ALBERT

Description: Studies on nasal T/natural killer (NK)–cell lymphoma have been hampered by its tendency to cause necrosis. Thus, the establishment of cell lines of this neoplasm would seem to be valuable. This study attempted to establish cell lines from primary lesions of this tumor, and successfully obtained 2 novel Epstein-Barr virus (EBV)–positive cell lines, SNK-6 and SNT-8, by means of high-dose recombinant interleukin 2. Flow cytometry showed that SNK-6 had an NK-cell phenotype, CD3−CD4−CD8−CD19−CD56+T-cell receptor (TCR) α/β− TCR γ/δ−, whereas SNT-8 was CD3+CD4−CD8−CD19−CD56+TCR α/β− TCR γ/δ+. These were consistent with immunophenotypes of their original tumors, and the cell lines had monoclonal EBV clones identical to ones in their original tumors. Thus, the cell lines developed from cells forming the primary lesions. Genotypic analysis showed that SNK-6 had unrearranged TCR and immunoglobulin heavy-chain genes, supporting the conclusion that SNK-6 was of NK-cell lineage. On the other hand, SNT-8 had rearranged TCR β-, γ-, and δ-chain genes, and together with its phenotype, SNT-8 proved to be a γδ T-cell line. This is the first report of the establishment of cell lines from primary lesions of nasal T/NK cell lymphomas, and the results demonstrated that there are at least 2 lineages, NK- and γδ T-cell, in this neoplasm. Moreover, it has been suggested that nasal T/NK cell lymphomas of these lineages may belong to the same clinicopathologic entity because both types of cases shared common clinical and histopathologic features.

Description: Less than 60% of patients with B-cell non-Hodgkin’s lymphoma (B-NHL) can be cured with contemporary therapy. Using artificial receptors it is possible to redirect the specificity of immune cells to tumor-associated antigens, a strategy that holds great potential as a novel cancer therapy. Since B-NHL cells invariably express CD19, we transduced human peripheral blood T lymphocytes with a recently developed receptor (anti-CD19-BB-ζ), which consists of the single-chain variable domain (scFv) of an anti-CD19 monoclonal antibody, the hinge and transmembrane domains of CD8α, and the signaling domains of CD3ζ and 4-1BB. CD3ζ delivers the primary stimulus upon receptor engagement, while 4-1BB delivers co-stimulatory signals that are crucial for T-cell cytotoxicity. It has been shown that elicitation of 4-1BB signaling enhances the immune response to tumors in vivo, even when an immune response cannot be induced by CD28 stimulation. Retroviral transduction led to anti-CD19-BB-ζ expression in T cells with high efficiency: median percent of transduced cells was 60.3% (range, 25.7%–83.4%; n = 9). T lymphocytes expressing anti-CD19-BB-ζ expanded more vigorously that T cells transduced with receptors lacking 4-1BB and exerted powerful cytotoxicity against the CD19+ B-NHL cell lines Raji, Daudi, RL, and HT in vitro: at a 0.5: 1 effector: target ratio, mean (± SD) cell specific lymphoma cell killing was 96.6% ± 4.6% after 5–7 days of culture (4 experiments in each cell line). Transduced T cells were also effective against freshly isolated cells from patients with diffuse large, follicular large, Burkitt, and mantle cell lymphoma cultured on bone marrow-derived mesenchymal cells: in 10 samples, cell killing was 93.6% ± 5.7% at a 0.5: 1 ratio after 5–7 days of culture. Sensitivity to anti-CD19-BB-ζ-mediated killing was observed regardless of high Bcl-2 expression. T cells expressing anti-CD19-BB-ζ were also effective in a xenograft model of NHL, in which NOD/SCID mice were inoculated subcutaneously with lymphoma cells (1 x 107). Subsequent inoculation of T cells (2 x 106) transduced with anti-CD19-BB-ζ receptors significantly suppressed tumor growth, whereas inoculation of T cells transduced with empty control vector had no effect (3 mice for each treatment). These results provide a rationale for clinical testing of autologous T cells modified with anti-CD19-BB-ζ receptors in patients with aggressive or relapsed B-NHLs refractory to conventional therapy.

