Low cerebrospinal fluid-to-plasma ratios of orally administered lenalidomide mediated by its low cell membrane permeability in patients with hematologic malignancies.

Lenalidomide is a synthetic analog of thalidomide formed by the removal of one keto group (plus the addition of an amino group); it has anti-tumor activities beneficial for the treatment of hematologic malignancies. However, lenalidomide distribution to brain in animal models is reportedly low compared with that of thalidomide. The aim of this study was to evaluate plasma and cerebrospinal fluid concentrations of lenalidomide in three patients with malignant hematologic malignancies. Lenalidomide was detected in plasma from the three Japanese patients 1.5 h following oral administration of 20 mg lenalidomide using liquid chromatography/mass spectrometry, despite the in vitro gastrointestinal permeability of lenalidomide being low. Clinically observed cerebrospinal fluid-to-plasma ratios of lenalidomide were low (1.3-2.4%). Observed influx permeability values for lenalidomide in monkey blood-brain barrier model and human placental cell systems were one order of magnitude lower than those of thalidomide and another second-generation drug, pomalidomide along with a positive permeability control, caffeine. Because of the low cell-barrier permeability of lenalidomide demonstrated in in vitro assays, clinically relevant pharmacokinetic profiles of lenalidomide resulted in low penetrability from plasma into cerebrospinal fluid in patients with hematologic malignancies. Lenalidomide is conclusively suggested to expert its favorable immunomodulatory effects via systemic exposures in the patients.

Clinical outcome of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly in the rituximab era.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of malignant lymphoma. The incidence of Epstein-Barr virus (EBV)-positive DLBCL in Asian and Latin American countries ranges from 8 to 10%. The prognosis of patients with EBV-positive DLBCL is controversial. To compare the clinical outcome of EBV-positive and EBV-negative patients with DLBCL in the rituximab era, we analyzed 239 patients with de novo DLBCL diagnosed between January 2007 and December 2011. The presence of EBV in lymphoma cells was detected using EBV-encoded RNA in situ hybridization, and it was found that 18 (6.9%) of 260 patients with diagnosed DLBCL tested positive. Among the 260 cases, 216 cases were treated with rituximab plus chemotherapy, as were 8 EBV-positive DLBCL patients. The median overall survival and progression-free survival times in patients with EBV-positive DLBCL were 8.7 months and 6.8 months, respectively. The median overall survival and progression-free survival could not be determined in EBV-negative DLBCL patients (P = 0.0002, P < 0.0001, respectively). The outcome of patients with EBV-positive DLBCL remains poor, even in the rituximab era.

Clinicopathological Analysis of 320 Cases of Diffuse Large B-cell Lymphoma Using the Hans Classifier.

The estimation of clinical prognosis for diffuse large B-cell lymphoma (DLBCL) with a quick, cost-efficient method is necessary because of the clinical heterogeneity of this disease, which leads to death, relapsed or refractory disease in approximately 40% of patients. We analyzed 320 cases diagnosed from 2007 to 2013 treated with R-CHOP therapy at Tokai University Hospital and associated institutions. DLBCL was classified according to the cell-of-origin using the Hans algorithm [germinal center B-cell-like (GCB) vs non-GCB subtypes], and into 6 subgroups derived from combinations of CD10, BCL6 and MUM1 markers. The percentage of GCB and non-GCB (NGCB) subtypes was 35% and 65%, respectively. GCB-DLBCL was characterized by lower BCL2 immunohistochemical expression, extranodal sites ＜1, better therapeutic response, and favorable overall survival (OS) and progression free survival (PFS) (P＜0.01). The most frequent subgroup was NGCB-1 (CD10-BCL6+MUM1+, 51%) followed by GCB-1 (CD10+BCL6+or-MUM1+, 21%), NGCB-2 (CD10-BCL6-MUM1+, 13%), GCB-2 (CD10+BCL6+or-MUM1-, 10%), GCB-3 (CD10-BCL6+MUM1-, 4%) and NGCB-3 (CD10-BCL6-MUM1-, 2%). In comparison with GCB-2 and GCB-3 (both MUM1-), the GCB-1 (MUM1+) was characterized by favorable PFS (5-year PFS 84% vs 65%, OR 0.368, P＜0.05), independent of high LDH (associated with unfavorable PFS, OR 7.04, P＜0.01) in the multivariate analysis. This predictive value of MUM1 was independent of CD10. Interestingly, triple-negative NGCB-3 tended to have a more favorable prognosis than the other NGCB subgroups. In conclusion, the Hans classifier is a valid method to evaluate the prognosis of DLBCL NOS. In the GCB subtypes, GCB subtypes, MUM1-positivity is associated with a more favorable outcome (PFS).

