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What's New In Research - March 25, 2003
03.25.03

Low transplant related mortality in older patients with hematologic malignancies undergoing autologous stem cell transplantation.
Villela L, Sureda A, Canals C, Sanz MA Jr, Martino R, Valcarcel D, Altes A, Briones J, Gomez M, Brunet S, Sierra J.
Servei d'Hematologia Clinica, Hospital de la Santa Creu i Sant Pau, Antoni Maria i Claret, 167, 08025 Barcelona, Spain.
Haematologica 2003 Mar;88(3):300-5

BACKGROUND AND OBJECTIVES: Patients over 60 years are frequently excluded from autologous stem cell transplantation (ASCT) programs due to a traditionally high rate of transplant-related mortality (TRM) in such indications. We evaluated the results of ASCT in a group of 49 patients >= 60 years of age [32 males, median age 63 years (range, 60 to 71)] autografted in our institution from January 1995 to December 2001.

DESIGN AND METHODS: There were 27 patients with multiple myeloma, 13 with non-Hodgkin's lymphoma, 3 with acute myelogenous leukemia, 3 with chronic myelogenous leukemia and 3 with other hematological malignancies. The Karnofsky score was >= 80% in 47 cases. The median time from diagnosis to ASCT was 12 months (range, 5 to 61). Twenty-four patients were autografted in an early disease phase and 25 (51%) in an advanced phase. Peripheral blood stem cells were used in 46 patients (94%), bone marrow in one (2%) and bone marrow plus peripheral blood in two (4%). Forty-one patients received chemotherapy-only conditioning regimens, while only 8 patients received total body irradiation.

RESULTS: Engraftment occurred in all but one patient. The median times to achieve a sustained absolute neutrophil count > 0.5x109/L and a sustained platelet count >20x109/L were 13 (range, 10 to 35) and 13 days (range, 8 to 62), respectively. The actuarial 2-year overall survival was 67% [95% confidence interval (CI), 52-82%). Four patients died without progression due to central nervous system (CNS) hemorrhage (n = 1), CNS toxicity (n = 1), fungal infection (n = 1) or toxoplasmosis (n = 1). One hundred-day and 1-year actuarial TRM were 4% (95% CI, 1-16%) and 8% (95% CI, 3-21%), respectively.

INTERPRETATION AND CONCLUSIONS: ASCT is a feasible procedure in selected elderly patients, with apparently similar rates of engraftment and TRM to those reported for younger patients.


Infection - an underappreciated cause of bone pain in multiple myeloma.
Desikan R, Barlogie B, Sethi R, Toor A, Spoon D, Angtuaco E, Vanhemert R, VijayaGopal A, Singhal S, Mehta J, Jagannath S, Munshi N, Zangari M, Fassas A, Tricot G, Anaissie E.
Myeloma Institute for Research and Therapy and Depa
rtment of Radiology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Br J Haematol 2003 Mar;120(6):1047-50

Bone pain, especially back pain, is a common presenting feature of myeloma patients. We report three multiple myeloma patients with exacerbations of back pain and referred shoulder pain resulting from vertebral infections. Two patients were treated with surgery, and one patient had computerized tomography-guided percutaneous needle aspiration for diagnostic purposes. All three patients received a prolonged course of antibiotics. Vertebral infection resolved with this treatment in all three patients without any recurrence. Previous dexamethasone therapy, together with an episode of bacteraemia, appears to be a predisposing factor for vertebral infection. Magnetic resonance imaging enabled the diagnosis in all three patients.


Clinical and pharmacokinetic phase II study of fotemustine in refractory and relapsing multiple myeloma patients.
Dumontet C, Jaubert J, Sebban C, Bouafia F, Ardiet C, Tranchand B, Berger E, Lucas C, Guyotat D, Coiffier B.
Service d'Hematologie, CHU de Pierre Benite, Lyon.
Ann Oncol 2003 Apr;14(4):615-22

BACKGROUND: Patients with relapsing or refractory multiple myeloma have poor prognosis. Few compounds are active in these patients and response duration remains short. We report the results of an open phase II trial evaluating the efficacy and safety of fotemustine monotherapy.

PATIENTS AND METHODS: Twenty-one patients with relapsing (17) or refractory (four) multiple myeloma received fotemustine 100 mg/m(2) on an outpatient basis on days 1 and 8 of the induction cycle, followed after a 6-week rest period by fotemustine 100 mg/m(2) every 3 weeks until progression or unacceptable toxicity. Fotemustine pharmacokinetics during the first day of induction was compared between patients with normal or abnormal renal function.

