Search

Objectives C-C chemokine receptor type 4 (CCR4) proteins are expressed on the neoplastic cells of adult T-cell leukemia/lymphoma (ATLL). As the mutation status of CCR4 gene is reported to correlate with significant clinical information such as prognosis and response to mogamulizumab, we aimed to establish a screening method that is suitable for clinical laboratory tests. Methods In 34 patients with ATLL, CCR4 mutation analysis, high-resolution melting (HRM) analysis, fragment analysis, and direct sequencing were performed using both genomic DNA and complementary DNA (cDNA). Furthermore, 38 cases of asymptomatic carriers of human T-cell leukemia virus type 1 (HTLV-1) were screened for CCR4 mutation. Results Mutation analysis by direct sequencing of 34 ATLL clinical samples detected CCR4 mutation in four genomic DNA samples and seven cDNA samples, and two novel mutations were identified. All CCR4 mutations detected by direct sequencing were positive for HRM analysis and/or fragment analysis. CCR4 mutation was not detected in the asymptomatic carriers of HTLV-1. Conclusions CCR4 mutation screening by a combination of HRM and fragment analysis using cDNA is a simple and practical method, and it will contribute to better decision making for a therapeutic strategy, providing a rapid CCR4 mutational status to clinicians.

The development of effective therapies has enabled long-term survival for many patients with multiple myeloma (MM). However, the administration of antibody drugs, such as daratumumab, which bind to plasma cell (PC) surface proteins, may prevent PC detection by flow cytometry. We propose VS38 as an alternative antibody for CD38. VS38 recognizes cytoskeleton-linking membrane protein 63 (CLIMP-63) on the rough endoplasmic reticulum, and this protein may be expressed in secretory cells. We investigated VS38 staining in normal hematopoietic cells from five control samples, as well as PCs from 21 patients with plasma cell disorder (PCD). In normal hematopoietic cells, although VS38-stained monocytes, myeloid cells, and a subpopulation of B cells, PCs were significantly and brightly stained by VS38. There was no significant difference in VS38 staining between normal and abnormal PCs obtained from five patients with monoclonal gammopathy of undetermined significance. Furthermore, PCs in 21 PCD cases were clearly identified by VS38 in all cases, in contrast to CD38, even in daratumumab-administered patients whose CD38 epitopes on PCs were masked. These results suggest that the use of the VS38 antibody in flow cytometry contributes to PC detection, independent of therapeutic treatment.

Background Multi-parametric flow cytometry (MFC) is a helpful tool for detecting neoplastic cells in malignant lymphoma; however, lymphoma cells can be difficult to detect when characteristic immunophenotypic abnormalities are not evident. We evaluated the stainability of VS38, which is used for multiple myeloma, in normal and abnormal B cells using MFC to develop a new strategy for detecting lymphoma cells. Methods We compared the median fluorescence intensity of VS38 staining in lymphocytes from patients without hematopoietic neoplasms and in B cells from 26 patients with B cell lymphoma (BCL). To evaluate the performance of VS38 gating, we compared VS38-positive B cells with the percentages of BCL cells, and with the mutation ratios of MYD88 L265P measured by droplet digital PCR in patients with lymphoplasmacytic lymphoma (LPL)/Waldenstrom macroglobulinemia (WM). Results CD27-positive memory B cells were stained with VS38, whereas normal lymphocytes were faintly stained. Lymphoma cells were stained with VS38 in 11 of 12 patients with LPL/WM, 3 of 3 with chronic lymphocytic leukemia, 3 of 5 with mantle cell lymphoma, 2 of 4 with follicular lymphoma, and 1 of 1 with splenic marginal zone lymphoma. The percentages of VS38-positive B cells in VS38-positive BCL were equivalent to those of lymphoma cells and the mutation ratios of MYD88 L265P in LPL/WM. Conclusions VS38 identified neoplastic cells in plasma cell disorders and BCL. This might improve the accuracy of BCL diagnosis, especially in patients with LPL/WM.

