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Abstract 4646 The common fragile sites (CFSs) are regions of profound genomic instability, and hot-spots for deletions and other alterations in cancer cells. We have previously shown that promoter of FHIT is frequently methylated in multiple myeloma (MM) and correlated with worse prognosis. WWOX is located at a CFS region on chromosome 16q23.3, this gene is also a target of alterations in multiple cancers. The objective of current study is to find if this tumor suppressor gene is altered in MM and MGUS. Six myeloma cell lines, bone marrow mononuclear cells (BMMNC) of 165 MM and 25 MGUS patients were subjected to this study after obtaining informed consent. Isolated CD138 positive plasma cells (PC) of 24 MM were examined to see if the alteration occurred really in myeloma cells. Using methylation specific PCR, WWOX promoter methylation was detected in 2 of 6 cell lines, 35% of MM patients and 8% of MGUS patients (p=0.01). The overall survival of the MM patients with methylated WWOX tended to be worse than with unmethylated WWOX (p=0.2). Using nested RT-PCR, aberrant short transcripts of WWOX lacking exons coding SDR domain were detected in 4 cell lines, 68% of MM and 60% of MGUS (p=0.51) In isolated PC, aberrant transcripts of WWOX were detected in 58% of MM and 25% of MGUS (p=0.02). Real time PCR demonstrated higher WWOX expression in isolated PC of MM and MGUS than of BMMNC other than plasma cells (p=0.0001), and those higher WWOX were aberrant type (p=0.001). As aberrant WWOX is known to function as dominant negative for wild type, our results imply that this aberrant WWOX is an oncogene as well as losing tumor suppressor function. Since WWOX is demonstrated to regulate beta-catenin and NF-kB pathway, high frequency of aberrant WWOX expression indicates an important role in myeloma development. Disclosures: No relevant conflicts of interest to declare.

Extramedullary plasmacytomas are occasionally seen in the course of multiple myeloma; however, primary plasmacytomas are unusual. Most of the primary plasmacytomas arise in the upper respiratory system, but a plasmacytoma of the lymph nodes is very rare. A case of a 56-year-old female with primary plasmacytoma in the lower mandibular, cervical, axillary, and para-aortic lymph nodes accompanied by IgA Kappa paraproteinemia is described. After ten courses of combination chemotherapy with cyclophosphamide and prednisolone, she obtained complete response. However, 6 years later, she had a relapse of plasmacytoma in the generalized lymph nodes. She received ten treatments of melphalan-prednisolone and again obtained complete response. She has remained well without chemotherapy or recurrent signs for 9 years. It is considered that conventional chemotherapy can be useful for primary plasmacytoma of generalized lymph nodes.

The ultrastructure of neutrophilic phagosomes containing autologous platelets in vivo from a woman with specific granule deficiency is described. This phagosome formation was observed only when she had developed pneumonia due to Staphylococcus aureus. Therefore, the phenomenon might be induced by severe bacterial infection under an abnormal neutrophilic condition accompanied by specific granule deficiency.

A rare case of non-Hodgkin's lymphoma (NHL) that showed transient spontaneous regression (SR) is described. After 6 months of remission, recurrence was noted in the lymph nodes, pleura and the spleen. Although transient improvement was observed following combination chemotherapy, the pleural effusion became refractory to chemotherapy and the patient died 9 months after the relapse.