RESULTS: Five of 20 eligible patients had an objective response giving an intention-to-treat response rate of 25% [95% confidence interval (CI) 6% to 44%] and a 35.7% response rate (95% CI 11% to 61%) in the 14 patients having received at least four injections of fotemustine. The median time to objective response was 8.9 months. The median times to progression and survival were 13.8 and 23.1 months, respectively, with a 2-year survival rate of 49%. The main toxicity was myelosuppression with grade 3-4 neutropenia and thrombocytopenia in 66% and 71% of patients, respectively. There was one toxic death by sepsis after induction. The pharmacokinetic parameters in renal-impaired patients were not significantly different from those in patients with normal renal function with a similar incidence of grade 3-4 toxicity in both groups.

CONCLUSIONS: Fotemustine as a single agent has definite activity in patients with relapsing or refractory multiple myeloma, with acceptable toxicity and can be administered at conventional doses in patients with mild or moderate renal impairment.


Cytogenetics of multiple myeloma: interpretation of fluorescence in situ hybridization results.
Harrison CJ, Mazzullo H, Cheung KL, Gerrard G, Jalali GR, Mehta A, Osier DG, Orchard KH.
Department of Haematology, Royal Free and University College Medical School, London, and Royal Bournemouth Hospital, Bournemouth, UK.
Br J Haematol 2003 Mar;120(6):944-52

The cytogenetic picture in multiple myeloma (MM) is highly complex, from which non-random numerical and structural chromosomal changes have been identified. Specifically, translocations involving the immunoglobulin heavy chain gene (IGH) at 14q32 and either monosomy or deletions of chromosome 13 have been reported in a significant number of patients from both cytogenetic and interphase fluorescence in situ hybridization (FISH) studies. Importantly, these abnormalities of chromosome 13 have recently been associated with a poor prognosis. In view of the highly complex nature of the karyotypes in MM patients, interphase FISH results may be difficult to interpret. In this study, cytogenetics and/or interphase FISH were carried out on bone marrow samples or purified plasma cells from 37 MM patients. Abnormal karyotypes, characterized by multiplex FISH (M-FISH) were found in 11 patients, all of which were highly complex. Interphase FISH revealed translocations involving the IGH locus in 16 (43%) patients. The IGH/cyclin D1 (CCND1) gene fusion characteristic of the translocation, t(11;14)(q13;q32), was seen in 12 (32%) of these patients and other rearrangements of IGH in four (11%) patients. Fourteen patients had additional copies of chromosome 11. Twenty patients (54%) had 13q14 deletions, 10 of whom also had t(11;14) or another IGH translocation. By comparing cytogenetic and FISH results, this study has revealed that significant chromosomal abnormalities might be hidden within highly complex karyotypes. Therefore, extreme caution is required in the interpretation of interphase FISH results in MM, particularly in relation to certain abnormalities, such as 13q14 deletions, which have an impact on prognosis.


Urinary N-telopeptide levels in multiple myeloma patients, correlation with Tc-99m-sestaMIBI scintigraphy and other biochemical markers of disease activity
M. G. Alexandrakis1 *, D. S. Kyriakou 2, F. H. Passam 1, N. Malliaraki 3, I. G. Vlachonikolis 4, N. Karkavitsas 5
1Department of Hematology, University Hospital of Crete, Greece
2Department of Hematology, University Hospital of Thessalia, Greece
3Department of Clinical Biochemistry, University Hospital of Crete, Greece
4Department of Biostatistics, Medical School of Crete, Greece
5Department of Nuclear Medicine, University Hospital of Crete, Greece
Hematological Oncology Volume 21, Issue 1, 2003. Pages: 17-24

Abstract Urinary cross-linked N-telopeptide of type I collagen (NTx) has been reported to be a sensitive and specific marker of bone resorption in multiple myeloma (MM). In this study, we measured the levels of NTx in 30 newly diagnosed MM patients and 25 controls. We examined its association with the overall score of skeletal involvement measured by Tc-99m-MIBI scintigraphy and other biochemical markers of bone disease (tumour necrosis factor a (TNF-a), serum calcium and creatinine). We further studied the correlation of NTx with the stage of disease (according to Durie-Salmon criteria) and bone marrow infiltration by plasma cells. High levels of NTx, bone marrow infiltration, TNF-, calcium and creatinine were noted at advanced stages of disease (p < 0.05). NTx and TNF-a were found at significantly higher concentrations in patients with a high overall score (3 and 4) in Tc-99m-sestaMIBI in comparison to a low score (0, 1 and 2; p < 0.05). Positive correlations were found between NTx and TNF-a, as well as between bone infiltration and TNF-a or calcium. In conclusion, NTx is a useful marker for the monitoring of bone resorption in MM and correlates with imaging findings on Tc-99m-sestaMIBI and other biochemical markers of disease activity.


