Koji Nagafuji

Expression of programmed cell death ligand 1 is associated with poor overall survival in patients with diffuse large B-cell lymphoma

Programmed cell death ligand 1 (PD-L1) is expressed on both select diffuse large B-cell lymphoma (DLBCL) tumor cells and on tumor-infiltrating nonmalignant cells. The programmed cell death 1 (PD-1)/PD-L1 pathway inhibits host antitumor responses; however, little is known about how this pathway functions in the tumor microenvironment. The aim of this study was to determine the clinicopathological impact of PD-L1(+) DLBCL. We performed PD-L1/PAX5 double immunostaining in 1253 DLBCL biopsy samples and established a new definition of PD-L1(+) DLBCL. We also defined the criteria for microenvironmental PD-L1(+) (mPD-L1(+)) DLBCL (ie, PD-L1(-) DLBCL in which PD-L1(+) nonmalignant cells are abundant in the tumor microenvironment). Of the 273 patients whose clinical information was available, quantitative analysis of PD-1(+) tumor-infiltrating lymphocytes (TILs) was performed. The prevalence rates of PD-L1(+) and mPD-L1(+) DLBCL were 11% and 15.3%, respectively. Both PD-L1(+) and mPD-L1(+) DLBCL were significantly associated with non-germinal center B-cell (GCB) type and Epstein-Barr virus positivity. The number of PD-1(+) TILs was significantly higher in GCB-type tumors and lower in mPD-L1(-) and PD-L1(+) DLBCL. Patients with PD-L1(+) DLBCL had inferior overall survival (OS) compared with that in patients with PD-L1(-) DLBCL (P = .0009). In contrast, there was no significant difference in OS between mPD-L1(+) and mPD-L1(-) DLBCL (P = .31). The expression of PD-L1 maintained prognostic value for OS in multivariate analysis (P = .0323). This is the first report describing the clinicopathological features and outcomes of PD-L1(+) DLBCL. Immunotherapy targeting the PD-1/PD-L1 pathway should be considered in this distinct DLBCL subgroup.

Human Flt3 Is Expressed at the Hematopoietic Stem Cell and the Granulocyte/Macrophage Progenitor Stages to Maintain Cell Survival

FLT3/FLK2, a member of the receptor tyrosine kinase family, plays a critical role in maintenance of hematopoietic homeostasis, and the constitutively active form of the FLT3 mutation is one of the most common genetic abnormalities in acute myelogenous leukemia. In murine hematopoiesis, Flt3 is not expressed in self-renewing hematopoietic stem cells, but its expression is restricted to the multipotent and the lymphoid progenitor stages at which cells are incapable of self-renewal. We extensively analyzed the expression of Flt3 in human (h) hematopoiesis. Strikingly, in both the bone marrow and the cord blood, the human hematopoietic stem cell population capable of long-term reconstitution in xenogeneic hosts uniformly expressed Flt3. Furthermore, human Flt3 is expressed not only in early lymphoid progenitors, but also in progenitors continuously along the granulocyte/macrophage pathway, including the common myeloid progenitor and the granulocyte/macrophage progenitor. We further found that human Flt3 signaling prevents stem and progenitors from spontaneous apoptotic cell death at least through up-regulating Mcl-1, an indispensable survival factor for hematopoiesis. Thus, the distribution of Flt3 expression is considerably different in human and mouse hematopoiesis, and human FLT3 signaling might play an important role in cell survival, especially at stem and progenitor cells that are critical cellular targets for acute myelogenous leukemia transformation.