Induction of cytomegalovirus-specific CD4+ cytotoxic T lymphocytes from seropositive or negative healthy subjects or stem cell transplant recipients.

OBJECTIVE: We generated cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) in vitro using dendritic cells (DC) pulsed with crude CMV antigens (Ag). PATIENTS AND METHODS: Mononuclear cells from healthy CMV-seropositive or seronegative volunteers and from stem cell transplant (SCT) recipients were cultured with CD14(+) monocyte-derived DC prepulsed with CMV Ag and then matured in vitro with lipopolysaccharide and tumor necrosis factor-alpha. After proliferation, cells were checked for phenotype (CD4/CD8), while killing activity was measured by 51Cr-release assay. RESULTS: CD4(+) T cells, the main proliferating cells from both seropositive and seronegative individuals, killed autologous Ag-pulsed DC but not vehicle-pulsed autologous DC or CMV-pulsed allogeneic DC. Similar CTL induction was accomplished from SCT recipients. Significant killing of autologous CMV-infected fibroblasts required 16-hour incubation as opposed to the standard 4-hour incubation, which was prevented by either a perforin inhibitor or anti-Fas ligand monoclonal antibody. CTL enhanced surface HLA-DR expression of CMV-infected fibroblasts, and their activity was neutralized by anti-HLA-DR monoclonal antibody. CONCLUSION: CMV-specific CD4(+) CTL were inducible with or without antiviral humoral immunity, even from immunosuppressed SCT recipients. These CTL showed perforin- and Fas/Fas ligand-mediated cytotoxicity after long-term (16-hour) contact with CMV-infected targets.

Umbilical cord blood dendritic cells are a rich source of soluble HLA-DR: synergistic effect of exosomes and dendritic cells on autologous or allogeneic T-Cell proliferation.

In this study, we compared soluble HLA-DR (sHLA-DR) production in the culture supernatants of various cell sources [T and B cells, monocytes and dendritic cells (DCs) either from adult peripheral blood (PB) or umbilical cord blood (UCB)]. DCs produced the highest amount of sHLA-DR molecules as compared to other cell sources, with UCB DCs producing the highest amount. Different kinetics of sHLA-DR production were found between immature and mature UCB DCs (mDC, iDC) (derived either from CD34(+) or CD14(+) cells). Maximum production of sHLA-DR was observed in 72-hour culture supernatants of both CD34- and CD14-derived mDCs, whereas it peaked in the 24-hour culture supernatants from iDC. sHLA-DR molecules were pelleted after sequential centrifugation from UCB CD34(+) DCs and were found to contain both 36 kD alpha-chain and 29 kD beta-chain of HLA-DR, CD86, and Fas molecules. These sHLA-DR containing vesicles/exosomes alone evoked weak proliferative responses from autologous and allogeneic T cells, but the immune response was significantly increased when vesicles/exosomes were presented with DCs.

The in vitro generation of Ph1+ ALL-specific HLA-A24-restricted cytotoxic T lymphocytes using a synthetic 16 mer minor bcr-abl peptide.

Sixteen mer peptide, which spans the junctional region of the acute lymphoid leukemia (ALL)-specific minor bcr-abl fusion protein and contains a motif that can bind to human leukocyte antigen (HLA)-A24, was constructed. We tried to generate Philadelphia chromosome 1 (Ph1) positive ALL-specific cytotoxic T lymphocytes (CTLs) from eight normal HLA-A24+ individuals with peptide-pulsed autologous dendritic cells (DCs). CTLs could be generated from the mononuclear cells (MNCs) of a single donor, which could kill peptide-pulsed autologous DCs and two A24+ ALL lines, while an HLA-A24+ CML line was only weakly killed and unpulsed DCs or the control lines Daudi or K562 were not recognized. Those CTLs consisted predominantly of CD8+ T cells whose cytotoxicity could be neutralized by monoclonal antibodies to HLA-class I or HLA-A24, and also produced interferon (IFN)-gamma after being stimulated with peptide-pulsed DCs.