Allogeneic transplantation for multiple myeloma: late relapse may occur as localised lytic lesion/plasmacytoma despite ongoing molecular remission.
Byrne JL, Fairbairn J, Davy B, Carter IG, Bessell EM, Russell NH.
Bone Marrow Transplant 2003 Feb;31(3):157-61

Allogeneic SCT for myeloma may be curative for young patients, but its role remains controversial because of a reported high TRM in some series. Since 1991, we have performed 25 allografts for myeloma using fully matched sibling donors. Of the 18 evaluable patients, 13 achieved CR at a median time of 2.5 months post-transplant. The five patients who were not in CR when assessed at 3 months received a short course of alpha-interferon and four subsequently achieved CR with this approach at a median of 82 days. One patient who failed to respond to IFN went on to achieve CR after four doses of DLI therapy, thus giving an overall CR rate of 72%. Seven patients have relapsed at a median of 4.7 years post-transplant (range 1.38-7.7 years) including two patients who had received IFN therapy. In five of these cases, relapse has been as a localised area of bone disease or isolated plasmacytoma with no evidence of marrow involvement by trephine biopsy or molecular analysis. All patients with localised relapse were treated with local radiotherapy +/-DLI and four are currently disease free despite two patients having had further treatment for a second localised lesion. Six patients died of TRM (24%) and the OS at 8 years is currently 69% with an EFS of 26%. These results suggest that allogeneic SCT for myeloma can be carried out with an acceptable TRM and a high CR rate. However, late relapses as localised disease may be a frequent finding and may represent foci of myeloma not eradicated by the conditioning. The use of pretransplant MRI scanning and top-up radiotherapy to involved areas may be useful in preventing this type of relapse.Bone Marrow Transplantation (2003) 31, 157-161. doi:10.1038/sj.bmt.1703810


Use of erythropoietin in patients with multiple myeloma
Egerer G, Harter C, Karthaus M, Ho AD, Goldschmidt H.
Medizinische Klinik und Poliklinik V, Ruprecht-Karls-Universitat Heidelberg, Germany.
Onkologie 2003 Feb;26(1):80-4

The prevalence of tumour anaemia in patients with multiple myeloma is greater than 80%. At the time of diagnosis 20% of these patients are already anaemic. In about 70% of patients with multiple myeloma, recombinant human erythropoietin (r-HuEPO) leads to a reduction in transfusion frequency, resulting in a drop in transfusion- related side-effects like infections and immune reactions, iron overload and hyperviscosity which often negatively influence the course of disease. A further reason for the use of erythropoietin is to achieve and maintain high haemoglobin levels (11-12 g/dl), which are of considerable prognostic significance in patients with multiple myeloma. Increasing Hb levels with r-HuEPO also improve the quality of life of patients, thus leading to better therapy compliance. The trade-off between high costs of an erythropoietin treatment and lower indirect costs (infusion material, personal equipment, patient transport costs, etc.) should be evaluated. Nevertheless, an exact definition of patients for whom the use of erythropoietin is beneficial is warranted. The pathogenesis of anaemia and the clinical experiences of erythropoietin in patients with multiple myeloma are discussed. Copyright 2003 S. Karger GmbH, Freiburg.


Nonmyeloablative Allogeneic Stem-Cell Transplantation for Hematologic Malignancies: A Systematic Review
Benjamin Djulbegovic, MD, PhD, Jerome Seidenfeld, PhD, Claudia Bonnell, BSN, MLS, Ambuj Kumar, MD, MPH
Read full article in the Cancer Control: Journal of the Moffitt Cancer Center

ABSTRACT

Background: Increasingly, clinicians advocate the use of nonmyeloablative allogeneic stem-cell transplants (NM-allo-SCTs, "mini-transplants") to manage hematologic malignancies. They hypothesize that NM-allo-SCT is equally efficacious to standard allo-SCT but produces less regimen-related toxicity.

Methods: To analyze available evidence on the benefits and harms of "mini-transplants," we identified 23 manuscripts, 1 abstract, and 1 letter that reported the outcome of mini-transplants in hematologic malignancies.