L-Asparaginase induced durable remission of relapsed nasal NK/T-cell lymphoma after autologous peripheral blood stem cell transplantation

A 60-year-old Japanese woman who presented with right nasal congestion and high fever was admitted to our hospital in March 1999. She was diagnosed with nasal NK/T-cell lymphoma clinical stage IVB. Because her NK/T-cell lymphoma was highly aggressive and chemo-resistant, she underwent autologous peripheral blood stem cell transplantation (PBSCT). The patient received a pretransplantation conditioning regimen of ranimustine, etoposide, carboplatin, and cyclophosphamide. On July 29,1999,1.0 X 106/kg CD34+ cells were infused. The patient achieved first complete remission. In January 2000, NK/T-cell lymphoma relapsed in the skin and fever developed. CHOP (cyclophosphamide, vincristine, doxorubicin, and prednisolone) was administered, resulting in partial regression of the skin lesions, but fever persisted. L-asparaginase (L-Asp) at a dose of 6000 U/m2 per day was administered for 7 days, resulting in the complete disappearance of the skin lesions and resolution of the fever. The patient has been in second complete remission for more than 18 months since the completion of L-Asp treatment (as of July 2001). The effect of L—Asp in this patient was dramatic. Several cases have been reported describing the effectiveness of L-Asp in patients with nasal lymphoma and cutaneous T-cell lymphoma. A front-line chemotherapy regimen containing L-Asp for NK/T-cell lymphoma may warrant further evaluation.

Human Herpesvirus 6 (HHV-6) Reactivation and HHV-6 Encephalitis After Allogeneic Hematopoietic Cell Transplantation: A Multicenter, Prospective Study

BACKGROUND The epidemiology of human herpesvirus 6 (HHV-6) encephalitis after allogeneic hematopoietic cell transplantation (HCT) and its relationship with HHV-6 reactivation have not been sufficiently characterized. METHODS This prospective, multicenter study of 230 allogeneic HCT recipients investigated the epidemiology of HHV-6 reactivation and HHV-6 encephalitis. Plasma HHV-6 DNA load was prospectively evaluated twice weekly until 70 days after HCT. RESULTS Cumulative incidence of positive HHV-6 DNA and high-level HHV-6 reactivation (plasma HHV-6 DNA ≥10(4) copies/mL) at day 70 after HCT was 72.2% and 37.0%, respectively. Multivariate analysis identified myeloablative conditioning (hazard ratio [HR], 1.9; P = .004), umbilical cord blood transplantation (UCBT) (HR, 2.0; P = .003), and male sex (HR, 1.6; P = .04) as risk factors for displaying high-level HHV-6 reactivation. HHV-6 encephalitis occurred in 7 patients, and cumulative incidence at day 70 was 3.0%. None of the144 patients without high-level HHV-6 reactivation and 7 of 86 patients (8.1%) with high-level HHV-6 reactivation developed HHV-6 encephalitis (P = .0009). Prevalence of HHV-6 encephalitis was significantly higher among patients receiving UCBT than in patients with other sources (cumulative incidence at day 70, 7.9% vs 1.2%, P = .008). In each of 7 patients with HHV-6 encephalitis, central nervous system (CNS) symptoms developed concomitant with peak plasma HHV-6 DNA (range, 21 656-433 639 copies/mL). CONCLUSIONS High levels of plasma HHV-6 DNA are associated with higher risk of HHV-6 encephalitis. UCBT is a significant risk factor for HHV-6 encephalitis. HHV-6 encephalitis should be considered if CNS dysfunction develops concomitant to high-level plasma HHV-6 DNA after allogeneic HCT.

Monitoring of human herpesviruses after allogeneic peripheral blood stem cell transplantation and bone marrow transplantation

Herpesviruses frequently cause serious complications after allogeneic bone marrow transplantation (allo‐BMT). Recent studies have shown more rapid immune reconstitution after allogeneic peripheral blood stem cell transplantation (allo‐PBSCT) compared with allo‐BMT. However, it has not been clarified whether the improved immune reconstitution after allo‐PBSCT is associated with a lower incidence of herpesvirus infections. We monitored the emergence of Epstein‐Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV‐6) and HHV‐7 DNA by a nested‐double polymerase chain reaction in peripheral blood leucocytes from 22 allo‐BMT and 16 allo‐PBSCT patients. Each virus had an unique temporal profile of detection. HHV‐6 DNA was detected most frequently at 3 weeks after transplantation, whereas CMV and EBV DNA were detected later (2–3 months). Detection rates of HHV‐6 DNA at 3 and 4 weeks after allo‐BMT were significantly higher than those after allo‐PBSCT (9/16 v 2/13 at 3 weeks, P < 0.01; 10/21 v 1/15 at 4 weeks, P < 0.01). Detection rates of the other three herpesviruses after the two types of allogeneic transplantation were not significantly different throughout observation period. Furthermore, detection of HHV‐6 DNA within the first 4 weeks was associated with delayed platelet engraftment after both allo‐BMT and allo‐PBSCT (P < 0.01). These results suggest an advantage for allo‐PBSCT over allo‐BMT in terms of suppression of HHV‐6 reactivation and prevention of subsequent complications.