Clinical effects of infusing anti-Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocytes into patients with severe chronic active EBV infection.

Immune cell therapy with autologous Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocytes (CTLs) or lymphokine-activated killer (LAK) cells was performed in 2 adults with severe chronic active EBV infection (SCAEBV). The patient in case 1, who had complications of pancytopenia, high fever, and massive splenomegaly, was treated with 13 doses of LAK cell infusion followed by 4 doses of autologous CTL infusion. The patient in case 2, who had liver dysfunction due to natural killer cell-type infection, was treated with 4 doses of autologous CTL infusion. In case 1, the LAK cell infusions were effective in lowering the viral load and improving several biochemical parameters (lactate dehydrogenase, soluble interleukin 2 receptor) and resulted in complete amelioration of the high fever. Subsequent infusions of autologous CTLs reduced the viral load only temporarily and were accompanied by an increase in frequency of EBV-specific T-cells in the blood. However, the patient's main problem of pancytopenia was not resolved. In case 2, infusion of autologous CTLs did not improve the patient's hepatic dysfunction or viral load but caused a significant increase in autoantibody levels. Thus the effect of auto-CTL treatment was limited or deteriorative in SCAEBV patients.

The generation of immunocompetent dendritic cells from CD34+ acute myeloid or lymphoid leukemia cells.

The ability of CD34+ leukemic cells to differentiate to dendritic cells (DCs) was investigated in 18 acute myeloid leukemia (AML) and 4 lymphoid leukemia (ALL) patients. The generation of DCs was determined by the expression of DC-associated CD1a or CD83 (more than 30%) with costimulatory molecules, by CD80 antigens (>20%), and by the exhibition of allostimulatory activity. In the AML patients, allostimulatory mature DCs were generated from 3 of 9 M0 or M1, 2 of 5 M2,2 of 4 M4 or M5, and 3 of 4 ALL (L2) cases. In total, DCs were more efficiently induced from cases expressing over 75% of CD34+ among whole bone marrow mononuclear cells (8 of 12), compared with those under 75% (2 of 10; P < .05). B-cell (CD19), natural killer (NK)-cell (CD56), or T-cell (CD7) lineage markers, which were aberrantly expressed on the blasts, were rarely found on leukemic DCs at the end of the culture period, and myeloid (CD13, CD33), not lymphoid (CD10), markers were shown on ALL-derived DCs. In Philadelphia chromosome-positive ALL or AML patients with t (8;21), DCs were confirmed to be of leukemic origin by fluorescence in situ hybridization analysis.

Phase I Clinical Trial of Intravenous L-ascorbic Acid Following Salvage Chemotherapy for Relapsed B-cell non-Hodgkin's Lymphoma.

PURPOSE: To determine the safety and the appropriate dose of intravenous l-ascorbic acid (AA) in conjunction with chemotherapy for patients with relapsed lymphoma. PATIENTS AND METHODS: Patients with relapsed CD20-positive B-cell non-Hodgkin's lymphoma, who were going to receive the CHASER regimen as salvage therapy, were enrolled and treated with escalating doses of AA administered by drip infusion after the 2nd course of the CHASER regimen. The target plasma concentration immediately after AA administration was >15 mM (264 mg/dl). RESULTS: A serum AA concentration of >15 mM was achieved in 3 sequentially registered patients, all of whom had received a 75 g whole body dose. No obvious adverse drug reaction was observed in the patients. The trial was therefore successfully completed. CONCLUSION: Intravenous AA at a whole body dose of 75 g appears to be safe and sufficient to achieve an effective serum concentration. A phase II trial to evaluate the efficacy of intravenous AA in relapsed/refractory lymphoma patients will now be initiated.

Secondary pulmonary alveolar proteinosis in a patient with chronic myeloid leukemia in the accelerated phase.

