Clinical significance of sIL-2R levels in B-cell lymphomas.

Elevated soluble interleukin-2 receptor (sIL-2R) in sera is observed in patients with malignant lymphoma (ML). Therefore, sIL-2R is commonly used as a diagnostic and prognostic marker for ML, but the mechanisms responsible for the increase in sIL-2R levels in patients with B-cell lymphomas have not yet been elucidated. We first hypothesized that lymphoma cells expressing IL-2R and some proteinases such as matrix metalloproteinases (MMPs) in the tumor microenvironment can give rise to increased sIL-2R in sera. However, flow cytometric studies revealed that few lymphoma cells expressed IL-2R α chain (CD25) in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and most CD25-expressing cells in the tumor were T-cells. Distinct correlations between CD25 expression on B-lymphoma cells and sIL-2R levels were not observed. We then confirmed that MMP-9 plays an important role in producing sIL-2R in functional studies. Immunohistochemical (IHC) analysis also revealed that MMP-9 is mainly derived from tumor-associated macrophages (TAMs). We therefore evaluated the number of CD68 and CD163 positive macrophages in the tumor microenvironment using IHC analysis. A positive correlation between the levels of sIL-2R in sera and the numbers of CD68 positive macrophages in the tumor microenvironment was confirmed in FL and extranodal DLBCL. These results may be useful in understanding the pathophysiology of B-cell lymphomas.

Oral eltrombopag for up to three years is safe and well-tolerated in Japanese patients with previously treated chronic immune thrombocytopenia: an open-label, extension study.

Eltrombopag is an oral, nonpeptide, thrombopoietin receptor agonist approved for treatment of chronic immune thrombocytopenia (ITP). The safety, tolerability, and efficacy of eltrombopag for up to 3 years were evaluated in 19 Japanese patients with chronic ITP who had completed a prior 6-month study. Patients received eltrombopag once daily at the last dosage received in the prior study (12.5, 25, or 50 mg). Dose adjustments and treatment interruptions were permitted to maintain platelet counts of 50,000-200,000/µL. Primary evaluations were safety and tolerability of long-term eltrombopag treatment. The median duration of exposure was 27.5 months (range, 9.9-32.3). Adverse events were similar to those reported with short-term use of eltrombopag, and none led to treatment discontinuation. Nine serious adverse events were reported. Median platelet counts began to increase after 1 week of treatment and remained above 50,000/µL for most assessments. Bleeding episodes decreased from 63 % at baseline to 21 % after 2 weeks of treatment and remained below baseline for all assessments. Of 15 patients receiving concomitant baseline ITP medications, 10 permanently discontinued or achieved a sustained reduction of at least one treatment without requiring rescue treatment. Long-term treatment with eltrombopag was safe, well tolerated, and effective in Japanese patients with chronic ITP.

The incidence of leukemia, lymphoma and multiple myeloma among atomic bomb survivors: 1950-2001.

A marked increase in leukemia risks was the first and most striking late effect of radiation exposure seen among the Hiroshima and Nagasaki atomic bomb survivors. This article presents analyses of radiation effects on leukemia, lymphoma and multiple myeloma incidence in the Life Span Study cohort of atomic bomb survivors updated 14 years since the last comprehensive report on these malignancies. These analyses make use of tumor- and leukemia-registry based incidence data on 113,011 cohort members with 3.6 million person-years of follow-up from late 1950 through the end of 2001. In addition to a detailed analysis of the excess risk for all leukemias other than chronic lymphocytic leukemia or adult T-cell leukemia (neither of which appear to be radiation-related), we present results for the major hematopoietic malignancy types: acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, adult T-cell leukemia, Hodgkin and non-Hodgkin lymphoma and multiple myeloma. Poisson regression methods were used to characterize the shape of the radiation dose-response relationship and, to the extent the data allowed, to investigate variation in the excess risks with gender, attained age, exposure age and time since exposure. In contrast to the previous report that focused on describing excess absolute rates, we considered both excess absolute rate (EAR) and excess relative risk (ERR) models and found that ERR models can often provide equivalent and sometimes more parsimonious descriptions of the excess risk than EAR models. The leukemia results indicated that there was a nonlinear dose response for leukemias other than chronic lymphocytic leukemia or adult T-cell leukemia, which varied markedly with time and age at exposure, with much of the evidence for this nonlinearity arising from the acute myeloid leukemia risks. Although the leukemia excess risks generally declined with attained age or time since exposure, there was evidence that the radiation-associated excess leukemia risks, especially for acute myeloid leukemia, had persisted throughout the follow-up period out to 55 years after the bombings. As in earlier analyses, there was a weak suggestion of a radiation dose response for non-Hodgkin lymphoma among men, with no indication of such an effect among women. There was no evidence of radiation-associated excess risks for either Hodgkin lymphoma or multiple myeloma.