A phase I-II trial of autologous peripheral blood stem cell transplantation in the treatment of refractory autoimmune disease

Objectives: To carry out a phase I-II trial to elucidate the feasibility and efficacy of high dose cyclophosphamide (CY) supported by autologous peripheral blood stem cell transplantation (PBSCT) in the treatment of severe and refractory autoimmune disease (AD). Methods: Peripheral blood stem cells (PBSCs) were mobilised during haematological recovery after relatively high dose CY (2 g/m2) for 2 days, followed by administration of granulocyte colony stimulating factor. After collecting PBSCs—more than 2×106 CD34+ cells/kg—by apheresis, CD34+ cells were immunologically selected and cryopreserved. Eight patients were enrolled—five had systemic sclerosis (SSc) alone, one had SSc with systemic lupus erythematosus, one amyopathic dermatomyositis (ADM), and one Wegener’s granulomatosis (WG). All of the patients were treated with high dose CY (50 mg/kg) for 4 days and autologous PBSCT. Results: Haematopoietic reconstitution was rapid and sustained. Toxicity due to the regimen included various infections such as pneumonia, sepsis, cystitis, herpes zoster, and acute heart failure. However, there was no treatment related mortality. Encouraging results were obtained after autologous PBSCT. Sclerosis of the skin was markedly improved in all of the patients with SSc. Interstitial pneumonia (IP), evaluated by Pao2, serum KL-6 levels, and pulmonary high resolution computed tomography, improved significantly. In a patient with ADM, severe and progressive IP also improved markedly. In a patient with WG, the size of the left orbital granuloma decreased substantially, resulting in reduction of the exophthalmos. Conclusions: These observations suggest that high dose CY with autologous PBSCT is feasible and may be effective in the treatment of severe and refractory AD.

Association of MBL gene polymorphisms with major bacterial infection in patients treated with high-dose chemotherapy and autologous PBSCT

A growing body of evidence indicates that genetic factors are involved in an increased risk of infection. We investigated whether mannose-binding lectin (MBL) gene polymorphisms that cause low levels of MBL are associated with the occurrence of major infections in patients, mainly bearing hematological malignancies, after high-dose chemotherapy (HDT) rescued by autologous peripheral blood stem cell transplantation (auto-PBSCT). A retrospective evaluation of 113 patients treated with HDT and auto-PBSCT revealed that the low-producing genotypes, B/B and B/LXA, were associated with major bacterial infection (P=0.0016, OR 7.9). We next performed a nation-wide large-scale study to assess the allele frequency of the MBL coding mutation in a total of 2623 healthy individuals in Japan. The frequency of allele B was estimated to be ∼0.2, almost the same in seven different areas of Japan. This common occurrence suggests that MBL deficiency may play an important role in the clinical settings of immunosuppression.

Cutting Edge: Tyk2 Is Required for the Induction and Nuclear Translocation of Daxx Which Regulates IFN-α-Induced Suppression of B Lymphocyte Formation

IFN-α inhibits B lymphocyte development, and the nuclear protein Daxx has been reported to be essential for this biological activity. We show in this study that IFN-α inhibits the clonal proliferation of B lymphocyte progenitors in response to IL-7 in wild-type, but not in tyk2-deficient, mice. In addition, the IFN-α-induced up-regulation and nuclear translocation of Daxx are completely abrogated in the absence of tyk2. Therefore, tyk2 is directly involved in IFN-α signaling for the induction and translocation of Daxx, which may result in B lymphocyte growth arrest and/or apoptosis.

