ALBERT

Description: Inflammatory bowel disease (IBD) is a group of conditions involving chronic relapsing-remitting inflammation of the gastrointestinal tract with an unknown etiology. Although the cause–effect relationship between gut microbiota and IBD has not been clearly established, emerging evidence from experimental models supports the idea that gut microbes play a fundamental role in the pathogenesis of IBD. As microbiome-based therapeutics for IBD, the beneficial effects of probiotics have been found in animal colitis models and IBD patients. In this study, based on the dextran sulfate sodium (DSS)-induced colitis mouse model, we investigated Lactobacillus rhamnosus strain LDTM 7511 originating from Korean infant feces as a putative probiotic strain for IBD. The strain LDTM 7511 not only alleviated the release of inflammatory mediators, but also induced the transition of gut microbiota from dysbiotic conditions, exhibiting the opposite pattern in the abundance of DSS colitis-associated bacterial taxa to the DSS group. Our findings suggest that the strain LDTM 7511 has the potential to be used as a probiotic treatment for IBD patients in comparison to L. rhamnosus GG (ATCC 53103), which has been frequently used for IBD studies.

Description: Interleukin-6 (IL-6) is a key growth factor and thus plays a pivotal role in the pathogenesis of multiple myeloma. IL-6 is absolutely necessary for the cellular signalling cascade via JAK/STAT and RAS/MAPK pathways involved in proliferation and viability. We found interleukin (IL)-6 induced JAK-2 phosphorylation and induced proliferation of multiple myeloma cell line. Also phosphorylation of Stat1 and Stat3 was increased by IL-6 stimulation. Silencing JAK2 expression by small interfering RNA (siRNA) abrogated IL-6 induced phosphorylation of STAT3. In addition, soluble interleukin-6 receptor (sIL-6R) antagonized the function of hIL-6 and efficiently induced the growth arrest and apoptosis of U266 cells in a dose-dependent manner. Increasing levels of sIL-6R suppressed the phosphorylation of JAK-2 and Stat3. This suggests a comprehensive knowledge of the signaling and survival pathways could help find additional molecular targets and lead to the development of novel and more effective treatment strategies in multiple myeloma.

Description: Abstract 3117 Poster Board III-54 Differences in the clinical course of secondary acute myeloid leukemia (s-AML) according to the type of the preceding disorders are not defined. We compared the outcomes of therapy-related AML (t-AML), AML following myelodysplastic syndrome (MDS-AML) and AML following myeloproliferative disorder (MPD-AML). We also intended to find prognostic factors in s-AML overall. Retrospective medical record review at Seoul National University Hospital was performed. We assessed response to induction chemotherapy and overall survival (OS). 95 s-AML patients (median age 56.4 years) were analyzed. 26, 57 and 12 patients had t-AML, MDS-AML and MPD-AML respectively. For patients receiving induction chemotherapy, complete remission (CR) rate was 47.5% and CR rate was different according to the type of the preceding disorders (p=0.004). Compared to t-AML (p=0.027) and MDS-AML (p=0.050), MPD-AML had shorter OS. In s-AML, presence of trisomy 8 had a prognostic impact (p=0.003) along with cytogenetic risk group (p=0.016). In multivariate analysis, the type of the preceding disorders (p=0.026), 5q deletion (p=0.015) and trisomy 8 (p=0.040) were independent prognostic factors. In conclusion, prognosis of s-AML was different according to the type of the preceding disorders with the worst prognosis in MPD-AML. Along with cytogenetic risk grouping, trisomy 8 had a prognostic impact in s-AML. Table 1 Characteristics of s-AML patients according to preceding disorders Characteristics t-AML AML-MDS AML-MPD p-value Age (median) 54.1 57.2 65.5 0.888 Gender 0.262 Male 13 39 8 Female 13 18 4 Latency period 31.2 6.7 92.6 0.008 White blood cell (median) (/μL) 5615 2905 12785 0.002 Hemoglobin (median) (g/dL) 8.0 8.4 8.7 0.543 Platelet (median) (/μL) 51500 32000 100500 0.509 Risk group 0.001 Favorable 6 0 0 Intermediate 12 37 10 Poor 5 12 2 5q deletion 0.731 Present 5 7 2 Absent 18 42 10 -7/7q deletion 0.107 Present 1 11 1 Absent 22 38 11 Trisomy 8 0.777 Present 6 13 2 Absent 18 36 10 Induction chemotherapy 0.619 Yes 23 47 11 No 3 10 1 Complete remission after induction 0.004 Yes 17 19 2 No 6 27 9 Stem cell transplantation 0.137 Yes 9 4 1 No 8 15 2 Figure 1 CR-1 (A) and OS (B) according to preceding disorders CR-1 was not affected by preceding disorders, however OS was significantly the shorter in AML evolving from MPD (black line) compared to AML evolving from MDS (orange line) and t-AML (green line). Figure 1. CR-1 (A) and OS (B) according to preceding disorders CR-1 was not affected by preceding disorders, however OS was significantly the shorter in AML evolving from MPD (black line) compared to AML evolving from MDS (orange line) and t-AML (green line). Disclosures No relevant conflicts of interest to declare.