Abstract 2531 Background: Immune thrombocytopenia (ITP) is an autoimmune disorder caused by production of autoreactive antibodies against platelet antigens. The association between Helicobacter pylori infection and a subgroup of ITP is now widely recognized. Although multiple dysfunctions in cellular immunity are considered to be important in the pathogenesis of ITP, little is known about the role of innate immune cells. Dendritic cells (DCs) play an essential role in innate and adaptive immune response by regulating the differentiation of naïve T cells and interacting with NK or NKT cells. DCs are composed of two subsets: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in human peripheral blood. PDCs play an important role in several autoimmune diseases such as systemic lupus erythematosus through their ability to produce large amounts of type I interferon. In the present study, we analyzed the number of circulating DCs, regulatory T (Treg) cells, Th17 cells, NK cells and NKT cells in untreated patients with primary and H. pylori-associated ITP and assessed the changes of DC subsets after treatment in order to elucidate the role of these cells in the pathogenesis of ITP. Methods: This study was approved by the local institutional review board. After obtaining written informed consent, we enrolled 46 untreated patients with chronic ITP (38 females and 8 males; median age: 52.5 years) and 47 healthy adult volunteers (43 females and 4 males; median age: 50.5 years). We investigated the percentage and the absolute number of circulating pDCs (Lineage marker (Lin)− CD123+ HLA-DR+) and mDCs (Lin− CD11c+ HLA-DR+) in whole blood, and NK cells (CD3− CD56+), invariant NKT (iNKT) cells (Vα24+ Vβ11+), Treg cells (CD4+ CD25+ Foxp3+) and Th17 cells (CD4+ Interleukin (IL)-17A+) among peripheral blood mononuclear cells (PBMCs) by flow cytometry. Intracellular IL-17A production in CD4+ T cells activated by phorbol 12-myristate 13-acetate (PMA) and ionomycin was assessed in order to detect Th17 cells. In addition, we consecutively analyzed the number of DC subsets in ITP patients before and after H. pylori eradication or the administration of prednisolone (PSL). Results: H. pylori was detected in 15 of 38 patients (39.5%), and 7 of 15 H. pylori-positive patients (46.7%) probably had secondary ITP (H. pylori-associated), as platelet count elevation was observed only after H. pylori eradication in these cases. Thirteen patients underwent H. pylori eradication and 9 patients underwent PSL. We found a significant reduction in the percentage and absolute number of pDCs in untreated ITP patients when compared with controls (p < 0.001), while the percentage and absolute number of mDCs tended to decrease in ITP patients; however, the differences were not significant. In addition, the frequency of circulating pDCs was significantly lower in H. pylori-positive patients when compared with controls (p < 0.05) and was equal to that in H. pylori-negative patients. The frequency of circulating Treg cells, Th17 cells, NK cells and iNKT cells in ITP patients was similar to that in controls. Interestingly, we observed increases in the frequency of pDCs after H. pylori eradication only in responders, while there were no changes or slight reductions after this treatment in non-responders. On the other hand, we were unable to elucidate the relationship between changes in the frequency of mDCs and H. pylori eradication. To investigate the differences with treatment type, we performed serial measurement of DC numbers in cases treated by oral administration of PSL, which is standard therapy in primary ITP patients. The frequency of both pDCs and mDCs in all cases treated with PSL was markedly reduced in 1–2 months after treatment and tended to increase with dose reduction for 3–8 months after treatment. Because there were no differences in this trend between responders and non-responders to PSL, and because the frequency of DC subsets appeared to be inversely correlated with PSL dose, we considered that these results were caused by PSL, in contrast to cases undergoing H. pylori eradication. Conclusion: We found for the first time that circulating pDCs were significantly reduced in untreated patients with both primary and H. pylori-associated ITP. In addition, the reduced number of pDCs was restored after eradication in H. pylori-associated ITP patients. This suggests that pDCs play an important role in the pathogenesis of ITP. Disclosures: No relevant conflicts of interest to declare.

Fluconazole, a new effective triazole antifunal agent, has been reported to cause fewer adverse reactions than amphotericin B. A patient who was diagnosed as having agranulocytosis and thrombocytopenia after treatment with fluconazole was investigated and recovered after withdrawal of the antifungal therapy. This case suggests the need for careful haematological observation during the treatment with fluconazole.

A 24-year-old Japanese male having ulcerative colitis and idiopathic thrombocytopenic purpura is described. When the symptoms of ulcerative colitis were manifest during his clinical course, the platelet life-span was shortened and the platelet associated IgG level was increased. Thus, causal association between the two diseases may be suggested.

