ALBERT

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: 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: Interleukin-2 receptor (IL2R) is usually expressed on activated T cells by antigen stimulation and on pre-B cells during B cell differentiation. Soluble IL-2R (sIL-2R) in serum is a truncated form of the 55-kDa chain of IL-2R, which is believed to be produced by cleavage by proteases. The concentration of sIL-2R in serum has been an index of tumor burden in adult T cell leukemia/lymphoma (ATL), in which CD4-positive T cells express IL-2R (CD25) on the cell surface. Subsequently, analysis of serum sIL-2R concentration has also been useful in predicting disease activity and response to treatment in B cell lymphoma. However, it is still unclear whether B cell lymphoma cells express IL-2R (CD25) or whether serum sIL-2R concentration is due to IL-2R on B cell lymphoma cells. Furthermore, it is unclear how sIL-2R is released from IL-2R in ATL. First, we examined whether serum sIL-2R concentration is a prognostic factor in previously untreated patients with DLBCL (n = 105, median age 67.0 (18–91 years)) or FL (n = 30, median age 60.0 (40–82 years)) diagnosed between January 2001 and December 2005, and who received six cycles of R + CHOP or R + THP-COP therapy. Patients who relapsed or had disease progression after R + CHOP or R + THP-COP received R + EDAP or R + ICE for DLBCL, and R + FND for FL. The 5-year OS rates for patients with sIL-2R levels of 〈 1500 U/ml and ⊠ 1500 U/ml were 76% and 62%, respectively (p 〈 0.05) in DLBCL, and 100% and 79.3%, respectively (p = 0.19) in FL. Next, we analyzed IL-2R (CD25) expression on lymphoma cells by flow cytometry. Nine of 25 patients with DLBCL and 4 of 11 patients with FL showed CD25 expression. Some T cells (CD3-positive cells) expressed CD25 in both lymphomas. On the other hand, 7 of 7 patients with MCL expressed CD25. There was no significant relationship between serum sIL-2R concentrations and CD25 expression on lymphoma cells or clinical stage in either DLBCL or FL. Metalloproteinase-9 (MMP-9) is reported to be an important protease for releasing sIL-2R from IL-2R. However, there was no significant relationship between MMP-9 and sIL-2R levels in sera from patients with DLBCL or FL. On the other hand, 7 of 7 patients with ATL showed high concentration of MMP-9 (〉 128 ng/ml) irrespective of sIL-2R levels. We then confirmed MMP-9 expression in 3 ATL cell lines by Western blotting, and addition of MMP-9 inhibitor to culture media of these cell lines significantly decreased sIL-2R levels in supernatants. On immunohistochemical staining (IHC) using anti-MMP-9 antibody, macrophages not lymphoma cells or T cells were positive for MMP-9 in DLBCL and FL. These findings suggest that high serum sIL-2R concentrations in ATL are due to the cleavage of IL-2R by MMP-9 produced by ATL cells. On the other hand, the main source of sIL-2R may be due to release from activated T cells in DLBCL and FL. Therefore, serum sIL-2R levels may indicate activity of neoplastic cells in ATL; however, its levels may reflex the activity of microenviromental non-neoplastic cells in DLBCL and FL.