Description: To characterize molecular mechanisms by which transition from chronic phase to blast crisis in chronic myeloid leukemia (CML) for developing novel therapeutic targets, we analyzed gene-expression profiles of leukemic cells from 12 patients in chronic phase and 9 patients in blast crisis using a 8.7K cDNA chip. We identified 89 genes that were up-regulated as well as 54 genes that were down-regulated in blast crisis of CML. The expression profile included oncogenes, tumor suppression genes, and human genes encoding proteins involved in transcription, signal transduction, metabolism, cell growth, differentiation, apoptosis and immune functions. 18 genes were selected among the up-regulated group for analysis using real-time PCR. Real-time PCR data indicated that the expression of FLT3 (p 〈 0.001), CD32 (p 〈 0.001), ERG (p 〈 0.001), uPAR (p 〈 0.001), MAD (p 〈 0.001) and TAP2 (p 〈 0.001) showed statistically significant difference between chronic phase and blast crisis. For further analysis, we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human FLT3. These small interfering RNA constructs significantly inhibited FLT3 expression at mRNA and protein levels in K562 cells. After treating both the FLT3 knockdown cells and control cells (FLT3 wild type) with STI571, MTT assay and the expression patterns of apoptosis related genes (PARP, caspase-3, Bax) were examined. MTT assay and caspase-3 activity assay showed that silencing of the gene for FLT3 significantly reduced cell viability and ultimately facilitated the induction of apoptotic cell death by STI571. These findings uncovered evidence of a complex signaling network operating down-stream of FLT3 that actively contributes to tumor progression. Thus, RNA interference-directed targeting of FLT3 can be a potential candidate anticancer agent in association with STI571 against chronic myeloid leukemia.