Introduction Acute myeloid leukemia (AML) is a heterogeneous disease associated with various genetic abnormalities. Somatic mutations in nucleophosmin 1 (NPM1), fms-related tyrosine kinase 3 (FLT3), and DNA methyltransferase 3 alpha (DNMT3A) are the most frequent mutations associated with AML. However, because DNMT3A mutations are broadly distributed, they are challenging to analyze in routine laboratory tests. Hence, we developed a rapid screening method for DNMT3A mutations by high-resolution melting (HRM) analysis for clinical use at the point of AML diagnosis. Methods The detection limit for DNMT3A mutations from exons 8-23 by an HRM analysis was investigated using plasmid mixtures. In 69 patients with AML, somatic mutations in NPM1, FLT3-internal tandem duplication (ITD), FLT3-tyrosine kinase domain (TKD), DNMT3A, and isocitrate dehydrogenase 1/2 were screened using HRM analysis, and direct sequencing was performed for positive samples. Results High-resolution melting analysis enabled complete mutation detection in samples with 20% mutant alleles in all regions. In a clinical laboratory test, DNMT3A mutations were detected in 12 cases (17.3%), and we identified five novel mutations. Simultaneous NPM1, FLT3-ITD, and DNMT3A mutations constituted the most common pattern (30%) in de novo cytogenetically normal AML. Conclusion High-resolution melting analysis has sufficient performance for the detection of DNMT3A mutations in AML. This approach can facilitate rapid AML genotyping at diagnosis in clinical settings.

Background SF3B1 (splicing factor 3B subunit-1) somatic mutation is specifically detected in myelodysplastic syndrome (MDS) with ring sideroblasts (MDS-RS). We investigated the sensitivity and utility of SF3B1 mutation analysis as a clinical laboratory test. Method Detection limit for SF3B1 mutations by high-resolution melting (HRM) analysis was investigated by plasmid mixture. In 67 MDS patients, we examined the association between SF3B1 mutation and prognostic evaluation using the Revised International Prognostic Scoring System and revalidated MDS classifications based on the revised 4th edition of the WHO classification. Results HRM analysis enabled mutation detection in the 12.5% SF3B1 mutant alleles. SF3B1 mutation was detected in 9 cases, mostly in the low-risk group. Cases of MDS with ring sideroblasts unrelated to SF3B1 mutation were detected in the high-risk group. Two cases were reclassified as MDS-RS after detecting SF3B1 mutation. Conclusions SF3B1 mutation analysis as an initial screening at diagnosis increases the accuracy of prognostic prediction and disease classification.

Here, we show a functional role of casein kinase I (CKI) epsilon in hematopoietic cell survival through the modification of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Introduction of wild-type (WT)-CKIepsilon into interleukin-3 (IL-3)-dependent 32D cells increased the sensitivity to genotoxic stresses, such as gamma-irradiation, etoposide, and IL-3 deprivation, whereas kinase-negative (KN)-CKIepsilon suppressed it. Contrary to KN-CKIepsilon, WT-CKIepsilon attenuated the IL-3-induced activation of Akt with the increase of PTEN activity. Similarly, the increase of Akt activation, as well as PTEN inactivation, was accompanied both by a decrease of CKIepsilon expression induced by all-trans retinoic acid and by the addition of a specific inhibitor for CKIepsilon in HL-60 cells. CKIepsilon seems to activate PTEN by physical interaction. These results suggest that the CKIepsilon-induced down-regulation of PI3K/Akt signaling through PTEN lead to amplified sensitivity to apoptosis. Thus, the suppression of CKIepsilon in many human leukemia cell lines may play a role in the cell immortalization.

A member of the largest family of receptor protein kinases, EphB6, lacks its intrinsic kinase activity, but it is expressed in normal human tissues. To investigate the physiological function of EphB6, we generated EphB6 deficient mice. EphB6(-/-) mice developed normally, revealed no abnormality in general appearance, and were fertile. Although a developmental increase of EphB6 in the fetal thymus was confirmed, T-cell development in various lymphoid organs of EphB6(-/-) mice was comparable to those of EphB6(+/+). Even in fetal thymus organ cultures, any developmental differences of EphB6(-/-) and EphB6(+/+) thymocytes were undetectable. The different binding characteristics to ephrin-Fc proteins between EphB6(-/-) and EphB6(+/+) thymocytes demonstrated that ephrin-B2 is the unique ligand for EphB6 among eight known ephrins. While EphB6 was a dominant receptor that binds to ephrin-B2 in adult thymocytes, fetal ones also expressed another EphB that binds to ephrin-B2. Overlapping expression of the EphB subfamily in the fetal thymus might compensate for the loss of EphB6 during the thymic development.

Although most kinase-defective growth factor receptor proteins are associated with pathogenic conditions, a kinase-defective Eph-family receptor protein, EphB6, is expressed in normal human tissues. We generated monoclonal antibodies specific for human EphB6 to characterize its expression on human hematopoietic cells. A very small population of normal human peripheral white blood cells (0.57 +/- 0.07%, n = 12) expressed EphB6. The EphB6-positive cells were CD2+, CD7+, CD3+ and CD4+ or CD8+ lymphocytes, but they did not express CD19 or CD11b. In human bone marrow, only 1.5 +/- 0.19% of lymphocytes expressed EphB6. Compared with the expression in peripheral lymphocytes, prominent expression of EphB6 protein was demonstrated in CD4+CD8+ double-positive mouse thymocytes. The T-cell lineage-specific expression was strictly conserved in human leukemia/lymphoma cells. Among T-cell-derived leukemia cells, the expression level of EphB6 seemed to decrease with maturation of the cells. These results suggest that EphB6 expression is regulated in T-cell development.

