Yuji Wano

Genetic variants in C5 and poor response to eculizumab.

BACKGROUND Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear. METHODS We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients. RESULTS Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G → A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C → T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab. CONCLUSIONS The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).

DNA Damage and Cell Killing by Camptothecin and Its Derivative in Human Leukemia HL‐60 Cells

Camptothecin (CPT) has heen recognized as a topoisomerase I (Topo I) inhibitor. However, the mechanism of cytotoxicity of this agent remains unknown. In the present study, we analyzed the kinetics of Topo I‐mediated DNA single‐strand breaks and internucleosomal DNA cleavage produced by CPT and its derivative, 7‐ethyl‐10‐hydroxycamptothecin (SN‐38), in HL‐60 cells. DNA single‐ strand breaks were detected using alkaline sucrose gradient centrifugation when HL‐60 cells were incubated with 10 μM CPT or 10 μM SN‐38 for 30 min. These DNA single‐strand breaks were rapidly repaired after drug removal, while the cytotoxic action of these drugs was sustained. Treatment of HL‐60 cells with CPT or SN‐38 for 3 h produced extensive degradation of DNA. Agarose gel electrophoresis showed a ladder of DNA fragments consisted of multimers of approximately 200 base pairs, characteristic of apoptosis. Interestingly, this type of DNA fragmentation was also induced within 4 h after repair of DNA single‐strand breaks, and subsequently loss of cell viability was observed. When zinc ion, a potent inhibitor of endonuclease, was added to drug‐free medium after treatment with CPT or SN‐38, internucleosomal DNA cleavage was abolished. Furthermore, addition of zinc ion reduced the loss of cell viability. These data suggest that Topo I‐mediated DNA single‐strand breaks may be necessary but are not sufficient for cell death, and the endonuclease involved in induction of internucleosomal DNA cleavage may play an important role in HL‐60 cell death induced by Topo I inhibitor.

Analyses for binding of the transferrin family of proteins to the transferrin receptor 2.

Transferrin receptor 2α (TfR2α), the major product of the TfR2 gene, is the second receptor for transferrin (Tf), which can mediate cellular iron uptake in vitro. Homozygous mutations of TfR2 cause haemochromatosis, suggesting that TfR2α may not be a simple iron transporter, but a regulator of iron by identifying iron‐Tf. In this study, we analysed the ligand specificity of TfR2α using human transferrin receptor 1 (TfR1) and TfR2α‐stably transfected and expressing cells and flow‐cytometric techniques. We showed that human TfR2α interacted with both human and bovine Tf, whereas human TfR1 interacted only with human Tf. Neither human TfR1 nor TfR2α interacted with either lactoferrin or melanotransferrin. In addition, by creating point mutations in human TfR2α, the RGD sequence in the extracellular domain of TfR2α was shown to be crucial for Tf‐binding. Furthermore, we demonstrated that mutated TfR2α (Y250X), which has been reported in patients with hereditary haemochromatosis, also lost its ability to interact with both human and bovine Tf. Although human TfR1 and TfR2α share an essential structure (RGD) for ligand‐binding, they have clearly different ligand specificities, which may be related to the differences in their roles in iron metabolism.

Epstein-barr virus-associated composite lymphoma composed of peripheral t-cell lymphoma and an anaplastic variant of a diffuse large b-cell type of non-hodgkin’s lymphoma and strongly expressing P53 protein

We report a case of composite lymphoma consisting of peripheral T-cell lymphoma and an anaplastic variant of diffuse large B-cell lymphoma (DLBCL) and associated with Epstein-Barr virus (EBV) infection and strong p53 expression. A 65-year-old Japanese woman developed fever and generalized lymphadenopathy.A biopsy of the cervical node revealed the morphology of malignant lymphoma with 2 kinds of lymphoma coexisting in 1 lymph node. One lymphoma type consisted of immunoblastic large cells with the T-cell marker phenotype CD3+, CD45RO/UCHL-1+, CD20/L26-, CD79-, CD10-, CD30-, and CD15-; the other type consisted of large cells with abundant cytoplasm and pleomorphic nuclei with the marker phenotype CD79+, CD20/ L26+, CD45RO/UCHL-1-, CD3-, CD10-, CD30+, NPM/ALK-, and CD15-. Therefore, the diagnosis was composite lymphoma of peripheral T-cell lymphoma and an anaplastic variant of DLBCL, stage IVB, because the patient had bone marrow involvement with peripheral T-cell lymphoma. The biopsy led to findings of latent type II EBV-associated lymphoma in both the peripheral T-cell lymphoma and the anaplastic variant of DLBCL as the result of positive signals for EBV small RNAs by in situ hybridization, positive immunostaining results for EBV latent membrane protein 1 antibody, and negative immunostaining results for EBV nuclear antigen 2. Immunostaining of the mass with p53 antibody also yielded positive results for both types of lymphoma cells. This case suggests that the immunocompromised state of this patient with EBV-related peripheral T-cell lymphoma allowed the emergence of an EBV-related anaplastic variant of DLBCL and suggests a close relationship between p53 expression and latent EBV infection.