Description: Abstract 4432 Introduction The clinical evidence of responsiveness to immunosuppressive therapy (IST) supports immune pathophysiology for acquired aplastic anemia (AA). Recent studies suggested that auto-reactive cytotoxic T-cells against hematopoietic cells play a key role in the pathogenesis of AA with various cytokines. The purpose of this study is to investigate whether single nucleotide polymorphisms (SNP) of cytokine genes are related to the risk of AA and, furthermore, the response to IST. Methods We analyzed 80 adult patients diagnosed as acquired AA. The 84 unrelated, age and sex matched healthy subjects served as a control group. In 3 cytokine genes (IFN-γ, TNF-α, TGF-β) and one FAS gene, we selected 10 polymorphisms on the basis of allelic frequency and the assumption of clinical relevance from previously reported associations. To assess the association between polymorphisms and risk of AA, we calculated statistical differences in allele, genotype, and haplotype distributions between patients and controls using chi-square test in 3 genetic models (dominant, recessive, and additive). For the association between polymorphisms and response to initial course of IST, we analyzed 44 patients who were treated with IST using a multivariate logistic regression model. Results Among 10 SNPs in 4 genes, one SNP and one haplotype in IFN-γ gene were significantly associated with the development of AA: IFN-γ -2353A/T; the presence of the T allele in dominant model was protective and was related to a 2.3-fold reduction in the risk for AA (P =.012); the presence of the IFN-γ TCA haplotype was related to a two-fold reduced risk for AA (P =.038). The IFN-γ -2353 T allele was shown to be resistant to IST; the presence of T allele and TCA haplotype in IFN-γ gene (dominant model) was related to a 13.2-fold reduced hematologic response at 6 months following initial IST (P =.034). However, 4 SNPs and 2 haplotypes in TNF-α gene did not show any significant associations with the response at 3 and 6 months. In terms of 2 SNPs in TGF-β gene, TGF-β P10L T/C was independently related; the T allele (recessive model) was related to a 4.3-fold reduced response to IST at 3 months (P =.038). Accordingly, the response was related to the TGF-β haplotype; the TC haplotype homozygote (recessive model) was related to a 4.6-fold reduced response to IST at 6 months (P =.036); the presence of CT haplotype (dominant model) was favorable to IST and was related to a 5.7-fold higher response at 3 months than the absence of the CT haplotype (P =.038). Conclusion This exploratory study found that the genetic polymorphism of IFN-γ was susceptible to the development of AA and was related to the hematologic response following initial IST. In case of TGF-β, the polymorphisms were related to the response to IST, though they were not be related to the disease susceptibility. Large studies with a prospective design are needed to better understand the determinants of risk of AA and responsiveness to IST. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1930 Serum levels of IL-6 and TNF-a were significantly increased in patients with active multiple myeloma (MM) when compared with normal controls. Furthermore, MM patients with advanced aggressive disease had significantly higher levels of IL-6 and TNFa than those with MM in plateau phase. However, the detailed mechanisms involving TNFa-mediated cell signaling pathways resulting in increased IL-6 secretion from MM cells are largely unknown. In our study, we found that MEK and AKT phosphorylation was induced by TNFa treatments. TNFa increased MM-derived Interleukin-6 (IL-6) production. TNF-a-stimulated IL-6 production was abolished by inhibition of JAK2 and IKKa, but not of MEK, p38, PI-3K, or by small interfering RNA (siRNA) targeting TNF receptor (TNFR). Also, TNFa increased phosphorylation of STAT-3, -5 including c-Myc and cyclinD1. Three different types of JAK inhibitors decreased the activation of the above pathways. In conclusion, blockage of JAK/STAT mediated NF-kB activation was highly effective in controlling the growth of MM cells and consequently, a new inhibitor of TNFa mediated IL-6 secretion may be a potential new therapeutic agent for MM. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4841 Introduction The PAX5 gene (Paired box gene 5) works as a guardian in B-lymphopoiesis. It activates essential components of B lymphopoiesis and represses transcription of genes for differentiation to other lineage. Thus, abrogation of normal function of PAX5 is believed to contribute the leukemogenesis in B-ALL. In this study, we evaluated the utility of the detection of PAX5 gene alteration in practice in relation to PAX5 immunoexpression, other common cytogenetic changes and survival in childhood and in adult B-ALL. Materials and Methods Interphase FISH using PAX5 split signal probe (DakoCytomation, Glostrup, Denmark), and probes for BCR-ABL1, ETV6-RUNX1 and MLL rearrangements, and CDKN2A deletion was performed in 112 CD19+ B-ALL patients (79 children, 33 adults). G-banding and immunohistochemical staining with anti-PAX5 (BC/24, BIOCARE MEDICAL, CA, USA) antibody was also performed. Result The incidence of translocation, deletion and amplification was 2.5%, 10.0% and 12.7% in children, and 0.0%, 18.2% and 6.1% in adults, respectively. Alteration of PAX5 gene was more common than BCR-ABL1 and MLL rearrangement in children (8.9% and 5.1%, respectively), and MLL rearrangement in adults (3.1%). None of the children with ETV6-RUNX1 rearrangement (22.8%) showed PAX5 gene alteration. Most cases of the deletion of PAX5 were accompanied by CDKN2A deletion. Positive immunoexpression in PAX5 IHC was observed in 74.4% in children and in 76.9% in adults Either the children with positive or negative PAX5 immunoexpression had PAX5 gene abnormalities. The presence of PAX5 gene alteration or negative immunoexpression of PAX5 did not have prognostic impact. Discussion Based on high incidence of structural alteration of PAX5 in dominant leukemic clone, we suggest PAX5 FISH as a useful tool for the diagnosis and the disease monitoring in B-ALL. Frequent CDKN2A deletion, but not ETV6-RUNX1 rearrangement with structural alteration of PAX5 implies the role of PAX5 in determining prognosis of B-ALL. No definite relationship between the structural alteration and immunoexpression of PAX5 might be resulted from allele specific regulation and haploinsufficiency. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1895 Background: Interleukin-6 (IL-6) is a potent pleiotropic cytokine that regulates plasma cell (PC) growth via IL-6 receptor (IL-6R). We hypothesized that upregulation of IL-6R in myeloma cells might confer the growth privilege to myeloma cells over bone marrow (BM) hematopoietic cells. We investigated the frequency and prognostic implication of increased copy number of IL-6R gene by fluorescence in situ hybridization (FISH) in patients with newly diagnosed multiple myeloma (MM). To confirm the role of IL-6R, the change of proliferative potentials after blocking of IL-6R by monoclonal antibody was observed in a myeloma cell line. Methods: 102 patients with newly diagnosed MM were enrolled. FISH study for IL-6R was performed using home-made probe for the locus. The BAC (bacterial artificial chromosome) clone RP11 350G8 (BACPAC Resources, Oakland, CA) was used as the IL-6R gene-positive clone and the FISH probe was labeled by a nick translation reaction. FISH signals were counted among BM plasma cells sorted by immunoglobulin light chain staining (cIg FISH). U266 myeloma cell line was treated with IL-6R monoclonal antibody (MRA, Chugai, Japan) at concentration of 0.06 to 1.25 μM and then cell viability test were performed. Results: The amplification of IL-6R was detected in 53/102 patients (52.0%). The 5-year overall survival (OS) rate of patients with IL-6R gene amplification was 41.3% versus 44.8% for those with a normal IL-6R (P = 0.425). In 44 patients treated with high-dose chemotherapy and autologous stem cell transplantation (ASCT), patients with 3.1 or more mean IL-6R gene copy number per PC showed worse 5-year OS compared to those who had less than 2.1 mean copies of IL-6R gene (44.4% versus 78.0%, P = 0.024). In multivariate analysis, the increase of IL-6R copy numbers (mean copy/PC ≥ 3.1) could be considered as an independent prognostic factor for MM patients underwent ASCT (hazard ratio, 30.9; 95% CI, 1.74–548.6; P = 0.020). Treatment of IL-6R monoclonal antibody on the U266 cell line induced marked reduction of cell viability compared with control, ranged from 7.1% to 98.7% according to the increase in treated antibody level. Conclusions: The gain of IL-6R gene is frequent in myeloma, showing an association with adverse prognosis in MM patients treated with ASCT. In vitro suppression of cell proliferation by IL-6R blocker suggests the potential role of IL-6R in myeloma cell growth and therapeutic implication of IL-6R blocker in the future. Disclosures: No relevant conflicts of interest to declare.