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Abstract 3140 NLRP3 is an intracellular trigger of IL-1β production that plays important roles in the regulation of inflammation and apoptosis. A single nucleotide variation in the 3'-untranslated region of the NLRP3 gene, rs10754558 (+29940G>C), is linked to several immunological diseases. When we examined the impact of the NLRP3 genotype in a cohort consisting of 392 pairs of patients with hematologic malignancies and their unrelated HLA 12/12 matched bone marrow donors transplanted through the Japan Donor Marrow Program, the recipient NLRP3 GG genotype was found to be associated with a significantly worse 5-year overall survival (OS) rate (34% vs. 50%, P=0.006) (Fig. 1) and a trend toward a higher transplant-related mortality (TRM) rate (39% vs. 27%, P=0.09) than the recipient CC or CG genotype. The recipient GG genotype remained statistically significant in the multivariate analysis for OS (hazard ratio [HR], 1.86; 95% confidence interval [CI], 1.22 to 2.22; P=0.004) and TRM (HR, 2.28; 95% CI, 1.20 to 4.35; P=0.01). The donor NLRP3 genotype did not significantly influence the transplant outcomes. Next, we investigated the functional relevance of the NLRP3 +29940G>C variant. When leukocytes from healthy individuals were stimulated in vitro with NLRP3 ligand, the leukocytes with the NLRP3 GG genotype produced significantly more IL-1β than those with the NLRP3 CC or CG genotype (Fig. 2). These findings substantiate the functional relevance of the NLRP3 variant, and suggest that the higher IL-1β secretion in the peri-transplant period by recipients with the NLRP3 GG genotype likely accounts for their poor transplant outcomes. NLRP3 genotyping could therefore be useful in predicting prognoses and creating therapeutic strategies for improving the final outcomes of patients who undergo allogeneic hematopoietic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 3197 Poster Board III-134 Small populations of glycosylphosphatidylinositol-anchored protein (GPI-AP)-deficient blood cells are often detectable in the peripheral blood (PB) of patients with aplastic anemia (AA) and refractory anemia (RA) of myelodysplastic syndromes defined by the FAB classification. Such PNH-type cells are thought to be derived from PIG-A mutant hematopoietic stem cells (HSCs) that avoid the immunological attack against HSCs. Inefficient T cell responses to PNH-type cells were indeed demonstrated by a murine study. However, there is no direct evidence in support of the escape theory concerning the expansion of PIG-A mutant HSCs in such patients with bone marrow (BM) failure. If the escape theory is true, the PNH-type cells should be detected in myeloid cells derived from HSCs that are targeted by the immune system attack. The PB of 527 patients with BM failure was examined for the presence of GPI-AP deficient cells in various lineages of cells including granulocytes, erythrocytes, monocytes, T cells, B cells, and NK cells using high sensitivity flow cytometry to verify this hypothesis. PNH-type cells were detectable in at least one lineage of cells from 228 (43%) patients. Although most of the positive patients showed PNH-type cells in two or more lineages including granulocytes or monocytes, 14 patients (13 with AA and 1 with amegakaryocytic thrombocytopenia) displayed PNH-type CD48-CD55-CD59- cells only in T cells at a frequency of 0.003-0.3% of the total T cells (Figure). The PNH-type T cells were undetectable in any of 25 healthy individuals. The CD48-CD55-CD59- T cells consisted of predominantly effector memory and terminal effector memory cells with naïve phenotype cells. The phenotypic pattern of the PNH-type T cells was very similar to that of CD48-CD55-CD59- T cells from 11 patients with florid PNH but was different from that of CD48-CD55-CD59- T cells (central and effector memory cells alone) detected in 4 marrow transplant recipients who received anti-CD52 antibody (alemtuzumab) therapy as conditioning. PIG-A gene analyses of CD48-CD55-CD59- T cells revealed a single mutation in 2 patients with PNH-type T cells alone, while two different mutations were revealed in 2 patients treated with alemtuzumab. BM failure patients with PNH-type T cells alone and other BM failure patients possessing PNH-type granulocytes or monocytes showed similar clinical features characterized by predominant thrombocytopenia and good response to immunosuppressive therapy, thus suggesting an increase in the number of PNH-type cells in both groups to be associated with a similar immune pathophysiology. The escape theory cannot account for the presence of PNH-type cells exclusively in T cells in immune-mediated BM failure because T cell precursors are not the target of the immune system attack in AA. Therefore, mechanisms other than the escape theory must be considered for the initial proliferation of PIG-A mutant HSCs associated with the development of AA, such as preferential activation of dormant PIG-A mutant HSCs or T cell precursors due to the deficiency of GPI-APs that transmit negative signal Disclosures No relevant conflicts of interest to declare.