Association of Epstein-Barr virus with human immunodeficiency virus-negative peripheral T-cell lymphomas in Japan.

Abstract:  The association of Epstein–Barr virus (EBV) with human immunodeficiency virus‐negative T‐cell lymphoma was examined in 68 patients using the polymerase chain reaction (PCR) with DNA obtained from formalin‐fixed paraffin‐embedded tissues and an in situ hybridization technique. EBV‐encoded RNA (EBER) was detected in 43 of 68 cases (63%) of peripheral T‐cell lymphoma: in 100% (11 of 11 cases) of NK/T‐cell lymphomas, 70% (14 of 20 cases) of angioimmunoblastic T‐cell lymphomas (AILT) and 49% (18 of 37 cases) of other types of peripheral T‐cell lymphoma. A positive band was also detected at high incidence (36 of 65 cases; 55%) in a PCR analysis using primers to detect the Bam HI‐W fragment of EBV. In the immunohistochemical analysis using a monoclonal antibody to latent membrane protein 1 (LMP‐1) of EBV, one of the EBV‐encoded latent gene products, LMP‐1, was found to be expressed in 13 of 64 cases (20%), but EBNA‐2 was not expressed in all the cases examined (0 of 59 cases; 0%). The 5‐yr survival rate was 28% for peripheral T‐cell lymphomas overall, 0% for NK/T‐cell lymphomas, 38% for AILTs and 28% for other types of peripheral T‐cell lymphoma. The difference in the overall survival rate between NK/T‐cell lymphoma and non‐NK/T‐cell lymphoma was significant (P = 0.0498 by Log‐rank test). Among peripheral T‐cell lymphoma patients overall, the group severely infected with EBV (EBER‐ISH ++) had a lower 5‐yr survival rate (8%) than the group slightly (EBER‐ISH +) or not infected (38%; P = 0.0013).

Incidence of Diffuse Large B-Cell Lymphoma of Germinal Center B-Cell Origin in Whole Diffuse Large B-Cell Lymphoma : Tissue Fluorescence In Situ Hybridization Using t(14 ; 18) Compared with Immunohistochemistry

Diffuse large B-cell lymphoma (DLBCL) can be divided into prognostically important categories such as germinal center B (GCB)-like and non-GCB-like groups. The t(14;18)(q32;q21) translocation defines a unique subset of DLBCL cases with a GCB gene expression profile. Two-color fluorescence in situ hybridization (FISH) analysis was applied to detect t(14;18) (q32;q21) in the nuclei of paraffin-embedded tissue sections from 61 patients with de novo DLBCL.Nine (15%) of 61 cases had a positive pattern. Fifty-seven cases were subclassified in an immunohistochemical study with anti-CD10, anti-bcl-6, and anti-MUM1 antibodies. In this classification, 21 cases (37%) were placed in the GCB group, and 36 (63%) were placed in the non-GCB group. There was a discrepancy between t(14;18) occurrence and bcl-2 protein expression. Bcl-2 protein expression was positive in 40 (67%) of 60 cases.The expression of bcl-2 protein in the GCB and non-GCB groups was not significantly different: 15 (71%) of 21 cases in the GCB group and 24 (67%) of 36 cases in the non-GCB group tested positive.We found no difference between the FISH-positive and FISH-negative groups in overall survival time (P = .6019, log-rank test).The overall survival rates of GCB and non-GCB groups did not differ significantly by immunohistochemical classification (P = .5399, log-rank test). Overall survival was significantly longer in the group with a low International Prognostic Index (IPI) score than in the group with a high IPI score (P = .0002, log-rank test).Our results suggest that immunohistochemical study and cytogenetic study with t(14;18) FISH cannot predict the clinical outcomes of DLBCL patients.Astudy with a larger number of patients may show a difference in clinical outcomes between FISH-positive and FISH-negative groups and between GCB and non-GCB groups.

Marked upregulation of Survivin and Aurora-B kinase is associated with disease progression in the myelodysplastic syndromes.

