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What's New in Research - November 12, 2002

Upregulation of matrix metalloproteinase-9 in murine 5T33 multiple myeloma cells by interaction with bone marrow endothelial cells
Els Van Valckenborgh 1, Marleen Bakkus 1, Carine Munaut 2, Agnes Noël 2, Yves St. Pierre 3, Kewal Asosingh 1, Ivan Van Riet 1, IMF Scientific Advisor Ben Van Camp 1, Karen Vanderkerken 1 *
1Department of Hematology and Immunology, Free University Brussels, Brussels, Belgium
2Laboratory of Tumor and Developmental Biology, University of Liège, Liège, Belgium
3Immunology Research Center, Institut Armand-Frappier, University of Quebec, Laval, Canada

International Journal of Cancer
Volume 101, Issue 6, 2002. Pages: 512-518


MM is a B-cell malignancy mainly characterized by monoclonal expansion of plasma cells in the BM, presence of paraprotein in serum and occurrence of osteolytic bone lesions. MMPs are a family of proteolytic enzymes that can contribute to cancer growth, invasion, angiogenesis, bone degradation and other processes important in the pathogenesis of MM. We investigated MMP-9 production in the 5T33MM murine model. Expression of MMP-9 protein in supernatant and cell extracts was analyzed by gelatin zymography. The in vitro, stroma-independent variant 5T33MMvt showed no protein expression of MMP-9 in contrast to in vivo growing MM cells, 5T33MMvv. However, when 5T33MMvt cells were injected into naive mice and isolated after tumor take (5T33MMvt-vv), they secreted a significant amount of MMP-9. These results were confirmed by specific staining of cytospins with an anti-MMP-9 antibody. The MMP-9 production by 5T33MMvt-vv cells disappeared when the cells were recultured in vitro. These data demonstrated that upregulation of MMP-9 occurs in vivo and that this process is dependent on the microenvironment. Cocultures of 5T33MMvt cells with STR10 BMECs induced MMP-9 in MM cells, as determined by both gelatin zymography and flow-cytometric analysis. In conclusion, our results demonstrate that MMP-9 production by MM cells is upregulated in vivo by the interaction of MM cells with BMECs. © 2002 Wiley-Liss, Inc.

BLOOD Reports Revimid™ Has Anti-Tumor Activity in Patients with Relapsed and Refractory Multiple Myeloma
Data Demonstrate Revimid Overcomes Drug Resistance and Is Well Tolerated

WARREN, N.J. -- November 1, 2002 -- The first peer-reviewed data of Revimid™ (CC-5013) in relapsed or refractory multiple myeloma was published in today's issue of Blood, the journal of the American Society of Hematology.

The article, titled "Immunomodulatory Drug CC-5013 Overcomes Drug Resistance and Is Well Tolerated in Patients with Relapsed Multiple Myeloma," reports data from a Phase I/II study of Revimid in relapsed or refractory multiple myeloma that was conducted by Celgene in partnership with the Dana-Farber Cancer Institute. The data from the trial demonstrate that 17 of 24 patients (71 percent) experienced at least a 25 percent reduction in paraprotein (a measure of their tumor burden).

"Revimid represents a novel, oral therapy for relapsed and refractory multiple myeloma that overcomes drug resistance and is well tolerated," said Kenneth C. Anderson, MD, of the Dana-Farber Cancer Institute at Harvard University.

The study published today in Blood evaluated 24 patients with relapsed and/or refractory multiple myeloma. Fifteen patients had undergone prior autologous stem cell transplantation and 16 had received prior Thalomid® (thalidomide) treatment. Patients received a daily oral dose of Revimid in one of the following fixed dose levels for four weeks: 5, 10, 25, or 50 mg/day. After the four-week formal trial, the extension phase permitted dose adjustment. Patients were evaluated by reductions in levels of paraprotein.

Paraprotein reductions of at least 25 percent were observed in 17 of 24 (71 percent) evaluable patients, including seven patients (29 percent) who achieved a greater than 50 percent reduction in paraprotein levels. Two patients experienced stable disease, which was defined by a less than 25 percent reduction in paraprotein levels. The majority of responses were observed at 25 mg/day and 50 mg/day of Revimid. Median duration of response was six (range 2-18) months.