Description: Abstract 1902 Poster Board I-925 Myeloproliferative disorders (MPD), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages; they are associated with a high frequency of the Janus kinase 2 (JAK2) mutation V617F (JAK2V617F). Some patients with MPD exhibit leukemic transformation (LT) after several years of disease, but the mechanism of LT has been a matter of some controversy due to insufficient insight into the underlying molecular pathogenesis. LT may be a natural sequela of these diseases, whereas treatment with alkylating agents, hydroxycarbamide or their combination may increase the risk of LT. Previous studies have reported that chromosomal abnormalities including -5/5q- and -7/7q- were frequently detected in patients with MPD at the time of LT, suggesting that the development of cytogenetic abnormalities may be associated with the LT of patients with MPD. However, the genes involved in LT still remain obscure. Therefore, we attempted to identify gene alterations involved in LT from patients in the chronic phase (CP) of MPD. Patients with MPD were diagnosed and classified according to the WHO criteria between 1985 and 2007. Patients with PV, ET, PMF, MPD unclassified (MPD-U) and LT of these diseases were examined as approved by the institutional review board at Hiroshima University. Patients gave written informed consent, according to the Declaration of Helsinki. Mutations of JAK2V617F, AML1, CEPBA, FLT3, N-RAS, c-KIT, PTPN11 and TP53 were screened and were identified. Among 417 patients with MPD, 18 (4.3 %) patients progressed to leukemia. At LT, 13 of the 18 patients showed additional cytogenetic abnormalities, including -7/7q- with trisomy 21, inv(3)(q21q26), i(17)(q10) and t(11;19)(q23;p13.3), which are known to be associated with therapy-related leukemia. JAK2V617F was detected in 10 of 14 patients at LT. No patients had a JAK2 mutation pattern that changed during CP to LT. We analyzed gene mutations that may play an important role in leukemogenesis and found five AML1 mutations, one PTPN11 mutation and one CEBPA mutation in patients at the LT, whereas no mutation was detected in patients at CP. The gene alterations were detected at LT in both JAK2V617F-positive and −negative MPD patients, raising the possibility that the hematopoietic stem cells (HSCs) may have been transformed into leukemic blasts as a result of gene abnormalities. Among these gene abnormalities in the MPN patients, we focused on AML1 mutations. To clarify the leukemogenic effect of AML1 mutants, the AML1D171N mutant was transduced into CD34+ cells from eight patients in the CP of MPD using retrovirus transduction methods. The effect of this mutant on cell differentiation/proliferation was assessed by CFC re-plating assays. The D171N plates contained fewer erythroid colonies and more myeloid colonies than the controls. After re-plating, new colonies were detected on all D171N plates but on only few control plates. The D171N-transduced cells contained more CD34+ cells and proliferated more strongly than the controls. These results indicate that the D171N mutant has the potential to increase the myeloid immature cells and to enhance their self-renewal capacity. Furthermore, D171N-transduced cells retained more CD34+ cells than the controls after long-term culture on MS5 stroma cells, and showed significantly more colonies in long-term culture-initiating cells experiments. In this study, AML1 point mutations were detected with high frequency in patients at the LT from both JAK2V617F-positive and -negative MPD. Furthermore, the AML1D171N mutant transduced into CD34+ cells from MPD patients promoted proliferation of primitive progenitors, i.e. leukemic stem cells. These results indicate that AML1 point mutations may have a leukemogenic potential in JAK2V617F-positive stem cells or in pre-JAK2 stem cells, and they may promote leukemic transformation in MPD. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3613 BMI-1 is essential for the self-renewal and proliferation of leukemic and hematopoietic stem and progenitor cells. Increased expression of BMI-1 is known to be an indicator for a poor prognosis in cancer patients. Analysis of the expression of BMI-1 and survivin in 6 patients with B-cell lymphoma (3 drug-resistant and 3 sensitive cases) showed that in the drug-resistant patients, high levels of BMI-1 and survivin were maintained even after drug administration in vitro. However, there observed was a down-regulation of both BMI-1 and survivin expression in the drug-sensitive patients. BMI-1 transduction induced the drug-resistance of two B-cell lymphoma cell lines, HT and RL, to the anti-cancer drugs etoposide and oxaliplatin, but not to irinotecan. The expression of survivin was clearly augmented in the cells transduced with BMI-1. Moreover, we detected sustained expression of survivin