Abstract 1935 Poster Board I-958 Introduction: Several reports have identified soluble-form IL-2 receptor á (sIL-2Rá) as a significant prognostic factor in patients with non-Hodgkin lymphoma treated using chemotherapy, particularly in rituximab-containing regimens. However, the clinical significance of sIL-2R is not fully understood, as only small populations have been studied to date. The rationale for increasing of serum level of sIL-2Rá in non-Hodgkin lymphoma is also unclear. Patients and Methods: We analyzed 409 patients newly diagnosed with diffuse large B-cell lymphoma (DLBCL) between January 2001 and July 2008. Treatment comprised CHOP-like regimen with (R-CHOP-like) or without rituximab. Levels of sIL-2R were evaluated with enzyme-linked immunosorbent assay at diagnosis. Overall survival (OS) and progression-free survival (PFS, death from any cause, relapse and refractory disease) were analyzed using Kaplan-Meier methods and survival was compared using log-rank tests. To estimate the survival impact of several factors, including sIL-2Rá level, PS, LDH, B symptoms, extranodal sites ≥2 and age, we performed multivariate analysis using Cox proportional hazards. In 166 of 409 patients, CD25 (IL-2Rá) expression on tumor cells was evaluated using a lymphoma sample from the lymph node, bone marrow, blood or other extranodal organ by flow cytometry. To estimate CD25 expression of tumor cell, CD45 bright cells (mature lymphocyte gate) were gated and considered positive if positivity was seen in >20% of the population excluding CD4-positive cells) using three-color flow cytometry. Results: Median age was 68 years (range, 17-91 years), males/females 1.18, and 28.9% of patients were treated with CHOP-like regimen and 60.2% with R-CHOP-like regimen. Clinical stage was I in 24.4%, II in 24.2%, III in 13.1%, and IV in 38.8%. International Prognostic Index (IPI) was Low in 33.5%, LI in 23.5%, HI in 18.7% and H in 24.3%. Median follow-up for CHOP-like and R-CHOP-like groups was 924 days (range, 16-2878 days) and 799 days (range, 29-2688 days), respectively. Median sIL-2Rá value was 1360 U/L (range, 170-59,500 U/L). For the entire population, CR rate was 71.9%, 3-year OS was 67.6% and PFS was 58.8%. OS differed significantly between sIL-2R >1000 U/L and ≤1000 U/L, between >2000 U/L and ≤2000 U/L and between >3500 U/L and ≤3500 U/L, (p1000 U/L and ≤1000 U/L, between >2000 U/L and ≤2000 U/L and, between >3500 U/L and ≤3500 U/L (p3500 U/L is extremely poor even if treated with R-CHOP (53.2%). We showed that one rationale for increasing level of serum sIL-2Rá level in DLBCL is to remove from the tumor cell surface. Disclosures: No relevant conflicts of interest to declare.

Background: Dendritic cells (DCs), natural killer (NK) cells, and invariant NKT (iNKT) cells play important roles in innate immune systems. These cells have recently been shown to be involved in immunoregulation, and some studies have suggested associations with various kinds of autoimmune disease. Conversely, regulatory T cells (Tregs) that are important for peripheral tolerance and Th17 cells that play a central role in maintenance of chronic inflammation are also associated with the pathogenesis of several autoimmune diseases. Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease mediated by anti-platelet autoantibodies, but relationships to innate immunity are unclear. In addition, the pathogenesis of ITP associated with Helicobacter pylori remains obscure. In particular, the regulation of immune responses by these cells in patients infected with H. pylori has not been investigated. This study analyzed DCs, NK cells, iNKT cells, Tregs and Th17 cells in patients with ITP. Methods: Subjects comprised 31 patients with ITP and 22 healthy donors. Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all subjects. Flow cytometry was used to investigate amounts of circulating plasmacytoid DCs (pDCs) (CD123+ HLA−DR+) and myeloid DCs (mDCs) (CD11c+ HLA−DR+) from whole white blood cells, and NK cells (CD3− CD56+), iNKT cells (Vα24+ Vβ11+), Tregs (CD4+ CD25+ Foxp3+) and Th17 cells (CD4+ IL−17A+) from mononuclear cells. The intracellular interleukin (IL)-17A production in CD4+ T-cells activated by phorbol 12-myristate 13-acetate (PMA) and ionomycin was assessed to detect Th17 cells. Results: Both the percentage and numbers of pDCs were significantly reduced in patients compared to healthy controls (p Conclusion: These results suggest that alterations in innate immunity as a reduction of pDCs could be associated with the pathogenesis of ITP. Furthermore, as in some autoimmune diseases that have been considered as Th1 diseases, Th17 cells may play an important role in ITP.

