ALBERT

Description: Abstract 2673 Introduction: Follicular Lymphoma International Prognostic Index 2 (FLIPI-2) is a widely accepted tool for risk assessment of follicular lymphoma (FL), which is based on age, hemoglobin level, presence of bone marrow (BM) invasion, tumor size, and b2-microgloblin levels. Although it is easy to evaluate in clinical practice, it is a combination of tumor burden and patient physical condition, and a simple and powerful biomarker reflecting the tumor burden and its character is still not established. LR11 (also called SorLA or SORL1) was identified and characterized as a regulator of uPAR function through complex formation with uPAR. We have identified that serum soluble LR11 (sLR11) levels are significantly elevated in patients with acute leukemia and B cell lymphomas, and are associated with tumor burden and BM invasion (Sakai et al 2012). We have also found that high sLR11 levels had a significant negative prognostic impact on progression-free survival (PFS) in FL. Therefore, we have retrospectively evaluated the clinical characteristics of sLR11 and its prognostic impact on FL, in a larger patient cohort. Patients and Methods: Sixty-one patients with FL treated at Chiba University Hospital and affiliated hospitals from 2002 to 2012 were evaluated. The majority of patients were treated by the R-CHOP regimen (rituximab 375 mg/m2 on day 1; cyclophosphamide, 750 mg/m2 on day 1; adriamycin, 50 mg/m2 on day 1; vincristine, 1.4 mg/m2 on day 1; and prednisolone, 100 mg/body on day 1–5). Serum sLR11 levels were measured by ELISA method. Patient laboratory data and treatment outcome were obtained retrospectively. Results: Serum sLR11 levels of patients with lymphoma were significantly increased (mean ± SD: 19.4 ± 17.1 ng/ml) compared with those of normal control subjects (8.8 ± 1.79 ng/ml, P

Description: Abstract 2717 Introduction: LR11 (also called SorLA or SORL1) is a type I membrane protein, from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding. LR11 plays a key role in the migration of undifferentiated vascular smooth muscle cells, and circulating sLR11 is known to be a biomarker of carotid intima-media thickness. Along with the fact that circulating sLR11 levels represent the accumulation of vascular immature cells, human CD34+CD38− immature hematopoietic precursors have been reported to express high levels of LR11 mRNA. We have recently found that LR11 is specifically and highly expressed on cell surface of acute leukemia cells in addition to normal leukocytes (unpublished data). These facts prompted us to evaluate the serum sLR11 level in patients with acute leukemia and other hematological malignancies to validate sLR11 as a novel circulating marker for treatment outcome and prognosis. Patients and Methods: Serum sLR11 levels were measured by ELISA method in 139 patients with acute leukemia and other hematological malignancies treated at a single institution from 1999 to 2010. Patients' laboratory data and treatment outcome were collected retrospectively in 43 acute myeloid leukemia (AML) and 23 acute lymphoblastic leukemia (ALL) patients. Results: sLR11 levels of acute leukemia patients were significantly increased [ALL, 73.5±93.5 ng mld−1 (range, 5.7–407.0), P

Description: Abstract 4834 Introduction: LR11 (also called SorLA or SORL1) is a type I membrane protein, from which a large extracellular part, sLR11, is released by proteolytic shedding. LR11 plays a key role in the migration of undifferentiated vascular smooth muscle cells, and circulating sLR11 is a biomarker of carotid intima-media thickness. In accordance with sLR11 levels correlating with the fraction of immature vascular cells, human CD34+CD38- immature hematopoietic precursors display high levels of LR11 mRNA. We investigated the expression of LR11 in normal leukocytes, leukemia cell lines and acute leukemia cells. Methods: A2-2-3 anti LR11 monoclonal antibody was used for immunoblotting. Biotinylated or FITC-conjugated anti LR11 monoclonal antibodies, M3 and R15 were used for flow cytometric analysis and immunohistochemistry. Normal mononuclear cells were obtained from healthy volunteer donors. Leukemia cells were obtained from patients' bone marrow or peripheral blood. LR11 protein levels and sLR11 in the culture supernatant of human leukemic cell lines were examined by Western blotting and ELISA using specific monoclonal antibody against LR11. The expression of LR11 mRNA of the cells was examined by Real-Time PCR. Flow cytometric analysis of cell surface LR11 was performed with desktop cell sorter JSAN (Bay Bioscience). Results: Most human leukemia cell lines expressed high level of LR11 mRNA and protein. sLR11 was also detected in the culture supernatant. The