Abstract 2305 Poster Board II-282 IL-17 plays an active role in autoimmune diseases and organ transplant rejection through its mediating proinflammatory responses and inducing inflammatory cytokine and chemokine production. This study examined the impact of donor and recipient polymorphism in the IL-17A gene on the clinical outcomes in unrelated HLA-fully-matched myeloablative bone marrow transplantation (BMT) through the Japan Marrow Donor Program. The IL-17A polymorphism (rs2275913, G197A) was retrospectively analyzed in a total 145 recipients with hematologic malignancies and their unrelated donors. In patients with standard risk disease, the donor IL-17A-197A genotype was associated with significantly improved OS (adjusted HR, 0.32; 95% CI, 0.13-0.79; p=0.01) and reduced transplant related mortality (TRM) (adjusted HR, 0.27; 95% CI, 0.11-0.67; p=0.005), but no impact on disease relapse or the development of grades II-IV acute GVHD or chronic GVHD. Patients with standard risk disease receiving transplants from donors with the 197A genotype had better 5-year OS (71% vs. 40%, p=0.02; Figure) and lower 5-year TRM rate (27% vs. 60%, p=0.02). The IL-17A polymorphism did not significantly influence the transplant outcomes in patients with high risk disease. These results suggest transplantation from IL-17A-197A positive donor is advantageous in improving OS and reducing TRM after HLA-matched unrelated BMT for standard risk hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Abstract 1489 Poster Board I-512 The hematopoietic stem cell (HSC) differentiation pathway in humans remains largely unknown due to the lack of an appropriate in vivo assay allowing the growth of HSCs as well as of clonal markers that enable the tracing of their progenies. Small populations of blood cells deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs) such as CD55 and CD59 are detectable in approximately 50% of patients with aplastic anemia (AA) and 15% of patients with refractory anemia (RA) of myelodysplastic syndrome defined by the FAB classification. Such blood cells with the paroxysmal nocturnal hemoglobinuria (PNH) phenotype (PNH-type cells) are derived from single PIGA mutant HSCs and their fate depends on the proliferation and self-maintenance properties of the individual HSCs that undergo PIG-A mutation by chance (Blood 2008;112:2160, Br J Haematol 2009 in press) Analyses of the PNH-type cells from a large number of patients on the diversity of lineage combination may help clarify the HSC differentiation pathway in humans because PIG-A mutant HSCs in patients with bone marrow failure appear to reflect the kinetics of healthy HSCs. Therefore, different lineages of peripheral blood cells were examined including glycophorin A+ erythrocytes (E), CD11b+ granulocytes (G), CD33+ monocytes (M), CD3+ T cells (T), CD19+ B cells (B), and NKp46+ NK cells (Nk) from 527 patients with AA or RA for the presence of CD55−CD59− cells in E and G, and CD55−CD59−CD48− cells in M,T, B, Nk with high sensitivity flow cytometry. Two hundred and twenty-eight patients (43%) displayed 0.003% to 99.1% PNH-type cells in at least one lineage of cells. The lineage combination patterns of PNH-type cells in these patients included EGM in 71 patients (31%), EGMTBNk in 43 (19%), EG in 37 (16%), T alone 14 (6%), EGMBNk in 11 (5%), G alone in 10 (4%), GM in 10 (4%), EGMNk in 7 (3%), EGMT in 7 (3%), EGMB in 6 (3%), EM in 5 (2%), EGMTB in 3 (1%), EGNk in 1 (0.4%), EGMTNk in 1 (0.4%), GMTB in 1 (0.4%), and GT in 1 (0.4%) (Table). All patterns included G or M, except for 14 patients displaying PNH-type T cells alone. No patients showed TB or TBNk patterns suggestive of the presence of common lymphoid progenitor cells. Peripheral blood specimens from 123 patients of the 228 patients possessing PNH-type cells were examined again after 3 to 10 months and all patients showed the same combination patterns as those revealed by the first examination. PIG-A gene analyses using sorted PNH-type cells from 3 patients revealed the same mutation in G and Nk for 1 patient and in G and T for 2 patients. These findings indicate that human HSCs may take a similar differentiation pathway to that of murine HSCs, the ‘myeloid-based model’ that was recently proposed by Kawamoto et al. (Nature 2008; 10:452), though the cases with PNH-type T cells alone remain to be elucidated. Table. Lineages of cells containing PNH-type cells in patients with AA or RA. The number in the parenthesis denotes the proportion of patients showing each combination pattern in the total patients possessing PNH-type cells. (+ ; presence of PNH-type cells) Disclosures No relevant conflicts of interest to declare.