In this study no dose-limiting toxicity was encountered within the first 28 days. Importantly, somnolence, constipation and neuropathy, which are common side effects of thalidomide treatment, were not seen. The most common adverse events observed during the first 28 days were fatigue, rash, leg cramps and neutropenia (decrease of the white blood cells). In the extension phase of the trial, 12 of 13 patients treated with 50 mg/day of Revimid developed grade three and four myelosuppression. All 12 patients were reduced to 25 mg/day of Revimid and tolerated the lower dose. Based on the adverse events observed in the extension phase of the study, the trial's lead investigators concluded that 25 mg/day is the maximum tolerated dose of Revimid.

Revimid, Celgene's lead IMiD (Immunomodulatory Drug), is an orally available compound that affects multiple pathways and multiple targets. In preclinical studies, Revimid demonstrated inhibition of inflammatory cytokines and growth factors such as tumor necrosis factor alpha (TNF alpha), basic fibroblastic growth factor (bFGF) and vascular endothelial growth factor (VEGF) that are involved in tumor development. Preclinical data indicates that Revimid enhances tumor cell death (apoptosis) and cell cycle arrest. Revimid boosts the immune system through stimulation of T-cell proliferation. Data from several Phase I/II trials suggest that Revimid has anti-tumor activity in hematological and solid tumor cancers.

"Revimid has demonstrated anti-tumor activity in preclinical and clinical studies and we believe it has significant potential in treating hematological and solid tumor cancers," said Sol J. Barer, Ph.D., President and Chief Operating Officer of Celgene Corporation. "To more fully evaluate the potential of Revimid, we have initiated pivotal programs for Revimid in multiple myeloma and metastatic melanoma and several Phase II trials in additional hematological and solid tumor cancers."

Pamidronate causes apoptosis of plasma cells in vivo in patients with multiple myeloma.
Gordon S, Helfrich MH, Sati HI, Greaves M, Ralston SH, Culligan DJ, Soutar RL, Rogers MJ.
Department of Medicine and Therapeutics, and Department of Haematology, University of Aberdeen Medical School, Aberdeen, and Haematology Department, Western Infirmary, Glasgow, UK.
British Journal of Haematology 2002 Nov;119(2):475-83

Anti-resorptive bisphosphonates, such as pamidronate, are an effective treatment for osteolytic disease and hypercalcaemia in patients with multiple myeloma, but have also been shown to cause apoptosis of myeloma cell lines in vitro. In this study, we found that a single infusion of pamidronate, in 16 newly diagnosed patients with multiple myeloma, caused a marked increase in apoptosis of plasma cells in vivo in 10 patients and a minimal increase in four patients (P < 0.05). The nitrogen-containing bisphosphonates pamidronate and zoledronic acid also induced apoptosis of authentic, human bone marrow-derived plasma cells in vitro. Apoptosis of plasma cells in vitro was probably caused by inhibition of the mevalonate pathway and loss of prenylated small GTPases, as even low concentrations (>/= 1 micro mol/l) of zoledronic acid caused accumulation of unprenylated Rap1A in cultures of bone marrow mononuclear cells in vitro. GGTI-298, a specific inhibitor of geranylgeranyl transferase I, also induced apoptosis in human plasma cells in vitro, suggesting that geranylgeranylated proteins play a role in signalling pathways that prevent plasma cell death. Our results suggest that pamidronate may have direct and/or indirect anti-tumour effects in patients with multiple myeloma, which has important implications for the further development of the more potent nitrogen-containing bisphosphonates, such as zoledronic acid, in the treatment of myeloma.

Combination therapy with thalidomide plus dexamethasone for newly diagnosed myeloma.
Rajkumar SV, Hayman S, IMF Scientific Advisor Gertz MA, Dispenzieri A, Lacy MQ, Greipp PR, Geyer S, Iturria N, Fonseca R, Lust JA, IMF Scientific Advisory Board Chair Kyle RA, Witzig TE. Division of Hematology, Mayo Clinic and Mayo Foundation, Rochester, MN.

J Clin Oncol 2002 Nov 1;20(21):4319-23

PURPOSE: Multiple myeloma is a malignancy of plasma cells and is characterized by increased marrow angiogenesis. Thalidomide, an agent with antiangiogenic properties, is effective in relapsed myeloma. We report the results of a study combining thalidomide and dexamethasone as initial therapy for myeloma.