levels of LR11 mRNA, the amount of cellular LR11 protein, and the amount of released sLR11 protein were significantly correlated with each other. Flow cytometric analysis of peripheral leukocytes using the anti-LR11 mAb M3, showed expression of LR11 in most CD14+ monocytes. LR11 was not significantly expressed on most T cells (CD4+, CD8+), B cells (CD19+), or granulocytes. However, the leukemia cell lines HL-60 (acute promyelocytic), CCRF-SB (lymphoblastic), and U937 (monocytic), but not K562 (chronic myelogenous) expressed LR11. Since LR11 is expressed by leukemia cells of different origins, we explored the expression of LR11 on the surface of patients' leukemia cells. We have examined 7 AML cases (M0, M1, M2, M3, M4, M5 and M6) and 3 ALL cases. Although expression level of LR11 differs among these cases, LR11 was detected in every case except one ALL case. The most dramatic M3-stained population was the clonally expanded CD19+ mononuclear fraction in MLL-AF4 positive early precursor B acute lymphoblastic leukemia (ALL). In addition, more than 50% blastic cells were positive for LR11 in a Philadelphia chromosome positive ALL patient. Over 50% of CD34+ mononuclear cells in AML (M0) were LR11-positive, whereas LR11-positive blasts predominated in the CD38- fraction. The majority of mononuclear cells in AML (M4) with high CD11b-expression were also LR11-positive. Thus, LR11 is specifically expressed on the surface of leukemic blasts in both ALL and AML. Furthermore, immunohistochemistry of bone marrow clot sections of AML and ALL revealed that cytoplasm of leukemia cells are specifically reacted against the anti-LR11 antibody. Thus, LR11 is expressed both in the cytoplasm and on the cell surface of acute leukemia cells. Conclusion: LR11 is specifically and highly expressed on cell surface of acute leukemia cells in addition to normal leukocytes. Together with our finding that sLR11 is a novel marker for acute leukemia, the identification of novel surface antigen sheds light on leukocyte biology and leukemia cell development. Disclosures: No relevant conflicts of interest to declare.

Description: We conducted a multicenter study to evaluate a new enumeration method of hematopoietic progenitor cells (HPC) using an automated hematology analyzer, the XN-series model (Sysmex Corporation, Japan). This method appeared to be very promising in a pilot study in a single institution [Tanosaki R, et al. Int J Lab Hematol 2013]. We herein discuss the final results of our study. PB or PBSC samples were taken from G-CSF- and/or chemo-mobilized subjects. The samples were divided into two tubes, one for an in-house assay where HPC were counted in each institution and one was shipped to a central laboratory (SRL, Inc., Japan) where the CD34+ cell count was examined. Firstly, we examined the frequency of invalid data. Secondly, the correlation between HPC and CD34+ cells was investigated. Thirdly, cut-off HPC values were determined to predict certain CD34+ cell counts, i.e. 20 cells/µL for PB and 1,000 cells/µL for PBSC. Finally, for the clinical application, we estimated values which predicted more than certain CD34+ cell counts with 95% probability by constructing a two-dimensional normal distribution model. This study was approved by the IRB of each institution, and informed consent was obtained from each participant before apheresis. One hundred and nine subjects (46 healthy donors and 63 patients undergoing PBSCT) were registered in 5 institutions, and 954 HPC measurements from 614 samples were performed. Thirteen (4.4%) HPC values in 297 PBSC assays were estimated to be invalid, which was identified by an asterisk automatically applied to the corresponding result. Next, the data of the first assay from each sample where both the HPC and the central laboratory-based CD34+ count were available were extracted, and 470 HPC-CD34+ pairs from 102 subjects (43 healthy donors and 59 patients) were used for further analyses. The median age was 50.5 years (range, 3-78), 60 males and 42 females, and diagnoses were malignant lymphoma 28 patients, multiple myeloma 27, and others. It was revealed that there was one mismatched result in the PBSC samples (arrow in Fig 1B; HPC 35,768/µL, CD34+ 399/µL). When this value was removed, strong correlations between the HPC and CD34+ cell counts were observed in both the PB (r=0.94) and PBSC (r=0.97) samples (Fig 1). Strong correlations were observed also at each institution. The HPC cut-off values to distinguish CD34+ cell counts below or above 20/µL in PB and 1,000/µL in PBSC samples were determined to be 25 (AUC 0.931, Youden Index 0.713) and 921 (AUC 0.946, Youden Index 0.778), respectively, by a ROC analysis (Fig 2). Finally, HPC of 56/µL was