level in the presence of etoposide in the BMI-1-overexpressing cells. By contrast, the mock-transduced cells succumbed in the medium with anti-cancer drugs with an accompanying decrease in the expression of survivin as well as BMI-1. Survivin has been reportedly implicated in resistance to chemotherapeutic agents. Intriguingly, survivin mRNA levels in BMI-1-overexpressing cells were consistent with those in controls. Also, the level of survivin was enhanced by treatment with a proteasomal inhibitor, MG132, suggesting that overexpression of BMI-1 stabilized survivin expression post-translationally. We further showed that sh RNA-mediated knockdown of BMI-1 or survivin restored sensitivity to etoposide in the HT cells overexpressing BMI-1. Our findings suggest survivin as a potential target for BMI-1. Thus BMI-1, by acting as an upstream regulator, may control the expression of survivin, facilitating drug resistance in B-cell lymphoma. Next, we examined whether B-cell lymphoma cells overexpressing BMI-1 are abrogated by immunotherapy with T cells containing anti-CD38 chimeric receptor in vitro. Interestingly, these B-cell lymphoma cells were effectively eliminated by specific T cells against B-cell lymphoma cells bearing CD38. These results suggest that the immunotherapy is useful for treatment of patients with B-cell lymphoma cells overexpressing BMI-1, which are refractory to chemotherapeutic reagents. BMI-1 may be an important prognostic marker as well as a future therapeutic target in the treatment of drug-resistant lymphomas. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2949 Adhesion of multiple myeloma (MM) cells to bone marrow stromal cells triggers cytokine-mediated tumor cell growth, survival, and drug resistance. In particular, integrin a4b1, very late antigen 4 (VLA-4)-mediated fibronectin adhesion confers a survival advantage to myeloma cells. One of the problems in treating patients with MM is that it is very hard to eliminate residual myeloma cells, even following high-dose chemotherapy followed by auto-stem cell transplantation. Importantly, cell adhesion-mediated drug resistance (CAM-DR) must be overcome in order to eliminate the minimal residual disease of MM. Here we characterized a multiple myeloma cell line, MSG1, which depends on HS23 stromal cells for its survival, was established from the pleural effusion of a patient with MM who expressed the M-protein of IgA-λ in his serum. During the first two months of culture, the myeloma cells survived on adhesive cells from the pleural effusion and subsequently, they continued to proliferate on HS23 stromal cells but not on HS27A stromal cells (both stromal cells were established by Torok-Storb B, Blood 1995). The phenotype of the established MSG1 cell line was: CD138+, CD38++, CD19−, CD56−, VLA-4+, VEGFR1+, and VEGFR2+. Furthermore, immunohistochemical staining demonstrated expression of IgA and λ chain in the cytoplasm. Karyotype analysis indicated complex chromosomal abnormalities, basically hypertriploidy including the deletion of chromosome 13 and 17, and c-myc translocation. MSG1 cells continued to proliferate, not only when co-cultured with HS23 cells, but also when cultured on fibronectin-coated plates with the supernatant of HS23 cells or RPMI1640 medium supplemented with 10% FBS (control medium) containing IL-6 (10 ng/ml). Notably, MSG1 could not survive in control medium containing IL-6 or in HS23 supernatant unless bound to fibronectin, which was also expressed on HS23 and HS27A cells. IL-6 and VEGF production were detected in the supernatants of both HS23 and HS27A stromal cells (36.8±4.5 pg/ml and 131±5.8 pg/ml; 13.2±1.9 pg/ml and 16664±418 pg/ml, respectively). Next, we analyzed the effect of tocilizumab, an anti-IL-6R antibody, and bevacizumab, an anti-VEGF antibody on MSG1 survival. Tocilizumab (50 μ g/ml) inhibited MSG1 survival when cultured on fibronectin-coated plates in control medium containing IL-6 (10 ng/ml), and tocilizumab (10 μ g/ml) inhibited MSG1 survival when cultured on HS23 stromal cells. However, bevacizumab (500 μ g/ml) did not show such inhibition. Therefore, MSG1 survival depends on HS23 stromal cells: in other words, it depends on binding to fibronectin and IL-6. If these factors induced CAM-DR in myeloma cells, MSG1 may be a unique myeloma cell line that will be useful for analysis of CAM-DR, and tocilizumab might be a useful drug for treatment of MM. Furthermore, since MSG1 could survive on irradiated HS27A cells, and since HS23 and HS27A express similar adhesion molecules (Torok-Storb B et al., Blood 1995), these data suggest that HS27A might secrete factors that are detrimental to MSG1 survival. The identification of such an inhibitory factors could be of interest in terms of the regulation of myeloma proliferation. Disclosures: No relevant conflicts of interest to declare.