Contribution of bone marrow-derived hematopoietic stem/progenitor cells to the generation of donor-marker⁺ cardiomyocytes in vivo.

Background Definite identification of the cell types and the mechanism relevant to cardiomyogenesis is essential for effective cardiac regenerative medicine. We aimed to identify the cell populations that can generate cardiomyocytes and to clarify whether generation of donor-marker+ cardiomyocytes requires cell fusion between BM-derived cells and recipient cardiomyocytes. Methodology/Principal Findings Purified BM stem/progenitor cells from green fluorescence protein (GFP) mice were transplanted into C57BL/6 mice or cyan fluorescence protein (CFP)-transgenic mice. Purified human hematopoietic stem cells (HSCs) from cord blood were transplanted into immune-compromised NOD/SCID/IL2rγnull mice. GFP+ cells in the cardiac tissue were analyzed for the antigenecity of a cardiomyocyte by confocal microscopy following immunofluorescence staining. GFP+ donor-derived cells, GFP+CFP+ fused cells, and CFP+ recipient-derived cells were distinguished by linear unmixing analysis. Hearts of xenogeneic recipients were evaluated for the expression of human cardiomyocyte genes by real-time quantitative polymerase chain reaction. In C57BL/6 recipients, Lin−/lowCD45+ hematopoietic cells generated greater number of GFP+ cardiomyocytes than Lin−/lowCD45− mesenchymal cells (37.0+/−23.9 vs 0.00+/−0.00 GFP+ cardiomyocytes per a recipient, P = 0.0095). The number of transplanted purified HSCs (Lin−/lowSca-1+ or Lin−Sca-1+c-Kit+ or CD34−Lin−Sca-1+c-Kit+) showed correlation to the number of GFP+ cardiomyocytes (P<0.05 in each cell fraction), and the incidence of GFP+ cardiomyocytes per injected cell dose was greatest in CD34−Lin−Sca-1+c-Kit+ recipients. Of the hematopoietic progenitors, total myeloid progenitors generated greater number of GFP+ cardiomyocytes than common lymphoid progenitors (12.8+/−10.7 vs 0.67+/−1.00 GFP+ cardiomyocytes per a recipient, P = 0.0021). In CFP recipients, all GFP+ cardiomyocytes examined coexpressed CFP. Human troponin C and myosin heavy chain 6 transcripts were detected in the cardiac tissue of some of the xenogeneic recipients. Conclusions/Significance Our results indicate that HSCs resulted in the generation of cardiomyocytes via myeloid intermediates by fusion-dependent mechanism. The use of myeloid derivatives as donor cells could potentially allow more effective cell-based therapy for cardiac repair.

JAK2 V617F uses distinct signalling pathways to induce cell proliferation and neutrophil activation

The acquired JAK2 V617F mutation is observed in the majority of patients with BCR‐ABL1 negative chronic myeloproliferative neoplasms (MPN). BCR‐ABL1 negative MPN displays myeloproliferation with an elevated leucocyte alkaline phosphatase (LAP) activity, a neutrophil activation marker. We tried to separate the downstream signalling of JAK2 V617F to stimulate myeloproliferation and LAP activity. NB4, a myeloid lineage cell line, was transduced with Jak2 V617F mutation or wild‐type Jak2. We found that Jak2 V617F mutation, but not wild‐type Jak2 enhanced LAP expression in NB4‐derived neutrophils and proliferation of NB4 cells. JAK2 V617F induces constitutive phosphorylation of STAT3 and STAT5, and uses signalling targets such as Ras/MEK/ERK and PI3K/Akt pathways. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, and STAT3 or STAT5 siRNAs, JAK2 V617F was found to specifically use the STAT3 pathway to enhance LAP expression, while STAT5, Ras/MEK/ERK and PI3K/Akt, but not STAT3 pathways, were able to stimulate cell proliferation. These data strongly suggest that JAK2 V617F uses distinct signalling pathways to induce typical pathological features of MPN, such as high LAP activity and enhanced cell proliferation.