[Radiation associated leukemia and myelodysplastic syndrome].

Leukemias including acute myeloid leukemia (AML), acute lymphocytic leukemia, and chronic myeloid leukemia as well as myelodysplastic syndrome (MDS), male non-Hodgkin lymphoma and MGUS are statistically significant radiation-associated hematopoietic neoplasms. Recently, MDS has been confirmed to increase among atomic bomb survivors. AML/RUNX1 is a critical transcription factor of differentiation and proliferation of hematopoietic stem cells. AML1 point mutations, especially N-terminal RUNT domain in-frame type, are frequently detected in radiaton-associated and therapy-related (rad-t-) MDS/AML. In addition, the point mutations, are frequently associated with additional mutations in receptor tyrosine kinase (RTK)-RAS pathway, including FLT3, N-RAS, SHP2 and NF1. The combination of AML1/RUNX1 mutation and RTK-RAS pathway mutation in hematopoietic stem cells is considered responsible for the oncogenesis of rad-t- MDS/AML.

[Successful treatment with bortezomib for a patient with plasma cell leukemia accompanied by severe hyperbilirubinemia].

A 59-year-old woman was admitted to our hospital with jaundice, renal dysfunction, anemia and hypercalcemia. Primary plasma cell leukemia (PCL) was diagnosed based on findings of IgA-λ type M-protein, 22% plasma cells in the bone marrow and 23.1% plasma cells of WBC in the peripheral blood. Because the total bilirubin (T.Bil) level increased even after the administration of prednisolone (PSL), dexamethasone and methylprednisolone, the patient was started on bortezomib (0.7 mg/m(2) on days 1, 4, 8 and 11 for 3 weeks) combined with PSL (40 mg/day). The level of T.Bil decreased and the patient's condition remarkably improved. We then increased the dose of bortezomib to 1.0 mg/m(2) in the second course, but discontinued treatment just after starting the third course because NCI-CTCAE Grade 3 peripheral neuropathy developed. According to the criteria of the International Myeloma Working Group, the response category was VGPR (=very good partial response) at 1 month after pausing treatment. We recommend these novel agents for PCL, which is an aggressive form of extramedullary plasma cell cancer.

Overexpression of BMI-1 correlates with drug resistance in B-cell lymphoma cells through the stabilization of survivin expression.

The expression of BMI-1 is correlated with disease progression in cancer patients. We showed that ectopic expression of BMI-1 in B-cell lymphoma cell lines, HT and RL, conferred resistance to etoposide and oxaliplatin, known to enhance sensitivity by targeting the survivin gene, but not to irinotecan, which is not relevant to the downregulation of survivin expression. The expression of survivin was not only augmented in cells transduced with BMI-1, but persisted in the presence of etoposide in cells overexpressing BMI-1. By contrast, the mock-transduced cells succumbed in the medium with anticancer drugs, with an accompanying decrease in BMI-1 and survivin expression. BMI-1 overexpression stabilized survivin post-translationally without an accompanying rise in the mRNA, suggesting survivin as a potential target for BMI-1. Knockdown of either BMI-1 or survivin restored sensitivity to etoposide in the BMI-1-overexpressing lymphoma cells. An analysis of six patients with B-cell lymphoma showed that in the drug-resistant patients, levels of BMI-1 and survivin were maintained even after drug administration. However, downregulation of both BMI-1 and survivin expression was observed in the drug-sensitive patients. Therefore, BMI-1 might facilitate drug resistance in B-cell lymphoma cells through the regulation of survivin. BMI-1 could be an important prognostic marker as well as a future therapeutic target in the treatment of drug-resistant lymphomas.

Morphologic analysis in myelodysplastic syndromes with del(5q) treated with lenalidomide. A Japanese multiinstitutional study.

