Diverse B-cell tumors associated with t(14;19)(q32;q13)/IGH::BCL3 identified by G-banding and fluorescence in situ hybridization.

We characterized 5 B-cell tumors carrying t(14;19)(q32;q13) that creates the IGH::BCL3 fusion gene. The patients' ages ranged between 55 and 88 years. Two patients presented with progression or recurrence of B-cell chronic lymphocytic leukemia (B-CLL)/small lymphocytic lymphoma (SLL), two with diffuse large B-cell lymphoma (DLBCL) of non-germinal center B-like phenotype, and the remaining one with composite angioimmunoblastic T-cell lymphoma and Epstein-Barr virus-positive DLBCL. The presence of t(14;19)(q32;q13) was confirmed by fluorescence in situ hybridization (FISH), showing colocalization of 3' IGH and 3' BCL3 probes on der(14)t(14;19) and 5' BCL3 and 5' IGH probes on der(19)t(14;19). One B-CLL case had t(2;14)(p13;q32)/IGH::BCL11A, and 2 DLBCL cases had t(8;14)(q24;q32) or t(8;11;14)(q24;q11;q32), both of which generated IGH::MYC by FISH, and showed nuclear expression of MYC and BCL3 by immunohistochemistry. The IGH::BCL3 fusion gene was amplified by long-distance polymerase chain reaction in 2 B-CLL/SLL cases and the breakpoints occurred immediately 5' of BCL3 exon 1 and within the switch region associated with IGHA1. The 5 cases shared IGHV preferentially used in B-CLL cells, but the genes were unmutated in 2 B-CLL/SLL cases and significantly mutated in the remaining 3. B-cell tumors with t(14;19)(q32;q13) can be divided into B-CLL/SLL and DLBCL groups, and the anatomy of IGH::BCL3 in the latter may be different from that of the former.

Two cases of primary diffuse large B-cell lymphoma of the CNS associated with t(8;14)(q24;q32) or t(3;14)(q27;q32) identified by G-banding and fluorescence in situ hybridization applied to metaphase spreads.

We describe two patients with primary diffuse large B-cell lymphoma of the central nervous system (PCNS-DLBCL). The first patient (case 1) was a woman in her late 70s who presented with a tumor in the left frontal lobe, whereas the second patient (case 2) was a man in his early 70s who presented with a left frontal lobe tumor associated with intratumoral hemorrhage. The histopathology of the tumor specimen disclosed the proliferation of large cells with centroblastic (case 1) or immunoblastic/plasmablastic (case 2) cytomorphology and an accumulation of the tumor cells within the perivascular space. The cells in both cases were positive for CD20, CD79a, BCL6, IRF4/MUM1, MYC, and BCL2 and negative for CD5 and CD10. G-banding revealed t(8;14)(q24;q32) in case 1, and the tetraploid-range karyotype including two or three copies of der(3)t(3;14)(q27;q32) and der(14)t(3;14)(q27;q32) in case 2. Fluorescence in situ hybridization applied to metaphase spreads confirmed colocalization of MYC and IGH (case 1) and BCL6 and IGH (case 2) hybridization signals on the relevant derivative chromosomes. Case 1 carried the MYD88L265P mutation. This case report provides clear evidence for the occurrence of t(8;14)(q24;q32) and t(3;14)(q27;q32) in PCNS-DLBCL using metaphase-based cytogenetic analysis.

cDNA-Based Mutation Screening Using a Combination of High-Resolution Melting Curve and Fragment Analysis Facilitates Efficient CCR4 Mutation Analysis in Adult T-Cell Leukemia/Lymphoma.

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.

VS38 as a promising CD38 substitute antibody for flow cytometric detection of plasma cells in the daratumumab era.

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.

Investigation of screening method for DNMT3A mutations by high-resolution melting analysis in acute myeloid leukemia.

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.

Evaluation of SF3B1 Mutation Screening by High-Resolution Melting Analysis and its Clinical Utility for Myelodysplastic Syndrome with Ring Sideroblasts at the Point of Diagnosis.

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.

[Autologous peripheral blood stem cell transplantation for treating primary central nervous system lymphoma: a single-center retrospective study].

Primary central nervous system lymphoma (PCNSL) is more difficult to treat than other lymphomas. Recently, it has been suggested that high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is effective for treating PCNSL. In the present study, we retrospectively analyzed 12 patients with PCNSL at our hospital. Five young patients with good performance status (PS) received upfront ASCT. The conditioning regimen prior to ASCT with busulfan ＋ cyclophosphamide ＋ etoposide showed good prognosis (complete remission rate of 100%). In addition, the PS improved in patients treated with high-dose chemotherapy followed by ASCT, while it worsened in those treated without ASCT. Further investigations are needed to clarify inclusion/exclusion criteria and optimize conditioning regimens for ASCT.

Rapidly progressive autoimmune pancytopenia successfully treated with steroids.