Pulmonary alveolar proteinosis (PAP) is a rare respiratory disease the character of which is accumulation of protein consisting of surfactant in alveolar spaces. PAP sometimes complicates with hematological malignancies, especially myeloid leukemia. As one of the cause of PAP, impairment of alveolar macrophage is considered. We experienced a case of PAP with chronic myeloid leukemia (CML). 41 years old woman having CML for nine years developed PAP, and was treated by bronchoalveolar lavage and imatinib. She died of respiratory failure in the end, but BAL fluid had been becoming gradually crystalline after induction of imatinib. We consider that we should try to treat to improve respiratory status not only PAP but also hematological disease.

Killing activity of human umbilical cord blood-derived TCRValpha24(+) NKT cells against normal and malignant hematological cells in vitro: a comparative study with NK cells or OKT3 activated T lymphocytes or with adult peripheral blood NKT cells.

PURPOSE: We aimed to determine the effects of human umbilical cord blood (UCB)-derived natural killer T (NKT) cells as immunological effectors against hematological malignancies, as well as auto- or allo-dendritic cells (DCs) or EB transformed cell lines (EBCLs). MATERIALS: TCRValpha24(+) Vbeta11(+) UCB- or PB-NKT cells were isolated by sorting and activated by alpha-galactosylceramide-pulsed autologous DCs. UCB-NK cells were induced from CD34(+) cells by stem cell factor plus IL-15. UCB-T cells were primarily activated by anti-CD3 monoclonal antibody. All those effectors were cultured with IL-2 (100 U/ml), and their cytotoxic activities were evaluated by (51)Cr-release assay. UCB-NKT cells were cultured with IL-12, IL-18 or higher dose of IL-2 (1000 U/ml), and again tested for the cytotoxicity against selected targets. RESULTS: UCB-NKT cells exhibited a pattern of killing activity against various hematological malignancies similar to that of UCB-NK cells, but could not kill K562, which was a vulnerable target for NK cells. The level of activity was quite similar to that of PB-NKT cells. In contrast, OKT-3-activated UCB-T lymphocytes showed a stronger and wider spectrum of killing compared with UCB-NK or NKT cells. IL-12, IL-18 or a higher dose of IL-2 upregulated the activity; however several targets, including fresh leukemic cells, still remained resistant. NKT cells killed auto- or allo-DCs at a level similar to that of T cells, but could not kill allo-EBCLs, which were efficiently killed by T cells. While NK cells showed only marginal or no killing against DC or EBCLs. DISCUSSION: The anti-cancer activity of human NKT cells depends on the concentrations or the combination of Th1-cytokines. Basically, those cells might not be contributing to the immune surveillance of hematological malignancies, as shown by a relatively low cytotoxicity against malignant cells, together with the quite strong killing against auto-DCs.

The effects of alphaGalCer-induced TCRValpha24 Vbeta11(+) natural killer T cells on NK cell cytotoxicity in umbilical cord blood.

PURPOSE: The first objective of this study was to investigate in vitro effects of alpha-galactosylceramide (alphaGalCer) on the proliferation of umbilical cord blood (UCB) natural killer T (NKT) cells and enhancement of their cytotoxicity. The second one is to examine whether purified NKT cells could affect the cytotoxicity of UCB-NK cells either in the presence or absence of dendritic cells (DCs). METHODS: Mononuclear cells (MNCs) from UCB were cultured for 2 weeks in the presence of IL-2 (100 U/ml), with or without alphaGalCer. The effect of neutralizing monoclonal antibodies (MoAb) against TCRValpha24 and CD1d was also examined. TCRValpha24 Vbeta11 double positive NKT cells were purified by FACS sorter and then cocultured with syngeneic isolated UCB(-)CD56(+)NK cells in either the presence or absence of DCs. The cytotoxicity against various malignant cell targets and cytokine production was determined. RESULTS: The addition of alphaGalCer induced human NKT cells to proliferate in UCB-MNCs to a greater extent than in adult PB-MNCs. However, it suppressed the cytotoxic activity against malignant cell targets. Anti-TCRValpha24 and CD1d MoAb recovered the cytotoxicity by inhibiting the proliferation of UCB-NKT cells. NKT cells cocultured with auto-DCs significantly increased NK cell cytotoxicity against K562, and Raji cells and produced IFN-gamma at much higher levels than UCB-NKT cells alone. CONCLUSION: In UCB samples, alphaGalCer-pulsed DCs and NKT cells acted together to enhance NK cytotoxicity in vitro.