Description: Antigen (Ag) immunization induces formation of the germinal center (GC), with large, rapidly proliferating centroblasts in the dark zone, and small, nondividing centrocytes in the light zone. We identified a novel nuclear protein, GANP, that is up-regulated in centrocytes. We found that GANP was up-regulated in GC B cells of Peyer's patches in normal mice and in spleens from Ag-immunized mice. GANP-positive cells appeared in the light zone of the GC, with coexpression of the peanut agglutinin (PNA) (PNA)-positive B220-positive phenotype. The expression of GANP was strikingly correlated with GC formation because Bcl6-deficient mice did not show the up-regulation of GANP. GANP-positive cells were mostly surrounded by follicular dendritic cells. Stimulation with anti-μ and anti-CD40 induced up-regulation of ganp messenger RNA as well as GANP protein in B220-positive B cells in vitro. GANP is a 210-kd protein localized in both the cytoplasm and nuclei, with a homologous region to Map80 that is associated with MCM3, a protein essential for DNA replication. Remarkably, GANP is associated with MCM3 in B cells and MCM3 is also up-regulated in the GC area. These results suggest that the up-regulation of GANP might participate in the development of Ag-driven B cells in GCs through its interaction with MCM3.

Description: Conventional, ex vivo culture of monocytes with recombinant proteins for their differentiation into DCs involves considerable manipulation under “Good Manufacturing Practices” conditions, and is not only more labor intensive but importantly, after ex vivo produced DCs are administered, they lack the stimulatory signals to keep them alive and functional and therefore are short lived. Because of these problems, we have evaluated an one-hit lentiviral transduction approach for genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature DCs. High-titer third generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) efficiently achieved simultaneous and persistent co-delivery of the transgenes into purified human CD14+ monocytes. Co-expression of GM-CSF and IL-4 in monocytes was sufficient to induce their differentiation into lentivirus-modified DCs (“DC/LVs”), as evidenced by their morphology, immunophenotype and immune-function*. Mixed lymphocyte reactions showed that the T-cell stimulating activity of DC/LVs was superior to that of DCs grown by conventional methods. DC/LVs displayed efficient antigen-specific, MHC Class-I restricted stimulation of autologous CD8+ T-cells, as shown by IFN-G production and CTL assays. Importantly, DC/LVs could be maintained metabolically active and viable in culture for 2–3 weeks in the absence of exogenously added growth factors, unlike conventional DCs *. We are now evaluating whether DC/LVs can be re-infused immediately after gene transfer to achieve stable and long-lasting differentiation in vivo. Additionally, the genetic engineering of monocytes is anticipated to generate DCs after one hit of lentiviral transduction, instead of the three consecutive steps for development of DCs (differentiation, maturation, gene delivery of tumor antigens). We have thus established a mouse model for testing DC/LVs in vivo for the treatment of melanoma. Bone marrow cells from C57BL/6 mice transduced with lentiviral vectors expressing GM-CSF and IL-4 recapitulated the same DC/LV morphology and immunophenotype obtained in the human system. Mouse DC/LVs were also more viable in vitro and outperformed conventional mouse DCs in pilot immunization assays as followed by CTL assays and IFN-G ELISPOT. We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Co-delivery of a gene for DC maturation (CD40L) and of gene encoding a tumor-associated antigens (MART-1) is being performed. Our goal is to evaluate the implications of simultaneous co-expression of GM-CSF/ IL-4/ CD40L/ MART-1 in DC/LV differentiation and migration to lymph nodes in vivo, immunopotency and safety. Once these pre-clinical considerations are addressed, we foresee a broad clinical application of genetically engineered DCs for vaccination purposes against cancer and chronic infectious diseases.