The FcγRllla (FCGR3A) 158V/V allotype displays a higher antibody-dependent cellular cytotoxicity compared to the FCGR3A-158V/F or 158F/F allotype, leading to susceptibility to rheumatoid arthritis, better clinical response to rituximab in NHL, and lower risk of periodontitis. We tested the hypothesis that FCGR3A polymorphism influences the development of GVHD and GVL, and TRM. The FCGR3A-158V/F genotype was determined in 49 recipient and donor pairs who underwent HLA-matched SCT in our institute. Subjects with the recipient FCGR3A-158V/V allotype and the donor FCGRA-158V/V allotype were almost one-third and twice as likely to develop chronic GVHD respectively (Table 1). The recipients with the FCGR3A-158V/V allotype had a 60% increase in probability of 3-year TRM. Multivariate analysis showed that the recipient FCGR3A-158V/V allotype and the donor FCGRA-158V/V allotype were independent risk factors for chronic GVHD (HR = 0.8 and 1.2, respectively), as well as the unrelated donor BMT (HR = 1.6) and the related donor PBSCT (HR = 2.4). The recipient FCGR3A-158V/V was an independent predictor for 3-year TRM (HR = 4.0) with the high risk disease (HR = 3.0) and the cord blood transplantation (HR = 5.0). We conclude that the recipient and donor FCGR3A polymorphism is associated with the development of chronic GVHD and TRM after HLA-matched SCT. These findings could therefore be useful in selecting the donor and creating therapeutic strategies for improving the final outcome of allogeneic SCT. Besides, these raise the question of whether the genetic risk factor in a recipient might also be involved in the mechanisms of chronic GVHD. Table 1 | FCGR3A allotype | 2–4 acute GVHD(HR) | Chronic GVHD (HR) | 3 YR TRM (HR) | |:---------------------------------:| ------------------ | ----------------- | ------------- | | *P

The pathophysiology of bone marrow failure syndromes (BMS) such as aplastic anemia (AA) and refractory anemia (RA) of myelodysplastic syndromes defined by the FAB classification involves immune mediated bone marrow injury and intrinsic defects of hematopoietic stem cells. The diagnosis of the immune pathophysiology is essential in providing the optimal treatment to patients with BMS. In order to identify a new diagnostic marker for the immune pathophysiology of BMS, the sera of AA patients characterized by the presence of small populations of paroxysmal nocturnal hemoglobinuria-type (PNH) cells were screened for the presence of antibodies (Abs) recognizing proteins derived from a megakaryocytic leukemia cell line, UT-7. Two-dimensional electrophoresis of UT-7 lysates followed by immunoblotting revealed a distinct spot of 65 kDa. Peptide mass fingerprinting identified this protein as a heterogeneous nuclear ribonucleoprotein (hnRNP) K. Although hnRNP K is known to be ubiquitously expressed, the expression levels based on a Western blotting analysis were greater in several leukemia cell lines such as UT-7, K562 and OUN-1 in comparison to peripheral blood mononuclear cells. When the sera of newly diagnosed 87 BMS patients (62 with AA and 25 with RA) were examined for the presence of anti-hnRNP K Abs using an enzyme-linked immunosorbent assay (ELISA) with recombinant hnRNP K protein, significantly higher titers of anti-hnRNP K Abs were detected in 13 (34%, 10 with AA and 3 with RA) of 38 patients with BMS displaying increased PNH-type cells (PNH+) and in 15 (31%, 8 with AA and 7 with RA) of 49 patients without increased PNH-type cells. The presence of hnRNP K Abs was significantly correlated with the presence of anti-diazepam-binding inhibitor-related protein-1 (DRS-1) Abs (r=0.7206) and anti-moesin Abs (r=0.8318). Of the 87 patients, 49 patients (39 with AA and 10 with RA) received antithymocyte globulin plus cyclosporine therapy after the Ab screening. All of the 22 patients with high anti-hnRNP K Ab titers responded to therapy while 10 (37%) of 27 patients without the high titer Abs responded (P < 0.005). When the three Ab titers were assessed, the response rate to immunosuppressive therapy in patients showing high titers in at least one Ab was 96%, while the response rate in patients not showing high titers in any of the auto-Abs was 39% (P < 0.005). These findings suggest that in patients with immune mediated BMS, a breakdown of immune tolerance to multiple self antigens including hnRNP K may take place and that the detection of these auto-Abs may help diagnose the immune pathophysiology of BMS.