Results: Data were compiled on 603 treated patients, with 118 transplants using stem cells from matched unrelated donors. All studies were small prospective case series, and most lacked concurrent or historical controls. Outcomes of interest were not uniformly reported. The studies were heterogeneous and used different patient selection criteria, conditioning regimens, and timing of transplant with respect to disease status. The transplant-related mortality rate was 32%, the relapse rate was 15%, and toxicities included acute and chronic graft-vs-host disease and veno-occlusive disease. The aggregate rate of complete remission was 45%. Survival at 1 year or longer ranged from 30% to 60% at 1 to 5 years of follow-up. All studies reported successful chimerism.

Conclusions: Disease-specific studies with longer follow-up are needed to evaluate this potentially promising therapy.


Pamidronate is superior to ibandronate in decreasing bone resorption, interleukin-6 and beta 2-microglobulin in multiple myeloma.
Terpos E, Viniou N, de la Fuente J, Meletis J, Voskaridou E, Karkantaris C, Vaiopoulos G, Palermos J, Yataganas X, Goldman JM, Rahemtulla A.
Department of Haematology, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
Eur J Haematol 2003 Jan;70(1):34-42

OBJECTIVES: Bisphosphonates have been found to reduce skeletal events in patients with multiple myeloma (MM). This is the first randomised trial to compare the efficacy of pamidronate and ibandronate, a third-generation aminobisphosphonate, in bone turnover and disease activity in MM patients.

METHODS: Patients with MM, stage II or III, were randomly assigned to receive either pamidronate 90 mg (group I: 23 patients) or ibandronate 4 mg (group II: 21 patients) as a monthly intravenous infusion in addition to conventional chemotherapy. Skeletal events, such as pathologic fractures, hypercalcaemia, and bone radiotherapy were analysed. Bone resorption markers [N-terminal cross-linking telopeptide of type-I collagen (NTX) and tartrate-resistant acid phosphatase type 5b (TRACP-5b)], bone formation markers (bone alkaline phosphatase and osteocalcin), markers of disease activity (paraprotein, CRP, beta 2-microglobulin), and interleukin-6 (IL-6) were also studied.

RESULTS: In both groups, the combination of chemotherapy with either pamidronate or ibandronate produced a reduction in bone resorption and tumour burden as measured by NTX, IL-6, paraprotein, CRP, and beta 2-microglobulin from the second month of treatment, having no effect on bone formation. TRACP-5b also had a significant reduction in the pamidronate group from the second month of treatment and in the ibandronate group from the sixth month. However, there was a greater reduction of NTX, IL-6, and beta 2-microglobulin in group I than in group II, starting at the second month of treatment (P = 0.002, 0.001, and 0.004, respectively) and of TRACP-5b, starting at the fourth month (P = 0.014), that being continued throughout the 10-month follow-up of this study. There was no difference in skeletal events during this period. A significant correlation was observed between changes of NTX and changes of TRACP-5b, IL-6, and beta 2-microglobulin from the second month for patients of both groups.

CONCLUSIONS: These results suggest that a monthly dose of 90 mg of pamidronate is more effective than 4 mg of ibandronate in reducing osteoclast activity, bone resorption, IL-6, and possibly tumour burden in MM. TRACP-5b has also proved to be a useful new marker for monitoring bisphosphonates treatment in MM.


Cyclophosphamide metabolism, liver toxicity, and mortality following hematopoietic stem cell transplantation
George B. McDonald, John T. Slattery, Michelle E. Bouvier, Song Ren, Ami L. Batchelder, Thomas F. Kalhorn, H. Gary Schoch, Claudio Anasetti, and Ted Gooley
The Clinical Research Division, Fred Hutchinson Cancer Research Center, and the Departments of Medicine and Pharmaceutics, University of Washington Schools of Medicine and Pharmacy, Seattle, WA.
Blood, 1 March 2003, Vol. 101, No. 5, pp. 2043-2048