A monoclonal antibody (MAb) specific for interleukin-6 (IL-6) was generated by fusing SP2/0 cells with spleen cells from a mouse immunized with rat spleen cell derived plasmacytoma growth factor (rat PCT-GF). This MAb inhibited the growth of an IL-6-sensitive murine plasmacytoma clone, MD90, in the presence of the immunogen, rat PCT-GF. More interesting, however, this MAb demonstrated species cross-reactivity by neutralizing murine (recombinant and P388D1, cell line-derived) and human (recombinant) IL-6. IL-6 neutralization activity was also established in other IL-6 bioassays, such as the proliferation of spleen cells, plasmacytoma T1165, and a B-cell hybridoma 7TD1. IL-6 neutralization was overcome partially by increasing the concentration of PCT-GF. The MAb had no effect on PCT-GF-independent plasmacytoma KI81 proliferation. Plastic petri dish-bound MAb removed rmIL-6 activity. These results suggest that this MAb specifically binds IL-6 and neutralizes bioactivity of various PCT-GF, rmIL-6, and...

Background: Many clinical trials have documented the efficacy of rituximab in B-cell lymphomas. However, it remains unclear whether rituximab will improve long-term prognosis of the patients. This multicenter retrospective cohort study was conducted to evaluate the clinical impact of the drug in the treatment of B-cell lymphomas. Study design: We retrospectively analyzed clinical characteristics, 2-year progression-free survival(2y PFS) and 2-year overall survival(2y OS) of all B-cell lymphoma patients who were newly diagnosed and initially treated with either CHOP-like regimen alone (R(−) group) or in combination with rituximab (R(+) group) between 2000 and 2004 at our 20 hospitals belonging to the National Hospital Organization. Since rituximab was approved in 2002 for indolent B-cell lymphomas and in 2003 for aggressive B-cell lymphomas in Japan, the patients were almost automatically divided into the 2 groups dependent on the year they underwent treatment. Results: Of the 1,072 patients enrolled, 335 were given rituximab, while 737 did not receive it for the initial induction therapy. 2y PFS was 74.3% and 61.2% for R(+) group and R(−) group, respectively(P Conclusion: The addition of rituximab to the chemotherapy regimen significantly improves the clinical outcome in patients with previously untreated B-cell lymphoma regardless of the clinical aggressiveness.

Introduction: Interleukin 10 (IL-10) has been reported as an anti-inflammatory cytokine and B-cell proliferation factor, and has been implicated in autoimmunity, tunorigenesis and stem cell transplantation tolerance. IL-10 is also reported to be involved in multiple myeloma (MM) cell proliferation and survival. The polymorphism of position-592, -819, and -1082 in the promoter of IL-10 gene is a strong determinant of IL-10 expression. However, it is unclear whether IL-10 polymorphisms alter the incidence and clinical outcome of MM. We examined the single nucleotide polymorphism (SNPs) located within the promoter region of IL-10 genes in patients with MM, monoclonal gammopathy of undetermined significance (MGUS) and healthy controls in Japan. Methods: Seventy nine patients with MM [age range, 40–83 years; stage I (n=9), stage II (n=20), stage III (n=50); IgG(n=47), IgA(n=12), IgD(n=1), non-secretory (n=3), Bence Jones(n=16)], 46 patients with MGUS (age range, 44–86 years), and 200 healthy controls were included. Fifteen patients with transformation of MGUS to MM were included in MM group. Genotyping in IL-10 -1082G/A, -819C/T, -592A/C was determined by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Genotype and allele frequencies were compared between the study groups using χ2-test. The characteristics and laboratory features of MM patients with each IL-10 promoter polymorphism were compared using χ2-tests and student t-tests. Probability values Results: IL-10-592 A/A, A/C and C/C genotype frequencies were not significantly different between MM patients (51%, 34%, and 15%) and controls (42%, 43%, and 15%). Higher frequency of −592A/A genotype was seen in our Asian control compared with Caucasian. The frequency of IL-10 -1082 and -819 were also not significantly different between MM patients and healthy controls. The IL-10-592A/C genotype was detected in 10 of 16 patients (63%) with transformation of MGUS to MM and 20 of 46 patients (43%) with MGUS who did not progress to MM (odds ratio [OR], 2.2; 95% confidence interval [95% CI], 0.7–7.0; p=0.15). In MM patients who received autologous stem cell transplantation (ASCT), the overall survival was similar among 3 genotype groups. However, in patients who did not received ASCT, patients with IL-10-592A/C and C/C genotype had shorter overall survival compared with patients with IL-10-592A/A genotype (median survival, 32 months vs. not reached). Conclusion: It is reported that IL-10-592 A/C and C/C genotype are high producer of IL-10. Our results suggested that the IL-10-592 A/C and C/C genotype was not associated with the susceptibility to MM, however associated with poor prognosis in MM patients who did not receive ASCT. In addition, IL-10-592 A/C and C/C genotype may contribute to transformation of MGUS to MM. According these data, IL-10 may be implicated in the progression of MM and MGUS, and SNPs of IL-10 is one of prognostic factors in patients with MGUS and MM.