determined to be the optimal value for predicting more than 20/µL of CD34+ cells in the PB samples with 95% probability. This study confirmed that there was a strong correlation between the HPC and CD34+ cell count in a larger number of patients with different diagnosis at different institutions. For the PBSC collection, HPC ≥ 25/µL suggested an appropriate timing, and HPC 56/µL indicated a more secure threshold. HPC may also be a useful marker to rapidly estimate the quantity of CD34+ cells in the apheresis product. In cases where the HPC value is invalid, this may be overcome by serially evaluating HPC in the clinical course. In conclusion, HPC may be a good surrogate or an alternative for the CD34+ cell count. Prospective studies are warranted to verify the usefulness of HPC in clinical practice. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare paraneoplastic disease due to an underlying monoclonal plasma cell (PC) dyscrasia. Despite of dynamic symptoms associated with highly elevated VEGF, monoclonal PCs are thought to be quite small, and pathogenic significance of these PCs remains undetermined. In this study, we performed whole exome sequencing (WES), target sequencing, and RNA sequencing of PCs in patients with POEMS syndrome in order to define its genetic profiles. Methods: Patients diagnosed with POEMS syndrome at Chiba University Hospital from July 2014 to June 2016 were enrolled. DNA was extracted from either PCs which were isolated from patients' bone marrow (BM) using CD138 MACS (Miltenyi) or buccal cells as controls. WES and target sequencing were performed using HiSeq2500 (Illumina) and MiSeq (Illumina), respectively. The data of WES and target sequencing were analyzed by Empirical Bayesian mutation Calling (EBCall). Copy number was analyzed using the data of WES. RNA sequencing of PCs isolated by MACS and FACS sorting was conducted using HiSeq 1500 (Illumina). PCs from some patients diagnosed with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) were also collected as controls for RNA sequencing. Results: Twenty POEMS patients (M:F 12:8, mean age 42.6, range 16-78; 15 newly diagnosed, 5 refractory or relapsed cases) were included in this study. Regarding the types of M protein, 55% (11/20) were IgA-λ, 25% (5/20) were IgG-λ, and each individual case of the following; IgA-λ+IgG-λ, BJP-λ, IgG-κ, and Castleman's variant with no M protein. The mean serum VEGF was 6,471 pg/ml (range 1,190-13,800), and the mean PCs percentage in the BM was 4.4% (range 0.8-10.5). WES was performed in 15 cases; a total of 359 somatic mutations in 334 genes were revealed in 93.3% of cases (14/15) with a mean number of 23.9 (range 0-119) in each. All these mutated genes were significantly enriched in several pathways related to cell adhesion. Importantly, frequently mutated genes in MM such as NRAS, KRAS, and TP53 were not identified. Among all mutations, 1.7% were frameshift insertions, 2.0% were frameshift deletions, 4.2% were stop gains, 0.8% were non-frameshift deletions, 60.2% were other non-synonymous single nucleotide variants (SNVs), 29.5% were synonymous SNVs, and 1.7% were splicing mutations which were within 2-bp of a splicing junction. Copy-number variations were detected in 33.3% of cases (5/10) including -13 (2 cases), +1q (2 cases), and hyperdiploidy (2 cases). To carry out target sequencing in all 20 cases, we defined 51 target genes which included recurrently mutated genes from our WES data, frequently mutated genes in hematopoietic and lymphoid tissues according to the database (COSMIC), and 15 frequently mutated genes in MM (NRAS, KRAS, TP53, BRAF, CDKN2C, FGFR3, BIRC3, DIS3, CYLD, KDM6A, LRP1B, FAM46C, COL6A3, DNAH5, and KRT6A). A total of 60 somatic mutations were revealed in 65% of cases (13/20), and 9 new somatic mutations were found in the cases in which WES was also performed. Ten recurrently mutated genes were identified; KLHL6 in 20% of cases (4/20), each of LTB, RYR1 in 15% of cases (3/20), and each of EHD1, EML4, HEPHL1, HIPK1, PCDH10, USH2A, and ZNF645 in 10% of cases (2/20). Among frequently mutated genes in MM, only 3 genes (FAM46C, LRP1B, and DNAH5) were mutated, each in a single case. We finally conducted RNA sequencing of the FACS-sorted PCs in 5 POEMS patients compared to 5 MGUS and 4 MM patients. Upregulated genes were significantly enriched in some gene sets, gene ontology terms, and pathways related to immune response and cell adhesion, whereas downregulated genes were related to tumorigenesis. Of note, VEGF was not significantly upregulated in POEMS patients. Principal component analysis distinguished the 3 disease groups of patients with marginal overlaps between POEMS and MGUS, and also MGUS and MM. Conclusions: Our data clearly demonstrate that the genetic profiles of PCs in POEMS syndrome are distinct from those in MM and MGUS. Notably, PCs may not be the main source of extremely elevated VEGF in POMES syndrome. On-going further investigation will help clarify the molecular pathogenesis of POEMS syndrome. Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Description: [Backgrounds] POEMS syndrome is a rare plasma cell dyscrasia and its pathogenesis including the significance of monoclonal plasma cells in disease progression is poorly understood. Monoclonal plasma cells only produce λ immunoglobulins, and genes encoding immunoglobulin λ light chain (IGL) V regions are derived from IGLV1-44 or IGLV1-40 germline sequences. Here we analyzed the clonality in IGLV gene rearrangements using next generation sequencing (NGS) to evaluate the significance of monoclonal plasma cell clone size and follow its changes in clinical course to understand the pathogenesis of POEMS syndrome. [Methods] Patients who were diagnosed with POEMS syndrome between November 2006 and October 2015 at Chiba University Hospital were included in the study. As positive controls, 3 multiple myeloma (MM) patients with λ-type monoclonal light chain, and 9 negative control patients were also analyzed. NGS libraries were constructed from genomic DNA samples, extracted from bone marrow mononuclear cells. The IGLV1 and IGLV2 genes were amplified by polymerase chain reaction (PCR) using a 5' primer for the IGLV1/2 framework 3 (FR3) region and 3ʹ consensus primers for the IGLJ1/2/3 joining regions. Multiple samples were pooled, and paired-end 2 × 250 base pair sequencing reactions were carried out using an Illumina MiSeq sequencer. The closest matched germline sequences were determined using the ImMunoGeneTics database. Subsequently, frequencies of each clonotype that were characterized by a unique V-J rearrangement, conserved complementarity determining region 3 (CDR3) anchors and a unique CDR3 amino acid sequence, were calculated. [Results] Twenty-eight patients with POEMS syndrome were enrolled. All the patients had λ-type M protein. The median follow up time of the patients was 24.4 months (range, 3.7 - 113.9). Firstly we analyzed the usage of IGLV germline genes in each case. In 8 cases, the POEMS syndrome-specific germline sequences, IGLV1-40 or IGLV1-44, were dominant; accounting for more than 40% of all germline sequence usage. However, other samples showed minimal or no differences from controls, indicating that the clonal expansion of monoclonal plasma cells is generally low in patients with POEMS syndrome. Analyzing frequencies of the most dominant rearrangement in each germline, the clonal IGLV gene rearrangements of POEMS syndrome-specific germline sequences were significantly increased in 10 POEMS patients (35.7%; IGLV1-44: n = 8, IGLV1-40: n = 2). Significant increase of clone sizes were not directly linked to the initial disease status (vascular endothelial growth factor [VEGF] level and percentage of plasma cells in the bone marrow), overall survival and progression-free survival of POEMS patients. In 12 patients that we were able to follow their clinical courses, the clone size of IGLVgene rearrangements correlated with disease course assessed with serum VEGF level in most cases (n = 8), as they decreased with serum VEGF levels in disease remission and increased with re-elevation of serum VEGF in relapse cases. Clone sizes without significant increase at diagnosis were constantly flat even after achieving disease remission (n = 3). In one case, clone size with significant increase at diagnosis was unchanged even after serum VEGF level decreased (n = 1). Further observation is needed in this case. [Discussion] Considering the cases with significant increase of IGLV rearrangement clones, it was confirmed that clonal light chain gene expression is restricted to the IGLV1-44 and IGLV1-40 germline sequences, as previously reported. IGLV gene rearrangement clone was not detected as significant increase in some cases. Therefore, we speculated that there are certain numbers of patients with POEMS syndrome with extremely low frequency of clone cells. On the other hand, significant increase of clone size did not reflect disease status, suggesting that disease status is not regulated by tumor burden alone. By contrast, in cases with significantly increased clones, clone sizes changed dependiing on the disease status. These data do demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome. [Conclusions] Our analysis of IGLV gene rearrangements has demonstrated the association between the size of IGLV gene rearrangement clones and the clinical courses in POEMS syndrome. Disclosures No relevant conflicts of interest to declare.