A 73-year-old woman was admitted to our hospital because of pancytopenia. Bone marrow aspiration showed increased cellularity with no dysplastic change. Laboratory tests revealed increased reticulated erythrocytes and reticulated platelets, positive direct Coombs test, and hemolysis. These findings led to the diagnosis of Evans syndrome. Relatively decreased mature neutrophils in the bone marrow aspirate raised the possibility of autoimmune neutropenia. Antineutrophil antibody was detected by the 6 cell-lineage immunofluorescence test, consistent with the diagnosis of autoimmune neutropenia. The patient had no underlying diseases, and was therefore considered to have idiopathic autoimmune pancytopenia. Due to rapid progression of the disease, prednisolone was administered at an initial dose of 0.5 mg/kg per day and the pancytopenia improved promptly.

Successful Treatment of TAFRO Syndrome, a Variant of Multicentric Castleman's Disease, with Cyclosporine A: Possible Pathogenetic Contribution of Interleukin-2.

Multicentric Castleman's disease is a systemic inflammatory disorder characterized by lymphadenopathy and excessive interleukin-6 production. A unique clinicopathologic variant of multicentric Castleman's disease, TAFRO (i.e., thrombocytopenia, anasarca, fever, renal failure or reticulin fibrosis, and organomegaly) syndrome, was recently proposed in Japan. Despite the successful use of anti-interleukin-6 therapy in some patients with TAFRO syndrome, not all patients achieve remission. The pathophysiological etiology of and suitable therapeutic strategies for this variant have not been established. Here, we present our experience of a unique case of TAFRO syndrome in a 78-year-old woman whose symptoms responded differently to several therapies. Tocilizumab, an anti-interleukin-6 receptor antibody, successfully induced remission of fever and lymphadenopathy. However, severe thrombocytopenia persisted and she developed anasarca, ascites, and pleural effusion shortly thereafter. Rituximab, an anti-CD20 antibody, and glucocorticoid therapy provided no symptom relief. In contrast, cyclosporine A, an immunosuppressive agent that blocks T cell function by inhibiting interleukin-2, yielded immediate improvements in systemic fluid retention and a gradual increase in platelet count, with complete resolution of disease symptoms. Excessive serum interleukin-2, when used as an anti-cancer agent, has been reported to cause side effects such as fluid retention, thrombocytopenia, and renal failure. Our case was unique because the anti-interleukin-2 therapy successfully improved symptoms that were not relieved with anti-interleukin-6 therapy. The present report therefore provides insight into the possible role of interleukin-2, in addition to interleukin-6, in TAFRO syndrome. This report will certainly help to clarify the pathogenesis of and optimal treatment strategies for TAFRO syndrome.

Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly complicated by the onset of acute myeloid leukemia.

We herein describe the case of a 62-year-old woman who presented with anemia and an 8-month history of weight loss. Bone marrow aspiration showed increased myeloblasts. The histopathology findings of biopsy specimens of the right cervical lymph node and intestinal mass indicated B-lymphoproliferative disorder (B-LPD) with Hodgkin lymphoma-like morphologic features and polymorphous diffuse large B-cell lymphoma (DLBCL), respectively. In addition, both types of lymphoma cells were positive for Epstein-Barr virus (EBV)-encoded small RNA-1. The patient was diagnosed with EBV-associated B-LPD and simultaneous acute myeloid leukemia (AML). This is the first case of a patient diagnosed with simultaneous EBV-positive DLBCL of the elderly and AML.

Primary hepatic lymphoma in a patient with rheumatoid arthritis treated with methotrexate.

Primary hepatic lymphoma (PHL) has rarely been reported in patients with immunosuppression. We herein describe a case of Epstein-Barr virus- (EBV-) positive PHL in a 67-year-old Japanese woman receiving methotrexate (MTX) treatment for rheumatoid arthritis (RA). The patient, who had been receiving MTX therapy for more than 6 years, presented with low-grade fever and abdominal pain. Initial laboratory tests showed mildly elevated liver enzymes with normal levels of alpha-fetoprotein and carcinoembryonic antigen, and computed tomography scans revealed multiple hepatic tumors with no lymph-node swelling. Examination of liver specimens obtained via ultrasonography-guided needle biopsy indicated EBV-positive diffuse large B cell lymphoma; therefore, she was diagnosed with PHL. MTX was discontinued, and she was carefully monitored thereafter owing to the prolonged history of MTX administration for RA. Rapid progression of PHL was observed; therefore 10 days after the PHL diagnosis, she received 6 cycles of R-THP-COP (rituximab, cyclophosphamide, pirarubicin, vincristine, and prednisolone) therapy and achieved complete remission for more than 1 year. Although MTX-associated lymphoproliferative disorders often show remission after withdrawal of MTX, early diagnosis and treatment are essential for PHL in patients with RA treated with MTX, because of the aggressive nature of the disease.

Development of donor cell leukemia mimicking hematogones after unrelated cord blood transplantation for relapsed acute lymphoblastic leukemia.

