Efficacy and safety of rituximab in Japanese patients with relapsed chronic immune thrombocytopenia refractory to conventional therapy.

Primary immune thrombocytopenia (ITP) is an autoimmune disease mediated by the production of autoantibody against platelets. Rituximab, an anti-CD20 antibody, is reported to be useful for treatment of ITP. In Japan, however, robust evidence on this treatment has not been accumulated. Hence, we conducted this open-label phase III clinical trial to confirm the efficacy and safety of rituximab, administered at 375 mg/m² once per week at weekly intervals for 4 consecutive weeks in Japanese patients with chronic ITP, who had relapsed and were refractory to conventional therapy. The primary endpoint was defined as the percentage of patients with a platelet count above 50 × 109/L at week 24 after the first dose of rituximab, which was 30.8% of 26 patients (95% confidence interval 14.3-51.8%). Although the lower confidence limit of primary endpoint failed to meet the pre-specified threshold of 20%, the clinical efficacy of rituximab is substantial in consideration of the 2% response rate in the placebo arm in other clinical studies in patients with chronic ITP. We conclude that rituximab is clinically useful and safe in the treatment of Japanese patients with chronic ITP, achieving the goal of maintaining platelet count and reducing risk of bleeding while minimizing treatment-related toxicity.

Clinical features of de novo CD25(+) diffuse large B-cell lymphoma.

CD25 (interluekin-2 receptor) expression in diffuse large B-cell lymphoma (DLBCL) cells has been not examined. To characterize CD25(+) DLBCL, 123 patients, who were newly diagnosed with DLBCL, were analyzed by single-color flow cytometry (FCM). CD25-positivity was significantly higher in DLBCL patients (n = 123; mean ± SD, 27.8 ± 30.6%) than in those with reactive lymphadenopathy (n = 16; mean ± SD, 8.6 ± 4.3%) and follicular lymphoma (n = 60; mean ± SD, 12.7 ± 12.4%). By two-color FCM, CD25/CD19 or CD25/CD20 dual positivity in DLBCL patients was shown: mean ± SD, 63.7 ± 25.5% (n = 13) and 55.0 ± 28.1% (n = 14), respectively. Eighty-two percent of the patients with DLBCL received rituximab combined with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy. A cut-off value of 60% with CD25-positivity clearly divided patients with DLBCL into two groups: CD25-high or CD25-low DLBCL. Although clinical and immunophenotypic features were not significantly different in both groups, the former showed a significantly poorer response and more inferior progression-free survival than the latter. CD25 may be a new prognostic marker and could be a therapeutic target in DLBCL.

Clinical outcome of non-surgical treatment for primary small intestinal lymphoma diagnosed with double-balloon endoscopy.

Primary small intestinal lymphoma (PSIL) is often treated with surgical resection, and therefore response to non-surgical treatment is rarely known. We retrospectively analyzed the clinicopathological features of 19 patients with PSIL, who had been diagnosed by double-balloon endoscopy (DBE) and had not received surgical treatment. The immunohistological phenotypes of 18 patients were B-cell lymphomas. Five patients had tumors within the jejunum, nine within the ileum and five in multiple sites including the duodenum. Most cases were in the low or low-intermediate risk group of the International Prognostic Index score. Seventeen patients received chemotherapy, with an overall response rate of 82.4%. The estimated overall survival at 5 years was 72.2%. Response to initial chemotherapy and levels of hemoglobin (Hb) and albumin (Alb) were identified as favorable prognostic indicators. We conclude that PSIL can be effectively diagnosed by DBE and shows a good prognosis with chemotherapy alone.

Flow cytometric analysis of kappa and lambda light chain expression in endoscopic biopsy specimens before the diagnosis of B-cell lymphoma.

Forty-eight patients with gastrointestinal (GI) tract B-cell lymphoma (BCL) were analyzed retrospectively. The diagnosis was based on the histological examination of specimens obtained by endoscopic biopsy. Before the diagnosis was made, single-color flow cytometry was performed to analyze the expression of light chains and B-cell antigens including CD10 in the specimens. Restricted light chain (RLC) expression, a marker of B-cell clonality, was defined as κ and λ ratios of either more than 3.0 or less than 0.5. The specimens from 30 patients (62.5%) showed RLC expression. No RLC expression or RLC expression not examined was divided into two groups : those showing CD10 positivity in more than 20% of cells (4 patients, 8.3%) and those showing no positivity (14 patients, 29.2%). The cell number analyzed in the latter group was significantly smaller than that in the other two groups. Abnormal karyotypes were found in the specimens from 8 patients (16.7%). These results indicate that the flow cytometric analysis of endoscopic biopsy specimens is useful when BCL is suspected if an adequate number of cells are obtained.

