ALBERT

Description: Abstract 1820 Poster Board I-846 T cells contribute to the immunomodulatory control of the tumor in patients with monoclonal gammopathies. We previously found that CD3+CD8+CD57+TCRVβ+restricted cytotoxic T cell expansions were present in 48% of patients with multiple myeloma (n=221) and conferred a significant favorable prognosis. We now report the presence of these expansions in 70% of patients with Waldenstrom's Macroglobulinaemia (WM) (n=20) with a wide spectrum of the TCRVβ repertoire represented. Previous nucleoside analogue (NA) therapy, known to be associated with an increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVβ expansions (χ2=11.6; p

Description: Previous studies have suggested that expanded T-cell clones are found in the blood of 59% of patients with multiple myeloma. These expanded T-cell clones are associated with prolonged overall survival and thus it has been suggested that they may have anti-tumor activity. We have previously reported similar T-cell clones exist in the peripheral blood of patients with Waldenstrom’s Macroglobulinemia (WM) by using flow cytometry to determine the T cell receptor (TCR) Vβ repertoire. Expanded T-cell clones were detected in 9 of 15 (60%) patient samples. Of the nine patients with TCR Vβ clones, four patients had multiple clones. The TCR Vβ clones were not identical, representing a variety of families across the TCR Vβ repertoire. We have previously found that while the TCRVβ+CD8+CD57 negative subset represents polyclonal populations, the CD57 positive subset represents either monoclonal or biclonal populations. By comparing the genetic profiling of these two subsets from a statistically significant gene list, two genes have been found to be highly upregulated in the CD57 negative polyclonal subset. These two genes are i.) SESN3, a member in the Sorting Nexin (SNX) protein family which is implicated in regulating membrane traffic capable of interaction with phosphatidylinositol-3-phosphate (10.4 fold, p=0.0241); ii.) Epstein-Barr virus induced gene 2 (lymphocyte-specific G protein-coupled receptor) EBI2 (7.4 fold, p=0.0207): This finding is in contrast to previous report that EBI2 is expressed in B-lymphocyte cell lines and in lymphoid tissues but not in T-lymphocyte cell lines or peripheral blood T lymphocytes. For the CD57 positive clonal T cell expansions, consistent with our previous reports, CD28 expression was found to be down regulated by 2.6 fold. There are two genes found to be highly upregulated. They are i.) Granzyme B (4.3 fold, p=0.0337) also called Cytotoxic T-lymphocyte proteinase 2. This enzyme is necessary for target cell lysis in cell-mediated immune responses through caspase-dependent apoptosis; ii.) Granzyme H, also called Cytotoxic T-lymphocyte proteinase and probably necessary for target cell lysis in cell-mediated immune responses. In summary, we have shown that CD57 positive clonal T cell populations exist in some patients with WM. Importantly, microarray results have indicated some genes and proteins that may related to better patients survival as previously demonstrated in patients with Multiple Myeloma.

Description: Although thalidomide has been reported to increase T cell activity and NK cell cytotoxicity in the blood of patients with multiple myeloma (MM) it is not known whether thalidomide stimulates tumour-specific T cells or causes a general lymphocytosis. We investigated the immunodulatory effects of thalidomide by studying peripheral blood samples from 64 patients in the Australian ALLG MM6 trial, a multicentre randomised phase III study of low-dose thalidomide, prednisolone and Zometa versus prednisolone and Zometa used as post-autologous stem cell transplant (ASCT) maintenance therapy in patients with MM. We have previously demonstrated that TCR Vβ clonal expansions are present in approximately 50% of patients with MM and that their presence correlates with a good prognosis. Sequencing has confirmed that these cells are a clonal expansion of CD3+CD8+CD57+CD28−CD27− late-differentiated cytotoxic effector cells. Flow cytometric analysis of TCR Vβ expansions (24 families) was performed on blood samples from 64 patients enrolled in the MM6 trial both prior to and 12 months post transplant. Prior to transplant, 59% of the 64 patients had TCR Vβ expansions. After thalidomide, T cell expansions were found in a further 8/34 patients compared with 3/30 in the control, no thalidomide group. In the thalidomide group, 70% of the patients had an expansion of more than one T cell clone compared with 32% in the control group (chi2=28.8; p