Description: Recent study analyzing the gene expression in myeloma cells using cDNA microarray showed that myeloma cells are divided into 7 groups. Two showed a high expression of cyclin D1 together with a low expression of cyclin D2, and they belong to the low-risk group when analyzed based on event-free and overall survival. Since cyclin D1 promotes the cell cycle progression, we sought to explain why cyclin D1 overexpression appears to be a favorable prognostic variable for multiple myeloma (MM) patients treated with high-dose chemotherapy and single or double autologous transplantation. It is not clear whether the down-regulation of cyclin D2 in these myeloma cells might offset cyclin D1 overexpression in cell biology. We have established a myeloma cell line (RPMI8226) with cyclin D1 overexpression by transfection of the cyclin D1 gene using a retrovirus vector to analyze biological changes in myeloma cells with cyclin D1 overexpression. In comparison with myeloma cells transfected with the vector only (mock), the analysis of gene expression by cDNA microarray showed the down-regulation of cyclin D2 and MCL-1 and the up-regulation of CED6 in cyclin D1-transfected myeloma cells. However, there were no significant changes in Bcl-related genes between them. And as we expected, cyclin D1-transfected myeloma cells showed high proliferative activity, increased number of cells in S-phase, and increased pRb protein. Next we analyzed their sensitivity for bortezomib (Millennium Pharmaceuticals, Inc., USA), immunomodulatory thalidomide analogs (IMiD1, D2, D3) (Celgene Corporation, USA) and dexamethasone. Bortezomib and dexamethasone induced apoptosis at an earlier time point (12hr) in cyclin D1 transfectant compared to mock transfectant, concomitant with decreased expression of MCL-1, but with increased expression of Bim. Furthermore, we confirmed in cell culture condition with a low concentration of FCS that these results were not due to just promotion of the cell cycle caused by cyclin D1 overexpression. Given that myeloma cells with cyclin D1 overexpression easily undergo apoptosis upon bortezomib or dexamethasone treatment, this may explain why patients with those myeloma cells are in the low-risk group. With this finding, we then analyzed the expression of cyclin D1 by RQ-PCR and immunocytostaining before and after 2 or 3 cycles of VAD (vincristine, doxorubicin, dexamethasone). There was no significant difference between the response to VAD and the reduction rate of cyclin D1 positive myeloma cells or the decrease of cyclin D1 expression in RQ-PCR. Interestingly, cyclin D2 expression increases relatively in progressive disease (PD) after chemotherapy containing high-dose melphalan followed by autologous stem cell transplantation compared with those of cyclin D1 and D3 by RQ-PCR. Therefore, cyclin D1-induced chemosensitivity may be due to the induction of Bim, which consequently inhibits the function of MCL-1. New strategies to down-regulate of MCL-1 might be useful in the treatment of MM.

Description: A high incidence of somatically acquired point mutations in the AML1/RUNX1 gene has been reported in poorly differentiated acute myeloid leukemia (AML, M0) and in radiation-associated and therapy-related myelodysplastic syndrome (MDS) or AML. The vast majority of AML1 mutations identified in these diseases were localized in the amino (N)–terminal region, especially in the DNA-binding Runt homology domain. In this report, we show that AML1 point mutations were found in 26 (23.6%) of 110 patients with refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEBt), and AML following MDS (defined these 3 disease categories as MDS/AML). Among them, 9 (8.2%) mutations occurred in the carboxy (C)–terminal region, which were exclusively found in MDS/AML and were strongly correlated with sporadic MDS/AML. All patients with MDS/AML with an AML1 mutation expressed wild-type AML1 protein and had a significantly worse prognosis than those without AML1 mutations. Most AML1 mutants lost trans-activation potential, regardless of their DNA binding potential. These data suggested that AML1 point mutation is one of the major driving forces of MDS/AML, and these mutations may represent a distinct clinicopathologic-genetic entity.