Next-generation sequencing of miRNAs in clinical samples of Epstein-Barr virus-associated B-cell lymphomas.

Epstein-Barr virus (EBV) encodes 49 microRNAs (miRNAs) in the BART and BHRF1 regions of its genome. Although expression profiles of EBV-encoded miRNAs have been reported for EBV-positive cell lines and nasopharyngeal carcinoma, to date there is little information about total miRNA expression, including cellular and viral miRNAs, in the primary tumors of EBV-associated B-lymphoproliferative disorders. In this study, next-generation sequencing and quantitative real-time reverse transcription-PCR were used to determine the expression profiles of miRNAs in EBV-infected cell lines and EBV-associated B-cell lymphomas, including AIDS-related diffuse large B-cell lymphoma (DLBCL), pyothorax-associated lymphoma, methotrexate-associated lymphoproliferative disorder, EBV-positive DLBCL of the elderly, and Hodgkin lymphoma. Next-generation sequencing revealed that EBV-encoded miRNAs accounted for up to 34% of total annotated miRNAs in these cases. Expression of three miR-BHRF1s was significantly higher in AIDS-related DLBCL and pyothorax-associated lymphoma compared with methotrexate-associated lymphoproliferative disorder and EBV-positive DLBCL of the elderly, suggesting the association of miR-BHRF1s expression with latency III EBV infection. Heat map/clustering analysis of expression of all miRNAs, including cellular and EBV miRNAs, by next-generation sequencing demonstrated that each EBV tumor, except methotrexate-associated lymphoproliferative disorder, formed an isolated cluster. Principal component analysis based on the EBV-encoded miRNA expression showed that each EBV tumor formed a distinguished cluster, but AIDS-related DLBCL and pyothorax-associated lymphoma formed larger clusters than other tumors. These data suggest that expression of miRNAs, including EBV-encoded miRNAs, is associated with the tumor type and status of virus infection in these tumors.

Fumagillin, a potent angiogenesis inhibitor, induces Kaposi sarcoma-associated herpesvirus replication in primary effusion lymphoma cells.

Kaposi sarcoma and primary effusion lymphoma cells are infected with Kaposi sarcoma-associated herpesvirus (KSHV), predominantly in the latent form, and KSHV replication is observed rarely. Angiogenesis plays a crucial role in the pathogenesis of both Kaposi sarcoma and primary effusion lymphoma. In this study, we found that fumagillin, a potent angiogenesis inhibitor, induced replication of KSHV in primary effusion lymphoma cell lines. The transcript and protein product of replication transcriptional activator (RTA) were induced by 1-10 µM fumagillin at 24 and 48 h, respectively. Western blot analysis demonstrated that 10 µM fumagillin induced not only RTA expression but also other KSHV-encoded lytic proteins. A real-time PCR array detecting KSHV gene expression demonstrated that the expression profiles of KSHV induced by fumagillin were similar to those induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), but the amounts of each transcript were lower than those induced by TPA. Finally, real-time PCR demonstrated an increase in that viral DNA copy number per cell in fumagillin-stimulated primary effusion lymphoma cell lines, indicating replication of KSHV. In addition to TPA, 10 µM fumagillin resulted in growth inhibition of primary effusion lymphoma cell lines. These observations suggest that an angiogenesis inhibitor is an agent with potent effects on cell growth and KSHV reactivation in primary effusion lymphoma cells.

[Second transplantation for graft failure after allogeneic hematopoietic stem cell transplantation--a retrospective survey by Kanto Study Group for Cell Therapy].

We retrospectively surveyed patients who received a second transplantation for graft failure (GF) after allogeneic hematopoietic stem cell transplantation (SCT) in hospitals participating in the Kanto Study Group for Cell Therapy. A second SCT was performed in 21 of 45 patients with primary GF and in 13 of 15 with secondary GF. The median time between the first and second SCT was 49 days (range, 18-1204 days). The diagnosis included 28 patients with hematologic malignancies and 6 with aplastic anemia. Non-myeloablative or reduced-intensity conditioning was performed in 30 patients. Cord blood was frequently used as the source of stem cells followed by related donor peripheral blood, and unrelated bone marrow. Engraftment was achieved in 23 patients (68%). Conditioning regimen including total body or total lymphoid irradiation, was significantly associated with a higher engraftment rate. Overall survival at 5 years in all patients who underwent second SCT was 34%. Prognostic factors for better survival after second SCT were a time to second SCT longer than 90 days, the performance status at second SCT with 0 or 1, and the administration of tacrolimus for GVHD prophylaxis. The major cause of death after second SCT was infection. Although the outcome of a second SCT for graft failure remains poor, these findings suggest that the selection of patients as well as transplant methods, such as conditioning and GVHD prophylaxis, may contribute to survival.