Liver toxicity caused by high-dose myeloablative therapy leads to significant morbidity after hematopoietic cell transplantation. We examined the hypothesis that liver toxicity after cyclophosphamide and total body irradiation is related to cyclophosphamide through its metabolism to toxins. Cyclophosphamide was infused at 60 mg/kg over 1 to 2 hours on each of 2 consecutive days, followed by total body irradiation. Plasma was analyzed for cyclophosphamide and its major metabolites. Liver toxicity was scored by the development of sinusoidal obstruction syndrome (veno-occlusive disease) and by total serum bilirubin levels. The hazards of liver toxicity, nonrelapse mortality, tumor relapse, and survival were calculated using regression analysis that included exposure to cyclophosphamide metabolites (as the area under the curve). Of 147 patients, 23 (16%) developed moderate or severe sinusoidal obstruction syndrome. The median peak serum bilirubin level through day 20 was 2.6 mg/dL (range, 0.5-41.1 mg/dL). Metabolism of cyclophosphamide was highly variable, particularly for the metabolite o-carboxyethyl-phosphoramide mustard, whose area under the curve varied 16-fold. Exposure to this metabolite was statistically significantly related to sinusoidal obstruction syndrome, bilirubin elevation, nonrelapse mortality, and survival, after adjusting for age and irradiation dose. Patients in the highest quartile of o-carboxyethyl-phosphoramide mustard exposure had a 5.9-fold higher risk for nonrelapse mortality than did patients in the lowest quartile. Engraftment and tumor relapse were not statistically significantly related to cyclophosphamide metabolite exposure. Increased exposure to toxic metabolites of cyclophosphamide leads to increased liver toxicity and nonrelapse mortality and lower overall survival after hematopoietic cell transplantation.


Thalidomide analogs inhibit Hs Sultan cells and angiogenesis in vivo
Leukemia, 2003;17(1):41-44) from Hematology Week via NewsRx.com

"We have previously shown that thalidomide and its potent immunomodulatory derivatives (IMiDs) inhibit the in vitro growth of multiple myeloma (MM) cell lines and patient MM cells that are resistant to conventional therapy. In this study, we further characterize the effect of these drugs on growth of B cell malignancies and angiogenesis," researchers in the United States report.

"We established a beige-nude-xid (BNX) mouse model to allow for simultaneous in vivo measurement of both antitumor and antiangiogenic effects of thalidomide and its analogs," said S. Lentzsch and colleagues, Harvard University, School of Medicine.

"Daily treatment (50 mg/kg/d) with thalidomide or IMiDs was nontoxic. The IMiDs were significantly more potent than thalidomide in vivo in suppressing tumor growth, evidenced by decreased tumor volume and prolonged survival, as well as mediating antiangiogenic effects, as determined by decreased microvessel density," said investigators.

"Our results therefore show that the IMiDs have more potent direct antitumor and antiangiogenic effects than thalidomide in vivo, providing the framework for clinical protocols evaluating these agents in MM and other B cell neoplasms," they suggested.

The contact person for this report is K.C. Anderson, Harvard University, School of Medicine, Dana Farber Cancer Institute, Jerome Lipper Multiple Myeloma Center, Department of Adult Oncology, 44 Binney St., Boston, MA 02115, USA.


Antisense p53 transduction lessens apoptosis in multiple myeloma
Leukemia Research, 2003;27(1):73-78) from Gene Therapy Week via NewsRx.com

According to recent research from the United States, "multiple myeloma is a malignant proliferation of plasma cells which fail to undergo apoptosis. To understand events associated with lack of apoptosis in these cells, we studied effect of antisense p53 gene transduction in a multiple myeloma cell line, ARH77."

"Adeno-associated virus was used as a vector to introduce p53 cDNA in an antisense orientation driven by a herpes virus thymidine kinase promoter," stated R. Iyer and colleagues, Dana Farber Cancer Institute.

"We observed, that an antisense p53 (p53as) transduced cell line showed marked reduction in p53 mRNA and protein expression and increased growth when compared to the control cell lines transduced with neomycin-resistance gene or untransduced cells," said researchers.

"There was a concomitant up-regulation of Bcl-2 expression by over fivefold in p53as-transduced cells compared with controls; while there was no significant change in expression of c-myc and IL-6, genes implicated in myeloma growth."

"We measured apoptosis in the transduced cells by DNA end-labeling reaction which revealed decrease in apoptosis from 15.6% in control cells to 1.6% in p53as-transduced cells. Additionally, the p53as cells over expressing Bcl-2 also showed resistance to killing by dexamethasone," Iyer and coauthors said.

"In summary, our data demonstrates that loss of p53 function leads to myeloma cell progression and resistant phenotype through Bcl-2-related mechanisms," they concluded.

For additional information, contact N.C. Munshi, Dana Farber Cancer Institute, 44 Binney St., M557, Boston, MA 02115, USA.


