ALBERT

Description: Abstract 3468 Poster Board III-356 Background Transcription factor RUNX1 is essential for normal hematopoiesis. RUNX1 mutations, mainly at Runt homology domain (RHD), have been described in patients with AML-M0 and were rarely found in non-M0 AML. Aim We aimed to analyze the RUNX1 mutations in AML patients with partial tandem duplication of MLL (MLL-PTD) and to investigate the biological functions of the mutants detected. Patients and methods Bone marrow samples from 93 patients with MLL-PTD were analyzed for RUNX1 mutations. MLL-PTD was screened by Southern-blot analysis followed by RT-PCR or detected by real-time quantitative PCR. Mutational analysis of RUNX1 gene was performed by sequencing of all RT-PCR products amplified from exon 3 through exon 8. Each mutation was reconfirmed with alternative primers. The wild-type, all mutants of RUNX1 (except those truncated at RHD or silent) and pcDNA3.1 were transiently transfected into Cos-7 cells. Immunoblot analysis after immunoprecipitation with anti-FLAG RUNX1 antibody and electrophoretic mobility shift assay were used to determine the interaction with CBFβ and DNA-binding ability of the RUNX1 mutants. Dual luciferase assay system was used to analyze the transactivation potential of RUNX1 mutants in K562 cells. Results RUNX1 mutations were detected in 23 patients (24.7%) at diagnosis, with 3 patients carrying double mutations; 14 mutations were located in RHD (exons 3-5) and 12 at C-terminal region (exons 6-8). In addition, one patient acquired a C-terminal mutation at relapse. The patterns of 27 mutations consisted of 6 missense mutations, 3 nonsense mutations, 17 frame-shift mutations, and 1 silent mutation; all were heterozygous. Of the 3 patients with double mutations, clonal analysis showed that one patient had combined missense and frame-shift mutations on the same allele, the other patient had two missense mutations on different alleles, and another patient had a missense mutation and a silent mutation on the same allele. Functional analyses revealed significant difference among mutants. Two missense RUNX1 mutants at RHD (G108D and R174L) and all of the frame-shift mutants in the transactivation domain (TAD) ( S287fsX571, S295fsX571, L300fsX570, V333fsX574, I339fsX569 and P355fsX572) exhibited lack of DNA-binding ability and inhibited transactivation activity of wild-type RUNX1 in a dominant-negative effect. All frame-shift mutants distal to the TAD generated termination codons within the 3'-untranslated region (H377fsX565, Q388fsX572, L414fsX569, L414fsX567 and V425fsX576), they all retained the normal transactivation activity as the wild-type. R177X and R205W retained the ability of heterodimerization with CBFβ but they had markedly reduced DNA-binding and no transactivation potential without inhibitory effect on wild-type RUNX1. L183fsX185 could bind DNA but lacked transactivation activity. S114P and Q370R had normal transactivation activity. Conclusions Our results showed that patients with de novo AML with MLL-PTD had a high frequency of frame-shift mutations at C-terminal region of RUNX1; those within TAD had dominant-negative effects whereas those distal to TAD retained the normal transactivation potential. Supported by grants NHRI-EX96-9434SI, NSC97-2314-B-182 -011-MY3 and MMH-E-96009. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 3064 Poster Board III-1 Both ETV6-RUNX1 (TEL-AML1)fusion and hyperdiploidy (〉50 chromosomes) of lymphoblasts are favorable outcome predictors in childhood acute lymphoblastic leukemia (ALL). In 433 children with ALL diagnosed at our hospitals between 1997 and 2007 in Taiwan, the frequency of ETV6-RUNX1 fusion was 15.8%, and the frequency of hyperdiploidy (〉50 chromosomes) was 14.1%, both were lower than those of the West. While ETV6-RUNX1 fusion had borderline favorable impact on outcome (p=0.053-0.061), hyperdiploidy showed significant favorable impact on event-free survival (91.1% vs 76.6 %, p= 0.016) in our patients. A meta-analysis from literature enrolled reports in which the case numbers and frequency of ETV6-RUNX1 fusion or hyperdiploidy in childhood ALL were described. It revealed that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East (Japan, Korea, Hong Kong, Chinese in Singapore, and Taiwan) was 14.2% (127/893, range 10-17%), significantly lower than 21.8% (152/697, range 19-27%) in the West (USA, Germany, Italy, France and Chile) (p 〈 0.0001). The frequency of hyperdiploidy in Japan and Taiwan was 15.2% (140/921, range 13-20%), significantly lower than 31.6% in the West (977/3,158, range 19-34%) (USA, UK and Germany) (p 〈 0.0001). So far as we know, there were several articles, including ours, addressing that the frequency of ETV6-RUNX1 fusion in childhood ALL was lower in a Far East country. This is the first meta-analysis to demonstrate that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East was lower than that in the West. There was no report on that the frequency of hyperdiploidy in Far East was lower than that in the West. This is also the first meta-analysis to demonstrate that the frequency of hyperdiploidy in childhood ALL in Far East is significantly lower than that in the West. The nature of these differences, probably due to racial, needs further study. In Far East, with both a lower frequency of ETV6-RUNX1 fusion, and a lower frequency of hyperdiploidy, it warrants renewed effort to cure a higher proportion of children with ALL. Disclosures No relevant conflicts of interest to declare.

Description: Snake envenomation is a serious public health issue in many tropical and subtropical countries. Accurate diagnosis and immediate antivenom treatment are critical for effective management. However, the venom concentration in the victims’ plasma is usually low, representing one of the bottlenecks in developing clinically applicable assays for venom detection and snakebite diagnosis. In this study, we attempted to develop a simple method for rapid enrichment of venom proteins from human plasma to facilitate detection. Our experiments showed that several major protein components of both Naja atra (N. atra) and Bungarus multicinctus (B. multicinctus) venoms have higher isoelectric point (pI) values relative to high-abundance human plasma proteins and could be separated via strong cation exchange–high-performance liquid chromatography (SCX-HPLC). Based on this principle, we developed an SCX tip column-based protocol for rapid enrichment of N. atra and B. multicinctus venom proteins from human plasma. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the identification of cytotoxin and beta-bungarotoxin as the major proteins enriched by the SCX tip column in each venom sample. The entire process of venom enrichment could be completed within 10–15 min. Combination of this method with our previously developed lateral flow strip assays (rapid test) significantly enhanced the sensitivity of the rapid test, mainly via depletion of the plasma protein background, as well as increase in venom protein concentration. Notably, the SCX tip column-based enrichment method has the potential to efficiently enrich other Elapidae snake venoms containing proteins with higher pI values, thereby facilitating venom detection with other assays. This simple and rapid sample preparation method should aid in improving the clinical utility of diagnostic assays for snakebite.