Prediction of progression from refractory cytopenia with unilineage dysplasia by analysis of bone marrow blast cell composition.

A retrospective analysis of 71 patients newly diagnosed with refractory cytopenia with unilineage dysplasia (RCUD) revealed that 12 developed refractory anemia with an excess of blasts or acute myeloblastic leukemia. Before the diagnosis of RCUD was made, phenotypes of cells in the bone marrow (BM) blast region were analyzed using flow cytometry. Patients with RCUD were divided into two groups ; those with no progression (Group A) and those with disease progression later on (Group B). The cell composition in the BM blast region differed significantly between the groups : Group A showed higher percentages of B lymphoid cells but lower percentages of myeloid cells. A cut-off value of 20 for the CD33/CD10 ratio in the BM blast region clearly separated Group A from Group B. These results suggest that cell composition in the BM blast region evaluated by flow cytometry may indicate the progression of RCUD.

Transforming growth factor-ß-stimulated clone-22 is a negative-feedback regulator of Ras / Raf signaling: Implications for tumorigenesis.

Transforming growth factor-ß (TGF-ß)-stimulated clone-22 (TSC-22), also called TSC22D1-2, is a putative tumor suppressor. We previously identified TSC-22 downstream of an active mutant of fms-like tyrosine kinase-3 (Flt3). Here, we show that TSC-22 works as a tumor suppressor through inhibiting Ras/Raf signaling. Notably, TSC-22 was upregulated by Ras/Raf activation, whereas its upregulation was inhibited by concurrent STAT5 activation. Although TSC-22 was normally retained in the cytoplasm by its nuclear export signal (NES), Ras/Raf activation caused nuclear translocation of TSC-22, but not TSC22D1-1. Unlike glucocorticoid-induced leucine zipper (GILZ/TSC22D3-2) previously characterized as a negative regulator of Ras/Raf signaling, TSC-22 failed to interact physically with Ras/Raf. Importantly, transduction with TSC-22, but not TSC22D1-1, suppressed the growth, transformation and tumorigenesis of NIH3T3 cells expressing oncogenic H-Ras: this suppression was enhanced by transduction with a TSC-22 mutant lacking NES that had accumulated in the nucleus. Collectively, upregulation and nuclear translocation of TSC-22 played an important role in the feedback suppression of Ras/Raf signaling. Consistently, TSC22D1-deficient mice were susceptible to tumorigenesis in a mouse model of chemically-induced liver tumors bearing active mutations of Ras/Raf. Thus, TSC-22 negatively regulated Ras/Raf signaling through a mechanism different from GILZ, implicating TSC-22 as a novel suppressor of oncogenic Ras/Raf-induced tumors.

Rituximab plus 70% cyclophosphamide, doxorubicin, vincristine and prednisone for Japanese patients with diffuse large B-cell lymphoma aged 70 years and older.

In the rituximab era, several large studies have suggested that full-dose rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) might be the best treatment for patients with diffuse large B-cell lymphoma (DLBCL) aged 60 years and older. However, it remains unclear whether this is also the case for those aged 70 years and older. Previously untreated patients with DLBCL aged 70 years and older (elderly) were treated with R-70%CHOP, and patients younger than 70 years (younger) were treated with full-dose R-CHOP every 3 weeks, for a total of 6-8 cycles. Complete remission (CR) rates in elderly versus younger patients were 75 vs. 78% (p = 0.7), respectively. The 3-year overall survival, event-free survival and progression-free survival of elderly versus younger patients were 58 vs. 78% (p < 0.05), 45 vs. 70% (p < 0.05) and 64 vs. 72% (p = 0.43), respectively. Severe adverse events were more frequent in the elderly, even with the dose reduction in that age group. Three-year PFS with R-70%CHOP for patients aged 70 years and older was not significantly worse than that with full-dose R-CHOP for younger patients, suggesting that R-70% CHOP might be a reasonable choice for patients with DLBCL aged 70 years and older, especially for those with comorbidities.

Regulatory T cells and IL-17-producing cells in graft-versus-host disease.

Graft-versus-host disease (GvHD), a major complication following allogeneic hematopoietic stem cell transplantation, is mediated by donor-derived T cells. On activation with alloantigens expressed on host antigen-presenting cells, naive CD4(+) T cells differentiate into T-helper cell subsets of effector T cells expressing distinct sets of transcriptional factors and cytokines. Classically, acute GvHD was suggested to be predominantly related to Th1 responses. However, we now face a completely different and complex scenario involving possible roles of newly identified Th17 cells as well as Tregs in GvHD. Accumulating data from experimental and clinical studies suggest that the fine balance between Th1, Th2, Th17 and Tregs after transplantation may be an important determinant of the severity, manifestation and tissue distribution of GvHD. Understanding the dynamic process of reciprocal differentiation of regulatory and T-helper cell subsets as well as their interactions will be important in establishing novel strategies for preventing and treating GvHD.

