to May 2009, 94 transplants from syngeneic donors have been performed in 32 BMT centers. The median age was 20 (1.5 to 51) years old. The diagnosis included AML (29 cases), SAA (26 cases), ALL (17 cases), CML (12 cases), lymphoma (3 cases), MDS (4 cases), neuroblastoma (2 cases), and large granular lymphocytosis (1 case). The main conditioning regimens were CYTBI or BUCY for malignant diseases, none or CY plus ATG for SAA. Bone marrow (BM, 34) or peripheral blood (PB, 49) or both BM and PB (11) as grafts were used. Five patients (SAA 2, AML 3) underwent the same donor's syngeneic BMT twice. One patient with large granular lymphocytosis and 1 case with SAA underwent the same donor's syngeneic BMT thrice. The median follow-up time was 28 months (1 month to 45 years). The median time for white blood cells 〉 1.0 × 109/L, and platelets 〉 20 × 109/L was 11 (2-30) days, 13 (0-122) days, respectively. Two patients (2.1%) had grade I acute GVHD (aGVHD), and 4 cases (4.3%) had grade II aGVHD. However, only one patient's specimen was consulted by pathologist. All aGVHD was controlled easily with low-dose steroid. No chronic GVHD was noted. Three-year disease-free survival (DFS) for the patients with nonmalignant disorders was 88.5%. Among them, the longest survivor was living and well for 45 years after transplant. Three-year DFS for the patients with malignant diseases was 62.9%. The overall survival rates at 3 years were 87.9%, and 69.5% for nonmalignant, and malignant diseases, respectively. 22 of 94 patients died after BMT (nonmalignant 3, malignant 19). The only cause of death for the patients with nonmalignant disorders was rejection. Relapse was the main cause of death in patients with malignancies (17/19). TRM was 2.1%. In conclusion, syngeneic BMT is a safe and effective therapeutic option for both nonmalignant and malignant hematologic disorders. Syngeneic donor, if available, should be the first choice in all cases of AA and hematological malignancies in general. The longest survivor of 45 years post-BMT is presented in this series. The good results and advantage of syngeneic BMT cast light on the potential utility of stored autologous placental-cord blood which is shared by the identical twin through the same placenta. Disclosures: No relevant conflicts of interest to declare.