Description: New International Prognostic Index (IPI) staging system of multiple myeloma (MM) is a combination of the level of serum β2-microglobulin and serum albumin. Particularly, good survival (median survival 〉5 years) is associated with absence of chromosome 13q deletion. Recently, correlations between molecular subtypes and prognosis have been identified as a good prognosis with t(11;14) and a poor prognosis with t(4;14) and t(14;16) besides chromosome 13 abnormalities. We have reported that some MM cases with cyclin D1 overexpression detected by competitive RT-PCR were not caused by t(11;14)(q13;q32) or extra copies of 11q13 (In J Oncol, in press). A recent report revealed that subtypes of MM cases with the translocation of cyclin D showed a close correlation with bone disease and high level of DKK1. We also have been studing about the correlation between bone disease and bone morphogenetic protein (BMP) 2, or connective tissue growth factor (CTGF) that is supposed to inhibit the VEGF binding to its receptor or modulate cell signaling by BMP. First, we analyzed IPI staging in 91 MM cases, and then analyzed the relation between IPI staging and existence of cyclin D1 overexpression, or t(11;14)(q13;q32) and extra copies of 11q13. Competitive RT-PCR was performed in 77 cases, and cyclin D1 overexpression was detected in 40/77 (52%). Deletion of chromosome 13q was detected in 32/87 (37%), and t(11;14)(q13;q32) or extra copies of 11q13 was detected in 11/50 (22%) and 7/50 (14%), respectively. There were no significant differences of those factors among IPI staging. And we analyzed the scale of bone lesion by bone x-ray in 81 cases. We could not detect the relation between bone disease and cyclin D1 overexpression or translocation of 11q13. Furthermore, we analyzed the expression of BMP2 and CTGF by quantitative real time-PCR in purified myeloma cells or in bone marrow mononuclear cells (BMMNC) reduced myeloma cells less than 5%. We have gotten results that MM cases have a tendency to show higher CTGF expression in BMMNC compared with that of normal BM, but there was no significant difference of BMP2 expression in BMMNC between them. And there was no correlation between cyclin D1 overexpression and BMP2 or CTGF expression. So far a cause of bone lesions in MM is supposed to be the activity of osteoclast, however, our preliminary examination by TRAP staining revealed that osteoclast differentiation from BMMNC in MM cases by adding M-CSF (25 ng/ml) and RANKL (50 ng/ml) decreased compared with that in normal BM, and osteoblast diffentiation also decreased in MM by cytochemical staining for alkaline phosphatase (AP). We guess that both osteoclast and osteobalst differentiation are suppressed in MM and CTGF is a candidate for the suppressor of osteoblast differentiation. We will be able to show the result of AP activity of osteoblast and the effect of recombinant CTGF on osteoblast in meeting.

Description: Imatinib mesylate (Glivec; Novartis, Basel, Switzerland), which is a selective inhibitor of the Bcr/Abl tyrosine kinase, is now widely used for the treatment of Ph-positive leukemia based on the remarkable efficacy. Treatment with imatinib mesylate is generally well tolerated, and the risk for severe adverse effects is low. Hepatic toxicity is less common and usually resolves with interruption of imatinib therapy. On the other hand, although it is well known that HBV reactivations are observed in cancer patients with chronic HBV infection during chemotherapies, imatinib mesylate-induced HBV reactivation has not been reported yet. Here, we report the first case complicated by fatal fulminant HBV reactivation during imatinib mesylate treatment for CML. A 54-year-old man was diagnosed as CML on October 2003. At that time, HBsAg, HBeAb, and HBcAb were positive, whereas HBsAb was negative. HCV Ab and HCV RNA were also positive. However, hepatic examinations revealed normal findings, except slightly elevated levels of AST (50 IU/L). On November 2003, imatinib mesylate was started at the dose of 400mg/day p.o. and continued without any hepatic adverse effects. In contrast, the patient was suffered from neutropenia (grade 2) and lymphocytopenia (grade 2) on December 2003. Although dose of imatinib mesylate was reduced (300mg/day), lymphocytopenia continued. Since May 6, 2004, the patient complained general fatigue. On May 11, AST, ALT, and total bilirubin were 125 IU/L, 95 IU/L, and 0.7 mg/dl, respectively. At that time, bone marrow cytogenetics using a FISH of Bcr/Abl detected 2.0% fusion gene. Then, hepatic function deteriorated despite imatinib mesylate administration was stopped. On May 27, the patient had severe hepatic dysfunction with high AST, ALT, and total bilirubin (2098 IU/L, 1574 IU/L, and 5.6 mg/dl, respectively). In addition, prolonged prothrombin time (13.6%) and deterioration of consciousness were observed. HBV DNA polymerase was extremely increased (〉 20000 cpm; normal range:

Description: Multiple myeloma is an incurable hematological malignancy of plasma cells despite advances in conventional chemotherapy or high dose chemotherapy with stem cell transplantation. Recent advances in the biological treatment such as thalidomide and bortezomib will offer great promise to improve the outcome of refractory or relapsed patients with multiple myeloma; therefore, more novel biologically based therapies targeting both myeloma cells and its bone marrow microenvironment are urgently required. Shikonin, a Chinese herb derived from purple gromwell, Boraginaceae Lithospermum erythrorhizon, is previously known to have anti-inflammatory, anti-tumor, and anti-pyretic properties. Several studies have shown that Shikonin induces p53-mediated cell cycle arrest and apoptosis in human malignant melanoma cells, and induces apoptosis in human myeloid leukemic HL60 cells. However, none of the studies have concerned in the effects of Shikonin on myeloma cells. In this study, we investigated the possibilities to develop Shikonin as a novel promising agent for multiple myeloma. Shikonin suppressed the cellular growth of RPMI8226 and IM9 myeloma cells, via induction of apoptosis in a dose (0–1 μM)- and time (0–24 h)-dependent manner. Apoptotic cell death was induced in both RPMI8226 and IM9 cells within 3 h for treatment with only 0.5 μM of Shikonin, and finally 78% of RPMI8226 cells and 91% of IM9 cells were dead by apoptosis within18 h. Treatment with 0.5 μM Shikonin rapidly increased the population of cells in the G0/G1 phase with reduction of cells in the S phase, and then induction of apoptosis was confirmed by the appearance of cells in the sub-G1 fraction. Shikonin-induced apoptosis was in association with the loss of mitochondrial transmembrane potentials, and activation of caspase-3. Shikonin-induced apoptosis was completely blocked by the treatment with pan-caspase inhibitor (20 μM, Z-VAD), suggesting that caspase plays an important role in Shikonin-induced cell death in myeloma cells. Expression of p53 and Bax proteins was increased with down-regulation of Mcl-1 protein, but no changes were confirmed of the expression of Bcl-2 and Bcl-XL. Recent investigations have shown that cytokines such as IL-6, insulin growth factor (IGF), VEGF, and TNF-α mediate myeloma cell growth, survival and migration. IGF-1 increases survival of myeloma cells by activating various down-stream signal transduction molecules. Recently, inhibitors of IGF-1 receptor demonstrate promising anti-multiple myeloma activity in preclinical studies. Shikonin has reported to be an inhibitor of protein tyrosine kinase such as EGFR, v-Src, and KDR/Flk-1. To address the mechanism of Shikonin-induced apoptosis in myeloma cells, we thus examined the effects of Shikonin on the phosphorylation of IGF-1 receptor. IGF-1 (100 ng/ml) stimulated the proliferation of both RPMI8226 and IM9 cells. Interestingly, Shikonin (0.5 μM) overcame IGF-1-induced cell proliferation, and inhibited proliferation of myeloma cells via induction of apoptosis. Shikonin inhibited phosphorylation of IGF-1 receptor as early as 30 min with inhibition of PI3K/Akt signaling. These results suggest that Shikonin-induced apoptosis in myeloma cells was mediated by inhibiting phosphorylation of IGF-1 receptor and modulating its down-stream signaling pathway. In conclusion, Shikonin inhibited cellular growth by inhibiting IGF-1 receptor signaling in myeloma cells, and may have a potential as a novel biologically based therapeutic agent for the patients with multiple myeloma..