Description: Abstract 5029 Multiple myeloma (MM), cancer of the plasma cells, remains incurable despite advancement in therapeutic regiments. Studies showed that the ‘side population (SP)’, a subpopulation enriched with CSC in various cancers, has higher drug transporter activity than the bulk tumor; suggesting the reason why these cells survived despite post-chemotherapy. The increasing evidence of cancer stem cells (CSC) suggests that anti-cancer drugs targeting this subpopulation may lead to eradication of the root of cancer recurrence. In this study, we explored the capability of kaempferide (KFD), a flavanoid in propolis (bee glue) that can reverse drug transporter activity, to combat SP cells in myeloma. KFD was one of the compounds in Brazilian propolis that reduced SP percentage in myeloma cell lines. We report for the first time that KFD is able to induce apoptosis in the SP cells in myeloma. The ability of KFD to inhibit growth of unfractionated KMS-11 cells was first investigated. Parthenolide (PAR), a natural product shown to inhibit growth of putative CSC in acute and chronic myelogenous leukemia was included as control. Unfractionated cells were seeded at 20000 cells per well in a 96 well plate. After 24h of incubation with various concentration of KFD, PAR, and DMSO (vehicle control), MTS solution was added and absorbance at 490 nm was determined. The IC50 of KFD and PAR in KMS-11 cells was 26 μM and 5 μM with 95% confidence intervals between 17.7 to 33.3 μM and 4.0 to 5.8 μM respectively. This is shown in the dose-response curve in figure 1A. No significant growth inhibition was observed in DMSO (0.1% to 0.5 % v/v) treated cells. We then examined if the KFD causes apoptosis in the sorted-SP cells. Sorted-SP cells were treated with 26 μM KFD, 5 μM PAR, or 0.2% v/v DMSO as mentioned above. After 1, 3, and 6 hour of incubation, cells were harvested and stained with Annexin V and propidium iodide and then analyzed using FACS Calibur. Result showed that percentage of Annexin V+ apoptotic cells in KFD-treated cells increased in a time series manner (1, 3, 6 h) (figure 1B). Because KFD was reported to be capable to reverse the activity of drug efflux transporter, further studies to investigate the synergistic effect of KFD with conventional drugs to treat myeloma will be carried out. Alternative medicines employing natural products have become increasingly sought after when conventional drugs cause immense side effects. Component in propolis that was reported to have anti-cancer properties, has low toxicity at high concentration, and spare normal cells, appeals to be a potential compound to combat myeloma. Exploitation of KFD that has the dual effect to induce apoptosis in putative CSC in myeloma and reverse drug transporter activity offers great opportunity in cancer drug development and future clinical trials in patients with myeloma. A B Annexin V-FITC (FL-1) Propidium iodied (FL-3) Figure 1. (A) Growth inhibition by KFD and PAR after 24h of incubation. Experiment was repeated at least 3 times. (B) KFD induced apoptosis in KMS-11 in a time-series manner. Cells were treated with 26 μM KFD or 5 μM PAR for 1, 3, and 6 hour and was stained with Annexin V and propidium iodide. KFD PAR Figure 1. (A) Growth inhibition by KFD and PAR after 24h of incubation. Experiment was repeated at least 3 times. (B) KFD induced apoptosis in KMS-11 in a time-series manner. Cells were treated with 26 μM KFD or 5 μM PAR for 1, 3, and 6 hour and was stained with Annexin V and propidium iodide. . / KFD . / PAR Disclosures: No relevant conflicts of interest to declare.