Two closely related casein kinase I (CKI) isoforms, CKIdelta and CKIepsilon, are ubiquitously expressed in many human tissues, but their specific biologic function remains to be clarified. Here, we provide the first evidence that CKIepsilon is involved in hematopoietic cell differentiation. CKIepsilon, but not CKIdelta, was down-regulated along with human granulocytic differentiation. The specific down-regulation was observed in granulocyte colony-stimulating factor (G-CSF)-induced cell differentiation of murine interleukin-3 (IL-3)-dependent myeloid progenitor 32D cells. Introduction of wild-type (WT)-CKIepsilon into 32D cells inhibited the G-CSF-induced cell differentiation, whereas kinase-negative (KN)-CKIepsilon promoted the differentiation. Neither WT- nor KN-CKIepsilon affected IL-3-dependent cell growth. Moreover, introduction of WT- or KN-CKIdelta did not affect the cytokine-induced cell growth and differentiation. While G-CSF-induced activation of signal transducers and activators of transcription 3 (STAT3) was sustained by KN-CKIepsilon, STAT3 activation was attenuated by WT-CKIepsilon. This may be explained by the fact that the suppressor of cytokine signaling 3 (SOCS3) was stabilized by its physical association with CKIepsilon. Such stabilization by CKIepsilon was also seen in IL-3-induced beta-catenin. The stabilization of downstream components of cytokine and Wnt signaling by CKIepsilon might be critical for integration of several intracellular signaling pathways to a cell-specific biologic response in hematopoietic cell self-renewal.

Purpose: The aim of this study was to investigate the efficacy of combination therapy of ionizing radiation (IR) and adenoviral p53 gene therapy and to evaluate its molecular mechanisms. Methods and Materials: Two human prostate cancer cell lines, DU145 and PC-3 cells, containing different types of p53 gene mutations, were investigated. The recombinant adenovirus vector containing the wild-type p53 gene (Ad5CMV- p53 ) was used for this study. Cells were irradiated (in 0, 2, 4, and 6 Gy, 300 cGy/min) and after 12 h of irradiation, the cells were infected with various doses of Ad5CMV- p53 (0–40 multiplicity of infection [MOI]). Cytotoxicity was determined by clonogenic assay. The molecular mechanisms were evaluated by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR), apoptotic cell detection, and cell cycle analysis. Results: The cell growth inhibition in DU145 ( p53 -mutated) cells by IR was strongly enhanced by additional Ad5CMV- p53 infection in a viral dose–dependent manner. In DU145 cells, IR alone induced minimal p53 mRNA expression. However, IR combined with Ad5CMV- p53 infection stimulated significant increase in p53 mRNA expression supplemented with Bax and p21 mRNA expressions. In PC-3 ( p53 -null), IR induced Bax and p21 mRNA expression, while the combination effects were observed in p53, Bax , and p21 mRNA expression. Apoptotic cell deaths were rarely observed after IR alone (DU145: 3%, PC-3: 5%). However, after combination therapy, the proportion of apoptotic cells greatly increased (sevenfold in DU145 cells, and twice in PC-3 cells). G1 cell cycle arrest was observed after Ad5CMV- p53 infection and the combination in both cell lines. Conclusion: In this study, we demonstrated that the combination of IR and Ad5CMV- p53 gene therapy resulted in remarkable synergistic effects in human prostate cancer cells. This combination therapy could be one of the optimal treatment strategies for radioresistant prostate cancer.

A 40-year-old man received blood transfusion in December 1998 because of gastric bleeding from a peptic ulcer. One month later, he developed febrile hemolytic anemia. Administration of high doses of glucocorticoid significantly reduced the hemolysis, but did not cure the disease. To investigate the cause of the hemolysis, the patient was transferred to our hospital in May 1999. Giemsa-stained peripheral blood smears showed Babesia parasites in the red blood cells (RBC), and PCR analysis confirmed the presence of Babesia microti DNA. The parasitemia disappeared hematologically after 2 weeks of quinine and clindamycin therapy. However, parasite DNA was still detectable in the RBC. Although treatment with oral atovaquone was given for 2 weeks, parasitemia and febrile hemolysis recurred within a month after the last treatment. Fortunately, complete remission was obtained after a second 12-week course of therapy with quinine and clindamycin. PCR analysis revealed asymptomatic Babesia infection in one of eight samples from the original blood donor. The initial steroid therapy given to the patient without an accurate diagnosis seemed to have delayed augmentation of the specific antibodies (IgG) against Babesia microti, thus prolonging the parasitemia after the initial acute stage of babesiosis.