Antisense-mediated downregulation of Bcl-2 protein chemosensitizes myeloma.
Leukemia, 2003;17(1):211-219) from Gene Therapy Week via NewsRx.com

According to recent research from Netherlands, "an antisense oligodeoxynucleotide (ODN) complementary to the first six codons of the Bcl-2 mRNA, G3139 (oblimersen sodium; Genasense), has been shown to downregulate Bcl-2 and produce responses in a variety of malignancies including drug-resistant lymphoma."

"Incubation of ex vivo purified plasma cells from patients with multiple myeloma (MM) with carboxyfluorescein (FAM)-labeled antisense ODNs resulted in a time-and dose-dependent uptake in the cytoplasm and nucleus. No major differences in uptake of Bcl-2 antisense ODNs were observed among patients' samples," wrote N.W.C.J. van de Donk and colleagues, University Utrecht, Med Center.

"Incubation of purified myeloma plasma cells with G3139, but not solvent or reverse polarity control ODNs, resulted in a reduction (>75%) of Bcl-2 mRNA levels after 2 and 4 days, as measured by real-time PCR. Treatment with G3139 led to a sequence-specific reduction of Bcl-2 protein levels within 4 days of exposure in 10 out of 11 clinical samples from patients with chemosensitive and multidrug-resistant disease, without significant reduction of alpha-Actin, Bax, Bcl-XL, or Mcl-1 proteins," they reported.

"This resulted in a significantly enhanced sensitivity of the myeloma tumor cells to dexamethasone or doxorubicin-induced apoptosis. G3139 can consistently enter myeloma cells, downregulate the expression of Bcl-2, and enhance the efficacy of myeloma therapy. These data support further clinical evaluation of G3139 therapy in multiple myeloma," researchers advised.

For additional information, contact H.M. Lokhorst, University of Utrecht, Medical Center, Department of Hematology, Heidelberglaan 100, NL-3584 CX Utrecht, Netherlands.


Oncolytic measles viruses displaying a single-chain antibody against CD38, a myeloma cell marker
Kah-Whye Peng, Kathleen A. Donovan, Urs Schneider, Roberto Cattaneo, John A. Lust, and Stephen J. Russell
The Molecular Medicine Program, Mayo Foundation, Rochester; and Division of Hematology and Internal Medicine, Mayo Clinic, Rochester, MN.
Blood 2003;101 2557-2562

Live attenuated measles virus (MV-Edm) has potent oncolytic activity against myeloma xenografts in mice. Therapy of multiple myeloma, a disseminated plasma cell malignancy, would require systemic administration of the virus. Thus, the virus should ideally be targeted to infect only myeloma cells to minimize collateral damage to normal tissues: viral binding to its natural receptors must be ablated and a new specificity domain that targets entry into myeloma cells be added. This study covers 2 critical steps toward generating such a retargeted virus: (1) a new specificity domain against the plasma cell marker CD38 was constructed in the form of a single-chain antibody (scFv) and (2) display of that scFv on the measles viral envelope glycoprotein successfully redirected virus entry through CD38 expressed on target cells devoid of the natural MV receptors. The anti-CD38 scFv was tethered to the C-terminus of the hemagglutinin (H) glycoprotein of MV-Edm through a Factor Xa protease cleavable linker. Immunoblot analysis demonstrated that the scFv was efficiently incorporated into recombinant viral particles. Replication of MV-CD38 was not hindered by the scFv, reaching titers comparable to MV-Edm. Chinese hamster ovary (CHO) cells were resistant to infection by MV-Edm and MV-CD38. In contrast, CHO cells expressing CD38 became susceptible to infection by MV-CD38 but not MV-Edm. Removal of the displayed scFv rendered MV-CD38 noninfectious on CHO-CD38 cells. Tumorigenicity of CHO-CD38 cells in immunocompromised mice was significantly attenuated by MV-CD38, resulting in enhanced survival of these mice compared with the control group.


CD40 induces human multiple myeloma cell migration via phosphatidylinositol 3-kinase/AKT/NF-kappa B signaling
Yu-Tzu Tai, Klaus Podar, Nicholas Mitsiades, Boris Lin, Constantine Mitsiades, Deepak Gupta, Masaharu Akiyama, Laurence Catley, Teru Hideshima, Nikhil C. Munshi, Steven P. Treon, and Kenneth C. Anderson
The Jerome Lipper Multiple Myeloma Center, Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA; and Department of Medicine, Harvard Medical School, Boston, MA.
Blood 2003;101 2762-2769

Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow and their egress into peripheral blood with progression to plasma cell leukemia. Our previous study defined a functional role of CD40 activation in MM cell homing and migration. In this study, we examine signaling events mediating CD40-induced MM cell migration. We show that cross-linking CD40, using either soluble CD40L (sCD40L) or anti-CD40 monoclonal antibody (mAb), induces phosphatidylinositol 3-kinase (PI3K) activity and activates its downstream effector AKT in MM.1S cells. CD40 activation also activates the MAP kinase (MEK) pathway, evidenced by phosphorylation of extracellular signal-regulated mitogen-activated protein kinase (ERK), but not c-jun amino-terminal kinase (JNK) or p38, in a dose- and time-dependent manner. Using pharmacologic inhibitors of PI3K and MEK, as well as adenoviruses expressing dominant-negative and constitutively expressed AKT, we demonstrate that PI3K and AKT activities are required for CD40-induced MM cell migration. In contrast, inhibition of ERK/MEK phosphorylation only partially (10%-15%) prevents migration, suggesting only a minor role in regulation of CD40-mediated MM migration. We further demonstrate that CD40 induces nuclear factor (NF)-B activation as a downstream target of PI3K/AKT signaling, and that inhibition of NF-B signaling using specific inhibitors PS1145 and SN50 completely abrogates CD40-induced MM migration. Finally, we demonstrate that urokinase plasminogen activator (uPA), an NF-B target gene, is induced by CD40; and conversely, that uPA induction via CD40 is blocked by PI3K and NF-B inhibitors. Our data therefore indicate that CD40-induced MM cell migration is primarily mediated via activation of PI3K/AKT/NF-B signaling, and further suggest that novel therapies targeting this pathway may inhibit MM cell migration associated with progressive MM.


Paracrine interactions of basic fibroblast growth factor and interleukin-6 in multiple myeloma
Guido Bisping, Regine Leo, Doris Wenning, Berno Dankbar, Teresa Padro, Martin Kropff, Christian Scheffold, Matthias Kroger, Rolf M. Mesters, Wolfgang E. Berdel, and Joachim Kienast
The Department of Medicine/Hematology and Oncology, University of Muenster, Germany.
Blood 2003;101 2775-2783

Myeloma cells express basic fibroblast growth factor (bFGF), an angiogenic cytokine triggering marrow neovascularization in multiple myeloma (MM). In solid tumors and some lymphohematopoietic malignancies, angiogenic cytokines have also been shown to stimulate tumor growth via paracrine pathways. Since interleukin-6 (IL-6) is a potent growth and survival factor for myeloma cells, we have studied the effects of bFGF on IL-6 secretion by bone marrow stromal cells (BMSCs) and its potential reverse regulation in myeloma cells. Both myeloma-derived cell lines and myeloma cells isolated from the marrow of MM patients were shown to express and secrete bFGF. Cell-sorting studies identified myeloma cells as the predominant source of bFGF in MM marrow. BMSCs from MM patients and control subjects expressed high-affinity FGF receptors R1 through R4. Stimulation of BMSCs with bFGF induced a time- and dose-dependent increase in IL-6 secretion (median, 2-fold; P < .001), which was completely abrogated by anti-bFGF antibodies. Conversely, stimulation with IL-6 enhanced bFGF expression and secretion by myeloma cell lines (2-fold; P = .02) as well as MM patient cells (up to 3.6-fold; median, 1.5-fold; P = .002). This effect was inhibited by anti-IL-6 antibody. When myeloma cells were cocultured with BMSCs in a noncontact transwell system, both IL-6 and bFGF concentrations in coculture supernatants increased 2- to 3-fold over the sum of basal concentrations in the monoculture controls. The IL-6 increase was again partially, but significantly, inhibited by anti-bFGF. The data demonstrate a paracrine interaction between myeloma and marrow stromal cells triggered by mutual stimulation of bFGF and IL-6.


SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma
Oliver Galm, Hirohide Yoshikawa, Manel Esteller, Rainhardt Osieka, and James G. Herman
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD; Medizinische Klinik IV, Rheinisch-Westfaelische Technische Hochschule (RWTH) Aachen, Aachen, Germany; and the Cancer Epigenetics Laboratory, Molecular Pathology Program, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain.
Blood 2003;101 2784-2788