Description: The recent development of a standardised proteomic microarray technique, DotScan, has allowed an innovative approach to the investigation of haematological disorders. In this study, mononuclear cells from 49 peripheral blood samples were studied to determine whether the technology could identify a differential consensus pattern of antigen expression for patients with monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). An automated reader simultaneously determined the expression of 82 different cell surface antigens by binding cells to antibodies on microscopic dots on the nitrocellulose-coated region of a microscope slide. The figure illustrates the image from a typical slide. A consensus pattern of antigen expression was analysed in duplicate by cross-validated discriminant analysis and a pilot database of disease groups was accumulated. Cross validated discriminant analysis successfully predicted patients with monoclonal gammopathy (98% success) and a discrete mosaic pattern could differentiate patients with MM who were treated with thalidomide (n=9). As expected, no single antigen could be used to discriminate between multiple myeloma (n=24), MGUS (n=14) and normal controls (n=11). Antigens with the highest ranking for differentiating the monoclonal gammopathies were CD25 (reduced after thalidomide), CD8 and CD57 (high in MM), CD28 (reduced in MM) and CD95 (reduced in MGUS) reinforcing the importance of immunomodulatory mechanisms in both MM and MGUS. Traditional flow cytometry was used to confirm these specific observations but also to demonstrate that the reduced CD28 expression was specific for CD8+ cells and the reduced CD95 expression was on CD8+ CD57+ cells. Three patients with MGUS were misclassified as MM but on review these 3 patients could have been classified as smoldering myeloma. There was no significant difference in the mosaic of 5 long term survivors of MM (〉 10 years). This study has demonstrated the potential of using disease-specific databases to compare the mosaic of antigen expression for the diagnosis of monoclonal gammopathies. Long term studies will be required to accurately determine the prognostic significance at diagnosis and the ability of consensus patterns to identify which MGUS patients develop MM. Figure Figure Specificity and sensitivity of proteomic array for monoclonal gammopathies MGUS MM MM Thal Normal Sensitivity % Specificity % MGUS prediction 5 3 2 4 35 100 MM prediction 0 15 0 0 100 91 MM Thal prediction 0 0 9 0 100 95 Normal prediction 0 0 0 11 100 89

Description: Limited response to idiotype vaccination in patients with myeloma suggests that there is a need to develop better immunotherapy strategies. It has been determined that the number of high-potency CMRF44+CD14−CD19−dendritic cells (DCs) in the blood of patients with myeloma (range, 0.03%-0.8% of mononuclear cells [MNCs]; n = 26) was not significantly different from that in controls (range, 0.05%-0.8% of MNCs; n = 13). Expression of the costimulatory molecules CD80 and CD86 on DCs from these patients (mean, 29%±17% of MNCs and 85%±10% of MNCs, respectively) was also normal (mean, 29%±17% and 86%±16% of MNCs, respectively). Up-regulation of CD80 expression in response to stimulation by human (hu)CD40LT + interleukin (IL)-2 was significantly reduced on the DCs of patients with myeloma during stable disease (n = 9) and was absent during progressive stages (n = 7) of disease. Similar effects were seen on B cells but not on monocytes of the same group of patients. CD86 expression on DCs was high before (86%) and after (89%) stimulation. Inhibition of CD80 up-regulation was neutralized by either anti–transforming growth factor (TGF)–β1 or anti–IL-10. Up-regulation of CD80 on DCs of controls was inhibited by rTGF-β1 in a dose-dependent manner. Serum TGF-β1 and IL-10 levels were normal in most patients studied. Cytoplasmic TGF-β1 was increased in plasma cells during progressive disease. Thus patients with myeloma have normal numbers of DCs, but CD80 expression may fail to be up-regulated in the presence of huCD40LT because of tumor-derived TGF-β1 or IL-10. Autologous high-potency DCs may have to be tested for CD80 up-regulation and biologically modified ex vivo before idiotype priming for immunotherapy.