[Refractory acquired hemophilia A in which factor VIII inhibitor was not detected at onset and thereafter deteriorated after total hip replacement arthroplasty].

A 58-year-old man was admitted to a general hospital to undergo total hip replacement arthroplasty for idiopathic osteonecrosis of the right femoral head. Pre-operative screening examination demonstrated isolated prolonged aPTT. Factor VIII (FVIII) activity was mildly decreased but there was no detectable FVIII inhibitor, so he was diagnosed as having congenital hemophilia. Surgery was performed safely with administration of FVIII preparation but 33 days postoperatively bleeding from the right hip joint appeared. It was not controllable with FVIII preparation and he was therefore admitted to our hospital.Laboratory examination at admission demonstrated prolonged aPTT (111.1 sec), reduced FVIII activity (3.0%), and the presence of FVIII inhibitor (17.0 B.U./ml). He was diagnosed with acquired hemophilia and administered Factor IX complex concentrates, prednisolone and cyclophosphamide. However, these treatments had only a temporary effect. Rituximab was administered every week and his condition soon improved. This case suggests that acquired hemophilia, in which there was no detectable FVIII inhibitor, may change to refractory disease after surgery. As a result, surgeons should pay careful attention to the preoperative presence of any mild coagulation abnormalities.

Successful autologous peripheral blood stem cell transplantation for a patient with primary adrenal lymphoma with hemophagocytic syndrome.

The adrenal glands are often an affected extranodal site in advanced non-Hodgkin lymphoma, but primary adrenal lymphoma (PAL) is extremely rare. Histologic examination of adrenal glands from patients with PAL reveals predominantly diffuse large B-cell lymphoma. Adrenal insufficiency is observed in about two thirds of patients. The prognosis of patients with PAL is poor: > 90% of patients die within 1 year of diagnosis, and long-term survivors are few. Effective therapeutic management of the condition has not been established. Herein, we report a 57-year-old man with PAL of diffuse large B-cell type that was later accompanied by hemophagocytic syndrome. He was initially treated with combination chemotherapy (1 cycle of CHOP [cyclophosphamide/doxorubicin/vincristine/prednisone] and 4 cycles of rituximab plus CHOP) and subsequently underwent autologous peripheral blood stem cell transplantation (auto-PBSCT) in his first complete remission. He has been well and disease free for 39 months since undergoing auto-PBSCT. The successful outcome of using auto-PBSCT in the treatment of a patient with PAL suggests that this therapeutic approach has value in treating this poor-prognostic disease.

Successful control of Epstein-Barr virus (EBV)-infected cells by allogeneic nonmyeloablative stem cell transplantation in a patient with the lethal form of chronic active EBV infection.

Chronic active Epstein-Barr virus infection (CAEBV) is a heterogeneous EBV-related disorder, ranging from mild/moderate forms to rapidly lethal disorders. The lethal form of CAEBV is characterized by multiple organ failure, hemophagocytic syndrome, and development of lymphomas. Allogeneic stem cell transplantation is considered as the only potentially curative treatment for the lethal form of CAEBV, but it is not always desirable because of the high incidence of regimen-related toxicities. A 17-year-old female with CAEBV, who was refractory to conventional therapies and considered to be unable to receive a myeloablative regimen because of multiple organ dysfunction, underwent allogeneic nonmyeloablative stem cell transplantation (allo-NST) before developing a hematological malignancy. She has been well without any signs of CAEBV for 27 months after allo-NST, and we confirmed that specific cytotoxic T lymphocyte activity against EBV was reconstituted. This outcome suggests that allo-NST can control CAEBV by reconstituting the host immunity against EBV.

A successful second unrelated BMT (UBMT) from a different unrelated donor to treat ALL that relapsed after the initial UBMT.

A 26-year-old male with acute lymphoblastic leukemia (ALL) in its second complete remission received an unrelated bone marrow transplantation (UBMT) following cyclophosphamide plus total body irradiation conditioning. The patient relapsed 7 months after the BMT. He received a second UBMT from a different donor 15 months after the initial UBMT. Conditioning for the second UBMT consisted of busulphan, melphalan, and anti-thymocyte globulin. The regimen was well tolerated, and engraftment was achieved. Both acute and chronic graft-versus-host diseases occurred but were successfully controlled with immunosuppressive drugs. He is alive and disease-free 29 months after the second UBMT. This is the first report of a successful second UBMT for ALL that had relapsed after the first UBMT and for which a different donor was used.