Description: Introduction LR11, which is also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding. We have demonstrated that serum sLR11 levels are significantly elevated in patients with acute leukemia and that sLR11 levels are associated with the peripheral blast percentage (Sakai et al. Clin Chim Acta. 2012). In addition, we found that high sLR11 levels have a significant negative prognostic impact on progression-free survival in patients with follicular lymphoma (FL; Kawaguchi et al. Br J Haematol. 2013). In this FL analysis, the immunohistological intensity of LR11 in lymph nodes of FL patients did not show a significant association with serum sLR11 levels. Therefore, a shedding mechanism is presumed to play a key role in the functions of LR11. A disintegrin and metalloproteinase 17 [ADAM17, also known as tumor necrosis factor (TNF)-α converting enzyme (TACE)] has been identified as the enzyme that cleaves TNF-α from its transmembrane precursor form, and it has been found to cleave the ectodomains of other cell surface proteins. LR11 is also shed by ADAM17. Tetraspanin CD9 has been recently shown to inhibit the shedding activity of ADAM17. Therefore, we investigated the role of CD9 in sLR11 release. Materials and methods First, we examined the gene expression levels of LR11 by quantitative real-time PCR and the cell surface expression of LR11 and CD9 by flow cytometric analysis in normal human peripheral-blood mononuclear cells (PBMCs). Second, we investigated the cellular expression and the released levels of LR11 and cellular CD9 in THP-1 monocytes, phorbol 12-myristate-13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1), and the B lymphoblastoid cell line CCRF-SB by immunoblot analysis. Furthermore, to assess the relationship between LR11 and CD9 in PMA/THP-1, we performed double immunofluorescence staining of these molecules followed by confocal microscopy analysis. Third, we examined the effects of ectopic neoexpression of CD9, anti-CD9 mAbs, and CD9 silencing. THP-1 cells stably interfered with CD9 were generated by the transfection of the shRNA expression vector that is specific for CD9. Results The gene expression levels of LR11 were significantly higher in CD3+ T cells than in CD14+ monocytes, and there was no difference between monocytes and CD19+ B cells. However, LR11 was only expressed on the cell surface of monocytes in normal PBMCs. CD9 was broadly expressed on the cell surface of every PBMCs. Because it has been reported that CD9 associates with ADAM-17 on the cell surface, we hypothesized that CD9 associates with the shedding of LR11 on the cell surface. To verify this hypothesis, we chose the monocytic cell line THP-1 because monocytes have both LR11 and CD9 on their surface. In addition, because the gene expression levels of LR11 were significantly higher in human monocyte-derived macrophages than that in monocytes, we investigated the levels in PMA/THP-1. LR11 was not expressed or released in undifferentiated THP-1 cells, but it was expressed or released in differentiated PMA/THP-1. CD9 was poorly expressed in THP-1 cells, and the CD9 expression levels were increased in PMA/THP-1. The confocal microscopy analysis showed colocalization of LR11 and CD9 proteins in PMA/THP-1. We next investigated the effects of ectopic neoexpression of CD9 in CD9-negative cells, neutralizing anti-CD9 mAbs (clone ALB-6) in CD9-positive cells, and CD9 silencing in THP-1 on the shedding of LR11. When CCRF-SB, LR11-positive and CD9-negative cells were transiently overexpressed with CD9, the amount of sLR11 released from CD9-overexpressing cells was decreased compared with that from control cells. The amount of sLR11 released from LR11-transfected THP-1 cells that were cultured with ALB-6 was significantly increased compared with that cultured with an isotypic control antibody. The PMA-stimulated release of sLR11 was significantly increased in CD9 shRNA-interfered THP-1 cells compared with cells that were transduced with the control shRNA. The increase in CD9 shRNA-interfered THP-1 was nullified by treatment with the metalloproteinase inhibitor GM6001 (Figure 1). Conclusion These results suggested that the tetraspanin CD9 inhibits ADAM17-mediated LR11 shedding in leukemia cells and that it may have a role in controlling the function of LR11. Disclosures: No relevant conflicts of interest to declare.