PATIENTS AND METHODS: Fifty patients with newly diagnosed myeloma were studied. Thalidomide was given at a dose of 200 mg/d orally. Dexamethasone was given at a dose of 40 mg/d orally on days 1 to 4, 9 to 12, and 17 to 20 (odd cycles) and 40 mg/d on days 1 to 4 (even cycles), repeated monthly.

RESULTS: Of all 50 patients, a confirmed response was seen in 32 patients yielding a response rate of 64% (95% confidence interval, 49% to 77%). Thirty-one patients (62%) proceeded to stem-cell collection after four cycles of therapy including 26 who underwent stem-cell transplantation and five who chose stem-cell cryopreservation. Major grade 3 or 4 toxicities were observed in 16 patients (32%), and the most frequent were deep vein thrombosis (six patients), constipation (four patients), rash (three patients), and dyspnea (two patients). Three deaths occurred during active therapy because of a pancreatitis, pulmonary embolism, and infection.

CONCLUSION: We conclude that the combination of thalidomide plus dexamethasone is a feasible and active regimen in the treatment of multiple myeloma. It merits further study as an oral alternative to infusional chemotherapy with vincristine, doxorubicin, and dexamethasone and other intravenous regimens currently used as pretransplantation induction therapy for myeloma.

Role of the bone marrow microenvironment in multiple myeloma.
IMF Scientific Advisor Roodman GD.
Center for Bone Biology, Division of Hematology/Oncology, University of Pittsburgh Cancer Institute, Pennsylvania 15261, USA.

J Bone Miner Res 2002 Nov; 17(11):1921-5

On June 26-27, 2001, the Sixth Research Roundtable in Multiple Myeloma, entitled "The Role of the Bone Microenvironment in Multiple Myeloma," was held and focused on the biology of cell-to-cell interactions, the mediators of bone disease, and novel treatment strategies for myeloma. Studies on cell-cell interactions showed that vascular cell adhesion molecule 1, expressed by local endothelial and stromal cells, binds to tumor cell surface integrins in which expression may be increased by tumor cell-derived chemokines such as macrophage inflammatory protein (MIP) 1alpha. These adhesive interactions increase production and release of vascular endothelial growth factor (VEGF). Studies on myeloma bone disease showed the ligand for receptor activator of nuclear transcription factor-kappaB (RANKL) was expressed on tumor cells and stromal cells associated with myeloma cells and was critical for osteoclast-induced osteolysis. Blockade of RANKL suppressed osteoclast maturation, bone resorption, and tumor development. Bisphosphonates, in addition to reducing osteoclast mobility and inducing osteoclast apoptosis, also decreased tumor cell adhesion to stroma. Immunomodulatory drugs such as thalidomide analogues targeted these tumor cell-stromal cell interactions, blocking both secretion of cytokines and activation of intracellular signaling pathways required for tumor survival and growth. These agents induced tumor cell apoptosis, decreased neovascularization, and potentiated natural killer cell activity. The proteasome inhibitor PS-341 also prevented expression of adhesion molecules and cytokines and triggered tumor cell apoptosis, even in drug-resistant cell lines, while showing minimal activity in healthy cells. In addition, potential therapeutic agents under investigation, which included RANKL antagonists, protein prenylation inhibitors, and osteoblast growth factors, were discussed.

Efficient transduction of normal human B lymphocytes and non-dividing myeloma B cells with HIV-1-derived lentiviral vectors."
Bovia F, Salmon P, Matthes T, Kvell K, Nguyen TH, Werner-Favre C, Barnet M, Nagy M, Leuba F, Arrighi JF, Piguet V, Trono D, Zubler RH.