Description: The occurrence of clonal T cells in multiple myeloma (MM), as defined by the presence of rearrangements in the T-cell receptor (TCR)–β chains detected on Southern blotting, is associated with an improved prognosis. Recently, with the use of specific anti–TCR-variable-β (anti–TCRVβ) antibodies, the presence in MM patients of expanded populations of T cells expressing particular Vβ regions was reported. The majority of these T-cell expansions have the phenotype of cytotoxic T cells (CD8+CD57+ and perforin positive). Since Vβ expansions can result from either a true clonal population or a polyclonal response, the clonality of CD8+TCRVβ+ T cells was tested by TCRVβ complementarity-determining region 3 length analysis and DNA sequencing of the variable region of the TCR. In this report, the CD57+ and CD57− subpopulations within expanded TCRVβ+CD8+ cell populations are compared, and it is demonstrated that the CD57+ subpopulations are generally monoclonal or biclonal, whereas the corresponding CD57− cells are frequently polyclonal. The oligoclonality of CD57+ expanded CD8+ T cells but not their CD57− counterparts was also observed in age-matched controls, in which the T-cell expansions were mainly CD8−. The CD8+CD57+ clonal T cells had a low rate of turnover and expressed relatively lower levels of the apoptotic marker CD95 than their CD57− counterparts. Taken together, these findings demonstrate that MM is associated with CD57+CD8+ T-cell clones, raising the possibility that the expansion and accumulation of activated clonal CD8+ T cells in MM may be the result of persistent stimulation by tumor-associated antigens, combined with a reduced cellular death rate secondary to reduced expression of the apoptosis-related molecule CD95.

Description: Multiple Myeloma (MM) is a B cell neoplasm in which malignant plasma cells accumulate in the bone marrow and produce excessive amounts of a monoclonal immunoglobulin. Among the large number of antigens expressed by plasma cells, CD38 is one of the highest expressed surface molecules, suggesting an alternative public tumour target. For instance, the myeloma cell line 8226 has a high surface expression of CD38. We plan to introduce genetically recombinant single-chain variable fragments (scFv) of anti-CD38 antibody, along with CD3 chain, into NK-92 a natural killer cell line which is highly cytotoxic against a variety of malignant cells (the only natural killer cell line being used in phase I/II clinical trials). It is likely that the retargeted NK-92 cells may specifically and efficiently kill CD38-expressing myeloma cells. By using a series of degenerate primer sets for mouse immunoglobulin variable region genes, VH and VL regions were isolated through reverse transcription PCR from OKT10 cell line. Further sequencing analysis provided novel information of the OKT10 immunoglobulin heavy and light chains. A PCR-based assembling strategy was employed to construct the scFv with FLAG sequence integrated as a tag. VH and VL domains were joined together with a hinge region of (Gly4Ser)3. The phagemid construct has been formed by subcloning the scFv fragment into the Sfi I/Not I cloning sites of the expression vector pHEN2, and subsequently transformed into the E. Coli HB2151. Five out of 7 transformed bacterial colonies screened did not contain the scFv fragment. For the remaining two clones with the correct-sized insert, we tested the specificity of the scFv fragments that were prepared through intracellular antibody capture as periplasmic proteins. We performed a competitive flow assay using periplasmic proteins with commercially available anti-CD38 PE conjugated antibodies on 8226 myeloma cell line. We found that one of these two clones (clone 6) showed a reduction in CD38 staining and presented as double peaks; while this pattern was not seen in the clone 7. Subsequent DNA sequencing revealed that there was a 100-bp truncation in the heavy chain region in clone 7; while the clone 6 has a complete productive heavy chain and light chain spanned by the linker region. We are going to transfect NK-92 cells with retrovial vector pL-scFv(clone 6) construct as the basis of an efficient approach for anti-myelome immunotherapy. (Dr. Daniel Sze is supported by the International Myeloma Foundation Senior Research Grant).