Lenalidomide is known to be effective in myelodysplastic syndromes (MDS) with del(5q) in improving anemia and suppressing del(5q) cells. MDS with del(5q) shows increase of nonlobulated megakaryocytes. However, histopathology of MDS with del(5q) treated with lenalidomide has not been fully studied. We investigated the morphologic changes in lenalidomide treated low- or intermediate-1-risk MDS with del(5q). All of evaluable patients showed high proportion of nonlobulated megakaryocytes. The nonlobulated megakaryocytes were markedly decreased in 6 patients during therapy in parallel with suppression of del(5q) cells. Our analysis suggests that single allele deletion of common deleted region inhibits nuclear lobulation of megakaryocytes.

Good's syndrome-associated pure red cell aplasia with myelodysplastic syndrome.

We report a case of Good's syndrome-associated pure red cell aplasia (PRCA) with myelodysplastic syndrome (MDS). In this case, effector memory T (T(EM)) cells were expanded in the bone marrow. It remains uncertain whether the development of MDS was caused by the basic marrow defects or radiation therapy. However, since CD8(+) perforin(+) T(EM) cells expanded in the bone marrow, as was previously described for 3 of our patients with thymoma-associated PRCA, it is highly possible that the pathogenic mechanism of PRCA that is accompanied by thymoma is related to the expanded CD8(+) perforin(+) T(EM) cells in this MDS-complicated case.

[Successful treatment with low-dose dasatinib in a patient with chronic eosinophilic leukemia intolerant to imatinib].

A 77-year-old man with cough and dyspnea was admitted to hospital. Chest X-ray demonstrated reticulated shadows in the bilateral inferior lung fields and marked eosinophilia was detected in peripheral blood. Although he received steroid pulse therapy, eosinophilia became more serious and he was referred to our hospital. Bone marrow examination demonstrated a hypercellular marrow that consisted predominantly of dysplastic eosinophils with differentiation. FISH analysis of bone marrow cells demonstrated 4q12 deletion and RT-PCR analysis detected FIP1L1-PDGFRA fusion gene, leading to the diagnosis of chronic eosinophilic leukemia (CEL). Treatment with low-dose imatinib was immediately initiated; however, drug-induced systemic edema was progressive and became intolerable. Therefore, we changed imatinib to low-dose dasatinib (20 mg/day), resulting in complete molecular response of CEL after 3 months without any severe adverse effects. This is the first report on the efficacy of low-dose dasatinib for the treatment of CEL. The peak level (Cmax) of dasatinib in this patient was 55.3 nM, which exceeded the concentration of dasatinib required to inhibit cells with FIP1L1-PDGFRA by 50%. Thus, low-dose dasatinib with therapeutic drug monitoring can be a useful therapy for imatinib-intolerant CEL even in elderly patients.

Establishment of an HS23 stromal cell-dependent myeloma cell line: fibronectin and IL-6 are critical.

A multiple myeloma (MM) cell line, MSG1, which depends on HS23 stromal cells for its survival, was established from the pleural effusion of a patient with MM who expressed the M-protein of IgA-λ in his serum. During the first 2 months of culture, the myeloma cells survived on adhesive cells from the pleural effusion and, subsequently, they continued to proliferate on HS23 stromal cells. The phenotype of the established MSG1 cell line was: CD138(+), CD38(++), CD19⁻, CD56⁻, VLA-4(+), VEGFR1(+) and VEGFR2(+). Immunohistochemical staining also demonstrated expression of the IgA and λ chain in MSG1 cytoplasm. Karyotype analysis indicated complex chromosomal abnormalities; hypertriploidy, including the deletion of chromosomes 13 and 17, and c-myc translocation. MSG1 cells continued to proliferate, not only when co-cultured with HS23 cells, but also when cultured only on fibronectin-coated plates with the supernatant of HS23 cells or with control medium containing IL-6. Tocilizumab, an anti-IL-6 receptor antibody, inhibited MSG1 survival under these conditions. Therefore, MSG1 may be a unique myeloma cell line that is useful for the study of cell adhesion-mediated drug resistance induced by adhesion molecules and IL-6 stimulation of myeloma cells.

Roles of Src-like adaptor protein 2 (SLAP-2) in GPVI-mediated platelet activation SLAP-2 and GPVI signaling.