Blood 2002 Oct 24; [epub ahead of print]

We studied the transduction of normal human B lymphocytes and myeloma cells with lentiviral vectors. In peripheral blood B cells which had been activated with helper T cells (murine thymoma EL-4 B5) and cytokines, multiply attenuated HIV-1-derived vectors pseudotyped with VSV G envelope protein achieved expression of green fluorescent protein (GFP) in 27 +/- 12% (mean +/- 1 SD, median = 27%) of B cells in different experiments. When compared in parallel cultures, the transducibility of B cells from different donors exhibited very little variation. The human CMV promoter gave 4- to 6-fold higher GFP expression than did the human elongation factor-1alpha promoter. A murine retroviral vector pseudotyped with VSV-G protein proved inefficient even in mitotically active normal B cells. B cells freshly stimulated with Epstein-Barr virus were also transducible by HIV vectors (24 +/- 9%), but B cells activated with CD40 ligand and cytokines resisted transduction. Thus, different culture systems gave very different results. Freshly isolated, non-dividing myeloma cells were efficiently transduced by HIV vectors; for six myelomas the range was 14 to 77% (median of 28%) GFP(+) cells. HIV vectors with a mutant integrase led to no significant GFP signal in normal B or myeloma cells, suggesting that vector integration was required for high transduction. In conclusion, HIV vectors are promising tools for studies of gene functions in normal human B cells and myeloma cells for the purpose of research and the development of gene therapies.

Response to induction chemotherapy is not essential to obtain survival benefit from high-dose melphalan and autotransplantation in myeloma.
IMF Scienfic Advisor Singhal S, IMF Scientific Advisor Powles R, Sirohi B, Treleaven J, Kulkarni S, IMF Scientific Advisor Mehta J.
Myeloma Unit, The Royal Marsden Hospital, Surrey, UK.

Bone Marrow Transplant 2002 Nov;30(10):673-9

Two hundred and twenty-two myeloma patients autografted after 200 mg/m(2)melphalan were studied to examine the relationship between response to induction chemotherapy and outcome. Induction comprised cyclophosphamide, vincristine, doxorubicin and methylprednisolone (C-VAMP) every 3 weeks for one cycle beyond maximum response. 81% responded to C-VAMP (chemosensitive) with 40 complete (CR) and 139 partial (PR) remissions, and 43 did not respond (NR; <50% reduction in paraprotein; primary refractory). Overall, 130 patients (59%) attained or remained in CR post-transplant; including 40% of NR, 53% of PR, and 97% of CR after C-VAMP (P < 0.0001). Amongst these 130 patients, the 5-year OS was independent of response to C-VAMP (NR 79%, PR 74%, CR 60%; P = 0.69). Similarly, among the 69 patients in PR post-transplant, the 5-year OS was independent of response to C-VAMP. In Cox analysis, lack of response to C-VAMP did not affect outcome significantly. These data show that lack of response to induction therapy does not automatically predict poor long-term outcome in myeloma, since a substantial proportion of these patients attain CR after autograft and enjoy extended survival. Myeloma patients should not be disqualified from an autograft based upon lack of response to induction chemotherapy.

Case Report: Testicular plasmacytoma: Report of a case and review of the literature
Gabriel Anghel1 *, Nicola Petti 1, Daniele Remotti2, Carla Ruscio1, Fortunato Blandino1, Ignazio Majolino1

1Hematology and Bone Marrow Transplantation Unit, Azienda Ospedaliera S. Camillo/Forlanini, Rome,0 Italy
2Service of Pathology, Azienda Ospedaliera S. Camillo/Forlanini, Rome, Italy

Online ISSN: 1096-8652 Print ISSN: 0361-8609
American Journal of Hematology Volume 71, Issue 2, 2002. Pages: 98-104


Plasmacytomas of the testis are rare neoplasias; they may occur as isolated tumors or in concomitance with generalized multiple myeloma. We report the case of a 77-year-old man with previous clinical evidence of multiple myeloma involving skin, ribs, and lungs, and initially treated with surgery, radiotherapy, and chemotherapy attaining partial response. Fourteen months after the onset, the patient presented with left testicular enlargement due to plasmacytoma. Immunohistochemical stains showed monoclonal cytoplasmic IgA- in tumour cells; serum M component showed the same immunoglobulin. Following radiotherapy the tumour mass disappeared. Nonetheless, 2 months later while on chemotherapy, disease recurred with progressive increase of skeletal lesions. The patient is currently alive with disease progression 22 months after onset. On the basis of a review of the literature, the clinical significance of testicular myeloma localization is discussed.

EntreMed, Inc.'s (ENMD) Thalidomide Analog Receives FDA Orphan Drug Designation For Multiple Myeloma

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