Description: Recently, novel agents such as bortezomib and lenalidomide have been introduced for multiple myeloma (MM) treatment and have improved patients' survival drastically. However, dexamethasone remains a mainstay in the treatment of MM. Dexamethasone effectively induces tumor cell death when used for the initial treatment of MM. In addition, dexamethasone has a synergistic effect with novel agents and is hence used in combination with such agents. However, prolonged dexamethasone exposure may lead to drug resistance. To elucidate the mechanism of dexamethasone resistance, we generated a dexamethasone-resistant subline of the MM cell line RPMI8226. We cultured RPMI8226 cells with 1 µM dexamethasone for 7 weeks and established the dexamethasone-resistant cell line Dex-R. This cell line showed no difference in survival in the presence or absence of 1 µM dexamethasone. We then examined differences in gene expression between RPMI8226 and Dex-R cells using cDNA microarray. Expression of the FARP1 gene, which is a transforming growth factor beta (TGF-b) target gene in myeloma cells, was increased approximately 50-fold in Dex-R cells compared to that in RPMI8226 cells. In some myeloma patients who become chemoresistant, myeloma cells show high levels of FARP1 expression at the initial stage. FARP1 has a Rho-GEF domain and can associate with proteins on the cell membrane through the FERM domain. In the nervous system, FARP1 is involved in synaptogenesis via the activation of Rac1. Based on these observations, we hypothesize that Dex-R cells acquires dexamethasone resistance with an increase in the level of FARP1 expression via the activation of Rac1. To verify this hypothesis, we established inducible FARP1 knockdown Dex-R cells using the TET-ON lentiviral system. We cultivated these cells for 24 h with doxycycline and added 1 µM dexamethasone. A total of 48 h after adding dexamethasone, we measured cell viability using the MTS assay. We cultured Dex-R cells with a Rac1 inhibitor (NSC23766) and added dexamethasone 12 h later. FARP1 expression decreased to approximately 10% in FARP1 knockdown cells 24 h after the addition of doxycycline. Without dexamethasone, there was no difference in survival in the presence or absence of doxycycline. However, when cells were cultured with dexamethasone, the growth of FARP1 knockdown Dex-R cells was significantly inhibited compared with that of the control (Fig 1). Next, we examined the change in dexamethasone resistance on the addition of the Rac1 inhibitor. The number of cells increased after 96 h without dexamethasone. On the other hand, the number of cells significantly decreased when cultured with dexamethasone (Fig 2). These data suggest that resistance to dexamethasone in Dex-R cells was mitigated by the inhibition of Rac1. We conclude that the activation of Rac1 through FARP1 is one mechanism of dexamethasone resistance in MM. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4795 Introduction: We have recently reported that LR11 (also called SorLA or SORL1) is specifically expressed on the surface of acute leukemia cells, and the circulating levels of soluble form (sLR11) are highly associated with peripheral blast numbers in acute leukemia patients at diagnosis and decreased in remission (2012, Sakai et al). Although the functional role of sLR11 in the migration of undifferentiated vascular cells has been studied, the pathological significance of sLR11 has not been elucidated for the development of leukemia blast properties. The expressions of WT1, an oncogene for the various malignant tumors, are associated with poor prognosis of patients with acute leukemias, and the WT1 levels have been thought to indicate the blast properties. In this study, in order to know the significance of sLR11 for the oncogenic properties of leukemia cells, we first examined the correlations of circulating sLR11 levels with WT1 mRNA levels in the bone marrow of acute leukemia patients. Next, we analyzed the effects of sLR11 on WT1 mRNA expressions, particularly in association with GATAs, factors for the WT1 gene transcription, in leukemia cell lines. Materials and methods: Levels of serum sLR11 and bone marrow cell WT1 mRNA were measured by ELISA and real-time PCR, respectively, using samples of acute leukemia patients (AML n=6, ALL n=5). Statistical analyses were performed by Spearman rank correlation system. mRNA levels of GATA1, GATA2 and WT1 were analyzed using K562 cells with or without recombinant sLR11 and LR11-overexpressing K562 cells. Results: Figure 1 shows the relationship between sLR11 in serum and WT1 mRNA in bone marrow mononuclear cells in acute leukemia patients at diagnosis. The sLR11 levels were significantly positively