Use of cryoprotectant-depleted allogeneic peripheral blood stem cells for transplantation.

Transplantation with cryopreserved allogeneic peripheral blood stem cells (PBSCs) from related donors is widely conducted in Japan. To freeze PBSCs, a solution containing dimethyl sulfoxide (DMSO), which can have various adverse effects, is added. DMSO-depleted allogeneic PBSCs were transplanted into 21 patients. The cryoprotectant was manually removed from thawed PBSCs and the cells were mixed with a solution containing citrate dextrose as an anticoagulant and RPMI-1640 medium. DMSO-depleted PBSCs were immediately infused into patients subjected to conditioning. Infusion-related adverse effects were only observed in three patients. The median neutrophil recovery (⩾0·5×10(9)/l) and platelet recovery (⩾20×10(9)/l) were 13·0 and 14·0 days, respectively. Only one patient with mixed-lineage leukemia in non-complete remission did not show engraftment, likely due to a second transplantation and a two-antigen disparity in human leukocyte antigen system A. The results suggest the removal of DMSO from thawed PBSCs to be safe and useful for transplantation.

Mesenchymal stromal cells inhibit Th17 but not regulatory T-cell differentiation.

BACKGROUND AIMS: A previous study has demonstrated that mouse mesenchymal stromal cells (MSC) produce nitric oxide (NO), which suppresses signal transducer and activator of transcription (STAT) 5 phosphorylation and T-cell proliferation under neutral and T helper 1 cells (Th1) conditions. We aimed to determine the effects of MSC on T helper 17 cells (Th17) and regulatory T-cell (T-reg) differentiation. METHODS: CD4 T cells obtained from mouse spleen were cultured in conditions for Th17 or Treg differentiation with or without mouse MSC. Th17 and Treg differentiation was assessed by flow cytometry using antibodies against interleukin (IL)-17 and forkhead box P3 (Foxp3), a master regulator of Treg cells. RESULTS: MSC inhibited Th17 but not Treg differentiation. Under Th17 conditions, MSC did not produce NO, and inhibitors of indoleamine-2,3-dioxygenase (IDO) and prostaglandin E(2) (PGE2) both restored MSC suppression of differentiation, suggesting that MSC suppress Th17 differentiation at least in part through PGE2 and IDO. CONCLUSIONS: Our results suggest that MSC regulate CD4 differentiation through different mechanisms depending on the culture conditions.

Mesenchymal stromal cells for graft-versus-host disease : basic aspects and clinical outcomes.

Mesenchymal stromal cells (MSCs) have unique characteristics such immune suppression by inhibiting T cell proliferation, tissue-repair ability and acceleration of hemopoietic stem cell engraftment. The cells are rare in bone marrow, but easily cultured under standard culture conditions. Soluble factors and cells are implicated in the MSC-mediated T cell suppression and numerous clinical trials using MSCs to prevent and treat graft-versus-host disease (GVHD) have been reported. MSCs are suggested to suppress acute GVHD without impairing graft-versus-leukemia effects and increasing systemic infections. In this review, we focus on basic aspects of MSC-mediated T cell suppression and clinical trials using MSCs for GVHD and related conditions.

Incidental carcinomas detected by PET/CT scans in patients with malignant lymphoma.

According to the international working group response criteria for malignant lymphoma revised in 2007, 18F-fluorodeoxyglucose positron emission tomography (¹8FDG-PET) combined with or without computed tomography (CT) is recommended for pre-treatment staging and response assessment among patients with diffuse large B-cell lymphoma and Hodgkin lymphoma. Recently, along with the widespread use of PET/CT, unexpected uptake and accumulation of ¹8FDG has been reported. Discussed in the present report are patients with malignant lymphoma and second primary carcinomas that were incidentally found by PET/CT. A total of 497 consecutive PET/CT were performed on 290 patients with malignant lymphoma in our institution from April 2008 through March 2010. Eight patients (2.8%) had pathologically confirmed second primary carcinomas consisting of 4 colon cancers, 3 lung cancers, and 1 pancreatic cancer. Two cases were diagnosed at the initial staging, and the others were detected after treatment for lymphoma. It is noteworthy that PET revealed high accumulations of ¹8FDG in 5 (62.5%) of the 8 patients without corresponding tumors in conventional CT. All of the 4 patients with colon carcinoma underwent curative surgery. The present study suggests that incidental findings by PET in malignant lymphoma can lead to early detection and successful treatment of second malignancies.