BACKGROUND: Glycoprotein VI (GPVI) /Fc receptor gamma (FcRγ)-chain complex is one of the collagen receptors in platelets and responsible for the majority of the intracellular signaling events through a similar pathway to immune receptors. Src-like adaptor protein 2 (SLAP-2) is a recently characterized adaptor protein predominantly expressed in hematopoietic cells. In T cells, SLAP-2 was reported to associate with several tyrosine phosphorylated proteins, and function as a negative regulator of signaling downstream of T cell antigen receptor by virtue of its interaction with the ubiquitin ligase c-Cbl. But the data regarding the presence and role of SLAP-2 proteins in platelets is limited. OBJECTIVES: We describe the characterization of SLAP-2 in human platelets. METHODS: Human platelets were analyzed by Western blot analysis, immunoprecipitation, and pull down assay, etc. RESULTS: Immunoprecipitation revealed the presence of two forms of SLAP-2 with approximately 28 kD and 25 kD, and following stimulation of GPVI, the additional form with approximately 32 kD apppeared. We have found that upon GPVI activation, SLAP-2 translocated from the Triton X-100-soluble fraction to the Triton X-100-insoluble cytoskeleton fraction, with concomitant association with Syk, c-Cbl, and LAT. CONCLUSIONS: SLAP-2 appears to play a role in regulating signaling pathways by bringing important signaling molecules such as c-Cbl and Syk into proximity of cytoskeletal substrates. In platelets, SLAP-2 may have function as a negative regulator of GPVI-mediated signaling by interacting with c-Cbl, being similar to that reported in T cells.

Clinical and genetic features of therapy-related myeloid neoplasms after chemotherapy for acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a highly curable disease with excellent complete remission and long-term survival rates. However, the development of therapy-related myeloid neoplasms (t-MN) is being reported with increasing frequency in patients successfully treated for APL. We attempted to clarify the different clinical features and hematologic findings between t-MN and relapse cases, and to identify gene alterations involved in t-MN. We compared 10 relapse and 11 t-MN cases that developed in 108 patients during their first complete remission from APL. At APL diagnosis, t-MN patients had lower white blood cell counts than did relapse patients (P = .048). Overall survival starting from chemotherapy was significantly worse in t-MN patients than in relapse patients (P = .022). The t-MN cases were characterized as CD34(+)/HLA-DR(+) and PML-RARA(-), and 4 RUNX1/AML1 mutations were detected. T-MN is easily distinguished from APL relapse by evaluating these hematologic features, and it may originate from primitive myeloid cells by chemotherapy-induced RUNX1 mutations.

Synergistic and persistent effect of T-cell immunotherapy with anti-CD19 or anti-CD38 chimeric receptor in conjunction with rituximab on B-cell non-Hodgkin lymphoma.

Using artificial receptors, it is possible to redirect the specificity of immune cells to tumour-associated antigens, which is expected to provide a useful strategy for cancer immunotherapy. Given that B-cell non-Hodgkin lymphoma (B-NHL) cells invariably express CD19 and CD38, these antigens may be suitable molecular candidates for such immunotherapy. We transduced human peripheral T cells or a T-cell line with either anti-CD19-chimeric receptor (CAR) or anti-CD38-CAR, which contained an anti-CD19 or anti-CD38 antibody-derived single-chain variable domain respectively. Retroviral transduction led to anti-CD19-CAR or anti-CD38-CAR expression in T cells with high efficiency (>60%). The T cell line, Hut78, when transduced with anti-CD19-CAR or anti-CD38-CAR, exerted strong cytotoxicity against the B-NHL cell lines, HT and RL, and lymphoma cells isolated from patients. Interestingly, use of both CARs had an additive cytotoxic effect on HT cells in vitro. In conjunction with rituximab, human peripheral T cells expressing either anti-CD19-CAR or anti-CD38-CAR enhanced cytotoxicity against HT-luciferase cells in xenografted mice. Moreover, the synergistic tumour-suppressing activity was persistent in vivo for over 2 months. These results provide a powerful rationale for clinical testing of the combination of rituximab with autologous T cells carrying either CAR on aggressive or relapsed B-NHLs.

Pure red cell aplasia associated with imatinib-treated FIP1L1-PDGFRA positive chronic eosinophilic leukemia.

A 28-year-old man with marked eosinophilia is described. FIP1L1/PDGFRA mRNA showed multiple alternatively-spliced fusion transcripts. Sequencing analysis showed that the deduced DNA breakpoints were intron 10 in the FIP1L1 gene and exon 12 in the PDGFRA gene. Then, a diagnosis of chronic eosinophilic leukemia (CEL) was made. Whereas the response to the treatments with prednisolone and hydroxyurea were unsatisfactory, treatment with imatinib showed a rapid decrease of eosinophils. The hemoglobin level also dropped and bone marrow examination showed pure red cell aplasia. Continued administration of very low dose imatinib (100 mg every 5 days) led to and maintained complete molecular remission, with good tolerability.