The suppressor of cytokine signaling (SOCS) family of proteins has been implicated in the negative regulation of several cytokine pathways, particularly the receptor-associated tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathways of transcriptional activation. SOCS-1 (also known as JAB and SSI-1) inhibits signaling by many cytokines. Because of the previously observed hypermethylation-associated inactivation of SOCS-1 in hepatocellular carcinoma and the critical role of interleukin-6 (IL-6) as a survival factor in multiple myeloma (MM), we examined CpG island methylation of the SOCS-1 gene in MM cell lines and primary MM samples. Aberrant SOCS-1 methylation was found in the IL-6-dependent MM cell lines U266 and XG1, which correlated with transcriptional silencing. Treatment of these cell lines with the demethylating agent 5-aza-2'-deoxycytidine (DAC) up-regulated SOCS-1 expression. Methylation-associated inactivation of SOCS-1 in hematopoietic cell lines correlated with greater sensitivity to the chemical JAK inhibitor AG490. Using methylation-specific polymerase chain reaction (MSP), we found that SOCS-1 is hypermethylated in 62.9% (23/35) of MM patient samples. In contrast, methylation analysis of malignant lymphomas of various histologies revealed SOCS-1 hypermethylation in only 3.2% (2/62), and there was no methylation of SOCS-1 in normal peripheral blood leukocytes or bone marrow cells. We conclude that SOCS-1 is frequently inactivated by hypermethylation in MM patients. Silencing of the SOCS-1 gene may impair negative regulation of the Jak/STAT pathway and therefore result in greater responsiveness to cytokines, thus supporting survival and expansion of MM cells.


SCIENCE SCAN B LYMPHOCYTES ACTIVATE PLASMA CELLS TO GENERATE HUGE ANTIBODY QUANTITIES AND MULTIPLE MYELOMA
(research briefs)
David N. Leff, Science Editor
BIOWORLD Today Volume 14; Issue 46
(Editor's note: Science Scan is a roundup of recently published biotechnology-relevant research.)

Stressed out? Try this multiple-choice quiz:

How many antibodies can one B cell churn out per second? (a) 1, (b) 2, (c) 10, (d) 100, (e) 1,000, (f) 2,000, (g) a million.

If you guessed (f) you were right on the money. A single B lymphocyte can secrete 2,000 monoclonal antibodies into the bloodstream in a single second. But it isn't easy for that molecule. Immune system cells get stressed out, too. Generating that lightning production pace involves the B cell in a stressful avalanche of genes and proteins that make the 2,000-per-second proliferation happen. (B cells are the humoral arm of the mammalian immune defenses, which manufacture antibodies against invading antigens.)

Here's an abridged rundown of that dizzying process: Sensing an intruding infectious bacterium, say, the immune defenses dispatch a battalion of cytokines - such as interleukin-4 and -6 - to engage the enemy. "It turns out that the B cells need to be stressed," observed research immunologist Neal Iwakoshi at the Harvard School of Public Health in Boston. "That cellular cell response," he added, "which we call UPR - unfolding protein response - causes the terminal differentiation of B lymphocytes, turning them into antibody-producing plasma cells, the action site for generating antibodies to fight infection."

Iwakoshi is first author of a paper in Nature, released online March 3, 2003. The paper is titled: "Plasma cell differentiation and the unfolding protein response intersect at the transcription factor XBP-1." Its senior author is molecular immunologist Laurie Glimcher at Harvard Medical School.

"Plasma cells," explained Glimcher, "are antibody factories capable of synthesizing and secreting vast amounts of specific antibodies into the bloodstream. However, precursors of these cells need to be alerted to increase their production capacity. Our Nature article shows that B cells make small amounts of antibody, but these molecules fail to fold properly and signal the stress response. During this same split second, B cells increase their expression of the XBP-1 gene, which is the master switch for plasma cell differentiation. However, the form of XBP-1 message produced in the absence of the stress response triggers a nonfunctional protein.

"Our new data," she continued, "reports how this stress response triggers splicing [activating] of the message to encode its functional form. This then turns on genes required for plasma cell differentiation and increased antibody production. Thus, aberrant activation of XBP-1 might contribute to the development of multiple myeloma - the malignant counterpart of plasma cells."

Malignant myeloma is a relatively uncommon blood dyscrasia of unusual comportment. It afflicts roughly two to three individuals per 100,000, with males outnumbering females three to two. It occurs when plasma cells turn malignant and give rise to plasmacytomas - cancerous tumors. These punch perforations in skeletal bones - in particular, pelvis, spine, ribs and skull. This osteoporosis brings on severe pain, especially in the back and chest, plus kidney failure and recurrent bacterial infections. With medical treatment, more than half of multiple myeloma patients show improvement, though survival averages 2.5 to three years.


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