Background Myelodysplastic syndromes are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Survivin is a member of the inhibitor of apoptosis family and suppresses apoptosis. Survivin also functions as a subunit of the chromosomal passenger complex for regulating mitosis with Aurora-B. Survivin and Aurora-B play an important role in maintaining genome stability. The aim of this study was to determine the role of Survivin and Aurora-B kinase in disease progression and prognosis of myelodysplastic syndromes. Design and Methods We evaluated the expression levels of these two genes in CD34+ cells prepared from 64 patients with myelodysplastic syndrome or leukemic blasts from 50 patients with de novo acute myeloid leukemia using quantitative real-time PCR. Results Survivin and Aurora-B expression levels were highly correlated with the type of myelodysplastic syndrome, were much higher in refractory anemia with excess blasts-1, refractory anemia with excess blasts-2, and secondary acute myeloid leukemia following myelodysplastic syndrome than in normal control, and increased during disease progression. There was a significant correlation between these expression levels and the International Prognostic Scoring System. Interestingly, these levels were remarkably higher in patients with secondary acute myeloid leukemia following myelodysplastic syndromes than in those with de novo acute myeloid leukemia. Conclusions This is the first report showing that high levels of Survivin and Aurora-B kinase expression in CD34+ cells are distinctive molecular features of high-risk myelodysplastic syndromes and secondary acute myeloid leukemia following myelodysplastic syndrome. Marked upregulation of Survivin and Aurora-B kinase may contribute to genetic instability and disease progression of myelodysplastic syndromes. Our data may explain why patients with high-risk myelodysplastic syndromes frequently show complex chromosomal abnormality.

Epstein-Barr Virus-Associated B-Cell Type Non-Hodgkin’s Lymphoma with Concurrent p53 Protein Expression in a Rheumatoid Arthritis Patient Treated with Methotrexate

A Japanese male patient received various medications for his long-standing rheumatoid arthritis (stage IV, class II). He developed a mass on the right anterior chest wall after being treated with methotrexate (MTX) for 4 months. A biopsy of the mass showed it to be Epstein Barr virus (EBV)-associated lymphoma of B-cell phenotype stage IE (bulky mass), with positive EBV-encoded small RNAs (EBERs) in situ hybridization, EBV latent membrane protein-1 (LMP-1) negative, EB nuclear antigen-2 (ERNA-2) negative, CD20/L26 (+), CD45RO/UCHL-1 (-). A single band of the joined termini of the EBV genome was demonstrated in DNA extracted from the mass, suggesting a clonal disorder of the mass. Immunostaining of the mass with p53 antibody was also positive.With discontinuation of MTX and administration of chemotherapy, the tumor disappeared but recurred after 3 months. This case suggests that concordant p53 expression and latent EBV infection may play an important role in the pathogenesis of lymphomas arising in patients with rheumatoid arthritis who are immunosuppressed with MTX.

T-Cell type acute lymphoblastic leukemia following cyclosporin A therapy for aplastic anemia.

Cyclosporin A (CsA) is used to prevent rejection in transplantation and to treat autoimmune and hematologic diseases such as aplastic anemia. However, the tumor growth-promoting effect of CsA remains controversial. We report the case of a 24-year-old man who developed acute lymphoblastic leukemia of precursor-T-cell origin after 75 months of treatment with CsA for aplastic anemia. The surface antigen phenotype of his leukemic cells was CD2+, CD3+, CD5+, CD7+, CD4−, CD8−, CD10−, CD20−, CD34−, CD41−, and CD56−. Southern blot analysis revealed a monoclonal rearrangement of T-cell receptor—J nongermline fragments inHindIII digestion.

Double Ph‐positive megakaryoblastic transformation of chronic myeloid leukemia

Abstract: A 64‐yr‐old Japanese man presented with mild anemia, leukocytosis, and thrombocytosis in November 1999. A diagnosis of chronic myeloid leukemia was made with a positive Ph chromosome, and interferon α treatment was started, 6 million units a day. Two years later, in October 2001, the patient developed leukocytosis, an increased LDH level, and large blasts with basophilic cytoplasm with cytoplasmic projections appeared in the peripheral blood. Bone marrow aspiration revealed increased blasts (59.6%). These blasts were negative on peroxidase stain, positive on acid phosphatase, and weakly positive on α naphthyl butyrate esterase stain and periodic acid‐Schiff stain. Immunohistochemical staining with monoclonal antibodies revealed that these blasts were strongly positive with anti‐CD41 (glycoprotein IIb/IIIa), weakly positive with CD7, CD33, and CD34, and negative with other monoclonal antibodies. A diagnosis of megakaryoblastic transformation from chronic myeloid leukemia was therefore made. Two‐color fluorescence in situ hybridization (FISH) for portions of the major‐bcr and abl genes from bone marrow cells revealed two fused signals in 90.6% and one fused signal in 5.8% of 106 cells. A cytogenetic study revealed that bone marrow cells were 69, XYY, +6, −7, +8, −9, t(9;22)(q34;q11), +11, +13, −15, −16, dic(17;18)(p11;p11), −18, +19, +21, der(22)t(9;22) in six of nine examined cells. These findings confirmed that these megakaryoblasts originated from megakaryocytes of the chronic myeloid leukemia clone.