Description: Background: The mutational status of immunoglobulin VH genes (IgVH) is an important prognostic marker in chronic lymphocytic leukemia (CLL), but its determination remains unadapted to routine practice. Several reports have showed that ZAP-70, whose expression can be detected by flow cytometry, can be considered as a reliable surrogate marker of IgVH mutational status. We recently conducted a gene expression profiling study of 18 cases of CLL, which pointed out 2 other genes which might also discriminate CLL groups: the lipoprotein lipase (LPL) and the disintegrin and metalloprotease 29 (ADAM29) genes which were overexpressed preferentially in unmutated (UM) and mutated (MT) cases respectively. Methods: We analyzed frozen cells obtained at diagnosis for 127 CLL patients (87 Binet stage A, 40 stage B or C). LPL, ADAM29 and house keeping GAPDH gene expression were measured by real time quantitative polymerase chain reaction in 111 cases, and ZAP-70 protein by flow cytometry in 107 cases, both analyses being performed in 93 cases. LPL and ADAM29 were also quantified in peripheral blood mononuclear cells (PBMC, n=4) and purified B cells (n=3) of healthy individuals. We correlated the results with the previously determined IgVH mutational status and clinical outcome. Results: With a cut-off value determined to be 1 for the LPL/GAPDH copy number ratio, LPL expression had a positive predictive value (PPV) of 68% for UM cases and a negative predictive value (NPV) of 85% for MT patients. Alternatively ADAM29 expression (ADAM29/GAPDH 〉 3) had a PPV of 77% for MT cases and of 86% for UM cases. Combining LPL and ADAM29 RNA quantifications by a simple 1:1 ratio (L/A ratio; threshold=1) provided slightly better results than those obtained with ZAP-70 (positivity threshold = 20%) for PPV of UM status (90% vs 76%) and similar results for NPV of MT status (90% vs 91%). Simultaneaous usage of L/A ratio and ZAP-70 expression allowed an almost perfect (73/74) assessment of the IgVH status in 80% (74/93) of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). Serial measurements of L/A ratio and ZAP-70 expression showed that both parameters can change over time (median follow-up 38 months, range 6–159) in a fraction of patients (5/25 tested). ADAM29 was not detected while LPL was present at very low levels or absent in PMBC or purified B cells from healthy donors. The IgVH mutational status, ZAP-70 and L/A ratio were predictive of event-free survival for the whole cohort and for stage A patients. However only the L/A ratio was significantly associated with a shorter survival (p=0.03) for stage B/C patients. Conclusions: Combination of LPL and ADAM29 mRNA quantification provides a good surrogate marker of the IgVH mutational status in CLL patients. Used in association with ZAP-70, it represents an reliable alternative to the sequencing of IgVH genes. In addition, it might constitute a more powerful prognostic marker than IgVH mutational status or ZAP-70.

Description: Imatinib mesylate has drastically changed Philadelphia chromosome positive (Ph+) leukemia treatment. However, remissions induced in advanced phase Ph+ leukemias tend to be short-lived even treated with imatinib. Combined therapy of imatinib with an agent which inhibits downstream signaling of BCR/ABL such as Ras is intriguing. Phase I studies of farnesyl transferase inhibitors (FTIs), which targeted farnesylation of Ras, with imatinib against Ph+ leukemias demonstrated limited combination effects. We previously reported that the third-generation bisphosphonates (BPs) zoledronate (ZOL) synergized with imatinib mesylate to inhibit Ph+ leukemia. BPs which inhibit both farnesylation and geranylgeranylation of Ras proteins may be more potent than FTIs. However, three important questions have remained unanswered to promote this attractive combination to a clinical trial. Concentrations of 20-30 μM ZOL are required in vitro to induce apoptosis in leukemic cell lines. However, peak serum concentrations after infusion of 4 mg ZOL were 1–2 μM. Therefore, it is important to investigate how ZOL achieves effective concentrations in vivo. The second is whether ZOL augments the effects of imatinib against not only cell lines but also primary cells from Ph+ leukemia patients. In addition, the safety of this combination therapy must be established, because the dose of ZOL used in our previous study was high compared with clinical doses administered for inhibition of bone lesions. To answer these questions, we herein investigated about the combination therapy of ZOL plus imatinib using NOD/SCID mice engrafted with primary Ph+ leukemic cells with or without mutations in the ATP binding site. ZOL inhibited the geranylgeranylation of Ras proteins of leukemic cells in bone marrow but not in peripheral blood, indicating that sufficient concentration of ZOL could be achieved in bone marrow. ZOL synergized with imatinib to enhance survival of mice engrafted with primary Ph+ leukemic cells without mutations and this combination was well-tolerated without any severe adverse effects. These findings suggest that ZOL inhibits leukemia growth in bone marrow and the combination of ZOL plus imatinib is a potential therapy for Ph+ leukemia patients.