Altered effector CD4+ T cell function in IL-21R-/- CD4+ T cell-mediated graft-versus-host disease.

We previously showed that transplantation with IL-21R gene-deficient splenocytes resulted in less severe graft-versus-host disease (GVHD) than was observed with wild type splenocytes. In this study, we sought to find mechanism(s) explaining this observation. Recipients of donor CD4(+) T cells lacking IL-21R exhibited diminished GVHD symptoms, with reduced inflammatory cell infiltration into the liver and intestine, leading to prolonged survival. After transplantation, CD4(+) T cell numbers in the spleen were reduced, and MLR and cytokine production by CD4(+) T cells were impaired. These results suggest that IL-21 might promote GVHD through enhanced production of effector CD4(+) T cells. Moreover, we found that CD25 depletion altered neither the impaired MLR in vitro nor the ameliorated GVHD symptoms in vivo. Thus, the attenuated GVHD might be caused by an impairment of effector T cell differentiation itself, rather than by an increase in regulatory T cells and suppression of effector T cells.

Predictive factors of response and survival following chemotherapy treatment in acute myeloid leukemia progression from myelodysplastic syndrome.

OBJECTIVE: The progression of myelodysplastic syndrome to acute myeloid leukemia (MDS/AML) is generally incurable and its prognosis is extremely poor. It is important to determine the predictive factors of response and survival in diseases treated with chemotherapy. METHODS: Twenty-nine patients who had been diagnosed of MDS/AML and had undergone chemotherapy between April 2001 and March 2008 were retrospectively analyzed. RESULTS: Of the 29 patients, 21 patients had an abnormal karyotype. Among them, 13 had complex type abnormalities and/or monosomy 7. Twenty-four patients were administered a low-dose AraC containing regimen and 5 received an AML-like regimen as the initial chemotherapy. The responses were CR4/PR2/NR23. The response rate (RR) in the patients with a normal karyotype was significantly better than in those with an abnormal karyotype (62.5% vs. 4.8%, p=0.003). Univariate analyses showed that the hemoglobin level and cytogenetic abnormalities were factors that contributed to the overall survival. CONCLUSION: In MDS/AML, patients with a normal karyotype tended to have a better response to chemotherapy. The hemoglobin level and cytogenetic abnormalities were significant factors affecting the overall survival.

Correlation between flow cytometric identification of CD33-positive cells and morphological evaluation of myeloblasts in bone marrow of patients with acute myeloblastic leukemia.

Determination of the percentage of myeloblasts in bone marrow is important for the evaluation of acute myeloblastic leukemia (AML) and related disorders. Using flow cytometry with a CD45-blast gate (FCM/CD45), 226 bone marrow aspiration samples serially collected from 71 patients with de novo AML were analyzed. Bone marrow smears were evaluated independently by pathologists who did not know the corresponding flow cytometric data in advance. Patients received remission induction followed by consolidation. The CD33+ cell percentages evaluated by FCM/CD45 were strongly correlated to the myeloblast percentages determined by microscopic examination (r=0.8360, p<0.001). When only samples containing leukemic cells demonstrated by chromosomal or fluorescence in situ hybridization (FISH) analysis after induction were evaluated, positive correlations were found between CD33+ cell percentages determined by FCM/CD45 and myeloblast percentages determined by morphology (r=0.672, p<0.001). The identification of CD33+ cells by FCM/CD45 is useful for the evaluation of bone marrow myeloblasts in AML.

Cell and gene therapy using mesenchymal stem cells (MSCs).

Mesenchymal stem cells (MSCs) are considered to be a promising platform for cell and gene therapy for a variety of diseases. First, in the field of hematopoietic stem cell transplantation, there are two applications of MSCs: 1) the improvement of stem cell engrafting and the acceleration of hematopoietic reconstitution based on the hematopoiesis-supporting ability; and 2) the treatment of severe graft-versus-host disease (GVHD) based on the immunomodulatory ability. Regarding the immunosuppressive ability, we found that nitric oxide (NO) is involved in the MSC-mediated suppression of T cell proliferation. Second, tumor-bearing nude mice were injected with luciferase-expressing MSCs. An in vivo imaging analysis showed the significant accumulation of the MSCs at the site of tumors. The findings suggest that MSCs can be utilized to target metastatic tumors and to deliver anti-cancer molecules locally. As the third application, MSCs may be utilized as a cellular vehicle for protein-supplement gene therapy. When long-term transgene expression is needed, a therapeutic gene should be introduced with a minimal risk of insertional mutagenesis. To this end, site-specific integration into the AAVS1 locus on the chromosome 19 (19q13.4) by using the integration machinery of adeno-associated virus (AAV) would be particularly valuable. There will be wide-ranging applications of MSCs to frontier medical treatments in the near future.