Abstract 3648 Background: Radioimmunotherapy (RIT) with 90Y-ibritumomab tiuxetan has been used for the treatment of relapsed or refractory indolent B-cell lymphoma. Early prediction of response to the therapy may offer the potential to identify patients who will benefit this therapy. We evaluated the efficacy of fluorine 18 fluorodeoxyglucose (FDG) combined positron emission tomographic-computed tomographic (FDG-PET/CT) imaging for assessment of therapy and predicting outcome in 90Y-ibritumomab tiuxetan radioimmunotherapy. Methods: Radioimmunotherapy with 90Y-ibritumomab tiuxetan was preformed in 52 patients with relapsed or refractory indolent B cell lymphoma (follicular lymphoma, 45; mucosa-associated lymphoid tissue lymphoma, 5; lymphoplasmacytic lymphoma, 2) at Hyogo College of Medicine Hospital from June 2009 through August 2012. FDG-PET/CT scanning was performed at two weeks and the later after 90Y-ibritumomab tiuxetan therapy. Results: In FDG-PET at 2 weeks after 90Y-ibritumomab therapy, 26 (50%) showed complete response (defined as early CR), 20 (38%) showed partial response (PR-2W) and 6 (12%) showed non response (NR-2W). Further follow-up revealed 10 later CR (8 CR out of 20 PR-2W and 2 CR out of 6 NR-2W), showing 69 %(36) of overall CR rate. Relapse was occurred in 4(17%) of 26 early CR and in 6 (60%) of 10 later CR, indicating significantly low relapse late in early CR (P= 0.0074, by Chi-square test). Patients with early CR (CR in 2 weeks after the RIT) showed significantly better progression free survival than those with later CR (P=0.0046, by Logrank test). In contrast, analysis at six weeks after the RIT showed 36 CR patients (10 of which eventually relapse), but failed to discriminate patients who had high risk of relapse. Conclusion: Our results suggest that CR assessed by FDG-PET/CT at 2 weeks after 90Y-ibritumomab tiuxetan therapy discriminates good responder to the RIT and predicts better progression free survival in relapsed or refractory indolent B cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

Normal human hematopoietic progenitors as well as leukemia/lymphoma cells express kinase-defective EphB6 receptors. The only unique high affinity ligand for EphB6 among eight known mammalian ephrins, ephrins-B2 is expressed not only on hematopoietic malignancies, but also on mesenchymal stem cells. However, the biological functions of the receptor and its ligand in hematopoietic cells are largely unknown. In the present study, we showed that the interaction between EphB6 and ephrinB2 could initiate forward as well as reverse signaling in vitro. Both pre-clustered and unclustered ligands could trigger the signal transduction, but pre-clustered ones more rapidly down-regulated the signaling. We also examined the EphB6/ephrinB2 function in cell adhesion and migration. Figure Figure HEK-EphB6 cells placed in the upper chamber of a Transwell apparatus, in which the lower side of filter was coated with different concentrations of ephrin-B2-Fc or Fc, were allowed to migrate to the lower side at 37°C overnight. Vector-transfected cells were used as controls. The cells that had migrated to the lower side of filter were stained, photographed. A BSA-coated filter is shown as a control. EphB6 exerted biphasic effects in response to different concentrations of the ephrin-B2. EphB6 promoted cell adhesion and migration when stimulated with low concentrations of ephrin-B2, whereas it induced repulsion and inhibited migration upon stimulation with high concentrations of ephrin-B2. A truncated EphB6 receptor lacking the cytoplasmic domain showed monophasic positive effects on cell adhesion and migration, indicating that the cytoplasmic domain is essential for the negative effects. We further explored the signal transduction of the biphasic effects. Figure Figure The Src family kinase, Fyn was co-immunoprecipitated with anti-EphB6 antibody in the absence or presence of ephrin-B2 stimulation. High concentrations of ephrin-B2 induced tyrosine phosphorylation of EphB6 through a Src family kinase activity. These results indicate that EphB6 can both positively and negatively regulate cell adhesion and migration, and suggest that tyrosine phosphorylation of the kinase-defective EphB6 receptor by a Src family kinase acts as the molecular switch for the functional transition. Thus, EphB6 expressed on hematopoietic cells may play an important role in the regulation of cell homing to hematopoietic tissues as well as leukemia cell infiltration.