Background: Thalidomide is effective for treating refractory multiple myeloma; however, it induces many adverse events, including peripheral neuropathy, deep vein thrombosis, constipation, and neuropsychological symptoms. Hematological adverse events have rarely been reported in the United States and Europe. Some Japanese cases have been reported to reveal leucopenia during thalidomide therapy. We report here severe leucopenia, which has been observed in the JMSG phase II study “Low-dose thalidomide plus low-dose dexamethasone therapy for refractory myeloma”, and analyze the factors related to leucopenia. Patients and Methods: Patients with refractory myeloma were eligible. Thalidomide at 100 mg/day was administered on Days 1–7 and increased to 200 mg/day without severe adverse events, and continued until disease progression. Dexamethasone at 4 mg/day was administered on Days 1–28, decreased to 1 mg/day, and maintained at 1 mg/day. The response was assessed by the finding of a decline in paraprotein in serum or urine on two occasions at least 4 weeks apart. Results: Sixty-six patients (male/female ratio: 0.7; median age, 64.5 years; range, 40–74 years) were enrolled in the study. The myeloma subtypes were 48 IgG, 6 IgA, 1 IgD, and 11 light chain only. Forty-nine patients were refractory for conventional chemotherapy and 17 patients had relapsed disease after autologous stem cell transplantation. Responses were observed in 30 patients (4 near complete response, 9 partial response, and 17 minimal response) (45.5%). Median progression-free and overall survivals were 6.2 months and 23.3 months. The incidence rates of grade 2 or higher events were: peripheral neuropathy, 7.5%; hyperglycemia, 1.5%; skin rash, 4.5%; constipation, 4.5%; and deep vein thrombosis, 4.8%. Grade 1–2 incidence of leukopenia was 41% and of thrombocytopenia was 27%; Grade 3 or higher leucopenia was observed in 12%. One patient died from sepsis caused by severe leucopenia. Thalidomide-induced leucopenia was closely related to pre-treatment white blood cell count (p Conclusion: Low-dose thalidomide plus low-dose dexamethasone therapy was as effective as low-dose thalidomide plus high-dose dexamethasone therapy in patients with refractory multiple myeloma. The incidence of adverse events including deep vein thrombosis and hyperglycemia was lower than the data reported in the United States and Europe. However, leukopenia is one of the most serious adverse events in Japanese patients, especially in patients with low pretreatment white blood cell and platelet counts.

We describe a unique case of IgG lambda myeloma in which a plasma cell leukemia developed and the paraprotein changed from IgG lambda to lambda chain, the latter inducing fatal renal failure. In addition, J-chain was detected in the cytoplasm of the plasma cell leukemia cells.

Four patients with common acute lymphoblastic leukemia antigen (CALLA)-positive myeloma are presented. The subclasses of monoclonal protein were IgD kappa (1 case), IgA lambda (1 case), and IgA kappa (2 cases). Bence Jones proteinuria was seen in all cases. The clinical stages were determined as IIA (2 cases) and IIIA (2 cases). All patients died with a median survival time after diagnosis of 62 days due to rapid development of renal failure (3 cases), and renal insufficiency and pneumonia (1 case). According to light microscopic evaluation, these myelomas corresponded to plasmablastic (1 case), immature (2 cases), and intermediate (1 case) types. Both CALLA and a cytoplasmic immunoglobulin identical with the serum monoclonal protein were simultaneously detected in single cells from all cases using immunofluorescent double labeling. These findings suggest that CALLA-positive and plasma-blastic myelomas constitute clinically a subgroup characterized by extremely poor survival but they represent cytologically different subcategories.

The ultrastructure of multinucleated giant myeloma cells obtained from an IgA kappa myeloma patient was investigated. The characteristic findings were marked nucleocytoplasmic asynchrony, irregular nuclear indentation, branched mitochondria, and Golgi apparatus surrounded by nuclei. The patient died of renal failure only 6 months after diagnosis.