correlated with the WT1 levels in the patients. In order to know the pathological link between sLR11 and WT1 in leukemia cells in detail, we then analyzed the effects of sLR11 on the WT1 mRNA expressions in K562 cells. WT1 mRNA levels were gradually increased after the incubation with 1 ng/ml sLR11, and the levels at 4 h were elevated to 2.5-fold of those before the incubation (Figure 2). The levels were then decreased to the levels equivalent to those before the incubation. The mRNA levels of GATA1 and GATA2 were also drastically elevated at 4 h and then sharply decreased after adding sLR11. GATA1 mRNA levels were particularly increased by 5-fold of those before the incubation. Thus, sLR11 were suggested to cause the activation of transcription factor network of GATAs, and induce the WT1 gene transcription in the cells. We finally investigated the effects of sLR11 overexpression on the elevation of WT1 mRNA in K562 cells. The LR11-overexpressing cells showed significantly increased expression levels of GATA1 and GATA2 in addition to WT1 mRNAs. Conclusions: Circulating sLR11 levels were significantly correlated with the WT1 mRNA expression levels in acute leukemia. sLR11 induces the WT1 expression, possibly through the activations of GATAs in leukemia cells. These results suggest that sLR11 may be a novel marker indicating the malignant properties of leukemia cells. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Although thrombopoietin-receptor agonists have recently been introduced for the treatment of refractory immune thrombocytopenia (ITP), splenectomy is still the standard treatment option for patients with steroid-refractory ITP. However, open and laparoscopic splenectomy is associated with a mortality rate of 1.0% and 0.2%, respectively, and the major known long-term risk of splenectomy is severe bacterial sepsis. Partial splenic embolization (PSE) is an alternative to splenectomy for patients with steroid-refractory (Kimura et al., AJR 2002). PSE does not require general anesthesia and has not been associated with severe complications. In this study, we analyzed the efficacy and long-term outcomes of PSE in steroid-refractory ITP patients. Patients and Methods Between 1988 and 2009, 77 patients with chronic ITP [30 men and 47 women, 16.5–80.9 years old (median age, 46.5 years)] underwent PSE at Chiba University Hospital. Almost all patients did not respond to steroid treatment or other therapies including Helicobacter pylori eradication and immunosuppressive drugs. A 5-French catheter was introduced via the femoral artery to the middle splenic artery and a 0.089-cm guidewire was inserted coaxially. Embolization of intrasplenic arterial branches was performed by the injection of 2 x 2 mm fragments of Gelfoam and an antibiotic solution. Splenic embolization was monitored angiographically during the procedure, and embolization was stopped after 70% of the splenic parenchyma had been ablated. The therapeutic response to PSE was evaluated on the basis of the platelet (PLT) count at 1 year after PSE: a complete response (CR) was defined as PLT count of more than 10 x 1010/L without any medication and a partial response (PR) was defined as PLT count = 5–10 x 1010/L without any medication. Patients who required medication to maintain a PLT count of more than 5 x 1010/L or patients with a PLT count of less than 5 x1010/L were considered to have no response (NR). Results The average PLT count prior to PSE was 2.7 ± 2.4 x 1010/L. The mean time to achieve the peak PLT count was 10.2 ± 5.2 days after PSE. The PLT count was 11.1 ±10.3 x 1010/L (n = 74), 9.8 ± 8.3 x 1010/L (n = 70), 9.1 ± 8.2 x 1010/L (n = 65), and 9.1 ± 7.5 x 1010/L (n = 69) at 1 month, 3 months, 6 months, and 1 year, respectively. One year post PSE, CR or PR was obtained in 33 (58%) of the evaluable 57 patients and 24 patients (42%) had NR. The group in which the peak PLT count after PSE was over 30.0 x 1010/L showed a significantly higher PLT count than the group in which the peak count was under 30.0 x 1010/L after PSE treatment (Figure 1). The peak PLT count was significantly higher in CR and PR groups (37.4 x 1010/L) than in the NR group (15.0 x 1010/L) (p = 0.041). In addition, 52% patients did not require additional treatment for 10 years after PSE (Figure 2); in 16 patients, PSE was performed for the second time after recurrence; and CR or PR was obtained in 5 patients (31%). There were no severe PSE-related complications such as splenic abscess and no PSE-related deaths. Conclusions PSE might be a safe and effective alternative therapy to splenectomy for patients with steroid-refractory ITP as it generates long-term durable responses. Disclosures: No relevant conflicts of interest to declare.