A 26-year-old woman, who developed ALL when she was eighteen years old, achieved remission after chemotherapy. Her ALL relapsed when she was twenty-two years old. After re-induction therapy, she underwent cord blood transplantation. Her bone marrow examination on the 42nd day revealed a lymphoblast count of 16%. She was observed without any therapy, but her bone marrow blast count continued to be around 6% for three years without any symptoms. The bone marrow blast fraction originated from the cord blood. Surface marker analysis of the blast fraction initially revealed a pattern of hematogones that was CD10 and CD19 positive, but then showed a myeloblast pattern that was CD13 and CD33 positive. AML developed as donor cell leukemia. When blasts appear in the early phase after transplantation and persist, an observation period is necessary with molecular chimerism, morphology, and surface marker analysis of the blast fraction to consider relapse, hematogones, or donor cell leukemia.

Functional characterization of OX40 expressed on human CD8+ T cells.

In the present study, we investigated the expression of OX40 on human CD8(+) T cells with regard to expression induction, costimulatory function and possible involvement in cytotoxicity. Human CD8(+) T cells were purified from peripheral blood mononuclear cell (PBMC) of healthy donors and cocultured with allogeneic monocyte-derived dendritic cells. Flow cytometric analysis showed that expression of OX40 was induced on CD8(+) T cells within 1 day and increased to the maximum levels on day 3. An addition of anti-OX40 ligand (OX40L) mAb suppressed CD25 expression, proliferation and IFN-gamma production of CD8(+) T cells, suggesting that OX40 functions as a costimulatory molecule not only for CD4(+) T cells but also for CD8(+) T cells. In parallel, coculture of pre-activated CD8(+) T cells with OX40L-transfected murine epithelial cells (MMCE-OX40L) resulted in an increase in CD25 expression, proliferation and IFN-gamma producing cells, compared with that with the mock control (MMCE-mock). Finally, non-specific cytotoxic activity of preactivated CD8(+) T cells was examined using OKT3 hybridoma as target cells after coculture with these transfectants. Coculture with MMCE-OX40L induced slightly higher cytotoxicity of CD8(+) T cells than that with MMCE-mock. These results indicate that OX40 is induced transiently on CD8(+) T cells upon activation and its signals contribute to both clonal expansion and functional reinforcement.

Atrial natriuretic peptide polarizes human dendritic cells toward a Th2-promoting phenotype through its receptor guanylyl cyclase-coupled receptor A.

Atrial natriuretic peptide (ANP) is a cardiovascular hormone secreted mainly by the cardiac atria and regulates the volume-pressure homeostasis. The action of ANP is mediated by its receptor, guanylyl cyclase-coupled receptor A (GC-A). In this study, we explored the possibility that ANP and GC-A may play a role in the dendritic cell (DC)-mediated immune regulation. We first examined the expression of GC-A in human monocyte-derived DCs in comparison with monocytes and found that DCs but not monocytes express GC-A at both the mRNA and protein levels. DCs responded to ANP with an increase in intracellular cGMP in a dose-dependent manner, indicating that GC-A expressed on DCs is functional. Furthermore, treatment of DCs with ANP decreased production of IL-12 and TNF-alpha and conversely increased that of IL-10 upon stimulation with LPS. In accordance with this change of cytokine production, DCs treated with ANP plus LPS promoted differentiation of naive CD4(+) T cells into a Th2 phenotype. Finally, we presented evidence that ANP affected cytokine production of fresh whole blood stimulated with LPS in line with the above-mentioned results. These results indicate that ANP polarizes human DCs toward a Th2-promoting phenotype through GC-A and thus can regulate immune responses.

Costimulation through OX40 is crucial for induction of an alloreactive human T-cell response.

The alloreactive immune response is a series of events initiated by the interaction of T cells with allogeneic dendritic cells (DCs), involving alloantigen recognition and costimulatory signals. In this study, we investigated the role of OX40 in alloreactivity in vitro. We first demonstrated that anti-OX40 ligand (anti-OX40L) monoclonal antibody (mAb) could markedly suppress the mixed leucocyte reaction (MLR) of peripheral blood mononuclear cells (PBMC). To further define the contribution of the OX40/OX40L system to the MLR, we set up a co-culture system of CD4+ T cells and allogeneic monocyte-derived dendritic cells (DCs). After 2 days, OX40 expression was induced on CD4+ T cells and this induction was strongly inhibited by the addition of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Fc fusion protein, suggesting that the expression of OX40 during alloreaction is dependent on CD28 signalling. Next we examined the effects of anti-OX40L mAb, CTLA-4-Fc fusion protein and anti-human leucocyte antigen (HLA)-DR mAb on the proliferative response of CD4+ T cells to allogeneic DCs. The proliferation of T cells was almost completely suppressed by anti-OX40L mAb, which was comparable with that of CTLA-4-Fc. Measurement of interleukin-2 (IL-2) production in the culture supernatants showed that suppression of a proliferative response was at least in part ascribed to reduced IL-2 production. Furthermore, purified OX40L- allogeneic DCs could induce considerable proliferation of CD4+ T cells, which was suppressed by anti-OX40L mAb. These results suggest that the OX40/OX40L system is crucial for induction of the allogeneic T-cell response and the OX40/OX40L system is subsequent to and dependent on CD28 signalling, but is crucial for the end outcome of the human alloreactive T-cell response.