ALBERT

Description: The mononuclear cells in the blood of myeloma patients have been reported to contain a high proportion of phenotypically abnormal myeloma B lymphocytes. These cells have been proposed to constitute the drug-resistant proliferative myeloma cell compartment. To determine the extent of B lymphocyte involvement, the proportion of clonotypic cells among the CD19-expressing cells from myeloma patients was estimated by quantitative polymerase chain reaction analysis of the third complementarity determining region (CDR3). The results indicate that the B lymphocytes constitute, on average, 6% of blood mononuclear cells, and that only a minor fraction of these are clonally related to the myeloma cells. While the small number of circulating clonal cells is not incompatible with their proposed role as a reservoir of proliferating myeloma progenitors, the majority of the B cells appear not to be clonally related to the myeloma cells.

Description: Sickle cell disease (SCD) results in chronic hypoxia and secondarily increased erythropoietin concentrations. Leukocytosis and activated monocytes are also observed in SCD in absence of infection or vaso-occlusion (steady state), the reasons for which are unknown. We found that erythroid cells produced placenta growth factor (PlGF), an angiogenic growth factor belonging to the vascular endothelial growth factor (VEGF) family, and its expression was induced in bone marrow CD34+ progenitor cells in the presence of erythropoietin. Furthermore, the steady state circulating PlGF levels in subjects with severe SCD (at least 3 vaso-occlusive crises [VOCs] per year) were 18.5 ± 1.2 pg/mL (n = 9) compared with 15.5 ± 1.2 pg/mL (n = 13) in those with mild SCD (fewer than 3 VOCs per year) and 11.3 ± 0.7 pg/mL (n = 9) in healthy controls (P 〈 .05), suggesting a correlation between PlGF levels and SCD severity. In addition, PlGF significantly increased mRNA levels of the proinflammatory cytochemokines interleukin-1β, interleukin-8, monocyte chemoattractant protein-1, and VEGF in peripheral blood mononuclear cells (MNCs) of healthy subjects (n = 4; P 〈 .05). Expression of these same cytochemokines was significantly increased in MNCs from subjects with SCD at steady state (n = 14), compared with healthy controls. Of the leukocyte subfractions, PlGF stimulated monocyte chemotaxis (P 〈 .05, n = 3). Taken together, these data show for the first time that erythroid cells intrinsically release a factor that can directly activate monocytes to increase inflammation. The baseline inflammation seen in SCD has always been attributed to sequelae secondary to the sickling phenomenon. We show that PlGF contributes to the inflammation observed in SCD and increases the incidence of vaso-occlusive events.

Description: Although monoclonal antibody (MoAb) therapy of the human malignant lymphomas has shown success in clinical trials, its full potential for the treatment of hematologic malignancies has yet to be realized. To expand the clinical potential of a promising human-mouse chimeric antihuman B-cell MoAb (chCLL-1) constructed using the variable domains cloned from the murine Lym-2 (muLym-2) hybridoma, fusion proteins containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (chCLL-1/GM–CSF) or interleukin (IL)-2 (chCLL-1/IL–2) were generated and evaluated for in vitro cytotoxicity and in vivo tumor targeting. The glutamine synthetase gene amplification system was employed for high level expression of the recombinant fusion proteins. Antigenic specificity was confirmed by a competition radioimmunoassay against ARH-77 human myeloma cells. The activity of chCLL-1/GM–CSF was established by a colony formation assay, and the bioactivity of chCLL-1/IL–2 was confirmed by supporting the growth of an IL-2–dependent T-cell line. Antibody-dependent cellular cytotoxicity against ARH-77 target cells demonstrated that both fusion proteins mediate enhanced tumor cell lysis by human mononuclear cells. Finally, biodistribution and imaging studies in nude mice bearing ARH-77 xenografts indicated that the fusion proteins specifically target the tumors. These in vitro and in vivo data suggest that chCLL-1/GM–CSF and chCLL-1/IL–2 have potential as immunotherapeutic reagents for the treatment of B-cell malignancies.

Description: Syndecan-1 (CD138) is a transmembrane heparan sulfate–bearing proteoglycan expressed by most myeloma plasma cells that regulates adhesion, migration, and growth factor activity. In patients with myeloma, shed syndecan-1 accumulates in the bone marrow, and high levels of syndecan-1 in the serum are an indicator of poor prognosis. To test the effect of soluble syndecan-1 on tumor cell growth and dissemination, ARH-77 B-lymphoid cells were engineered to produce a soluble form of syndecan-1. Controls included vector only (neo)–transfected cells and cells transfected with full-length syndecan-1 complementary DNA that codes for the cell surface form of syndecan-1. Assays reveal that all 3 transfectants have similar growth rates in vitro, but cells expressing soluble syndecan-1 are hyperinvasive in collagen gels relative to controls. When injected into the marrow of human bones that were implanted in severe combined immunodeficient mice, tumors formed by cells expressing soluble syndecan-1 grow faster than tumors formed by neo-transfected cells or by cells expressing cell surface syndecan-1. In addition, cells bearing cell surface syndecan-1 exhibit a diminished capacity to establish tumors within the mice as compared with both neo- and soluble syndecan-1–transfected cells. Tumor cell dissemination to a contralateral human bone is detected significantly more often in the tumors producing soluble syndecan-1 than in controls. Thus, high levels of soluble syndecan-1 present in patients with myeloma may contribute directly to the growth and dissemination of the malignant cells and thus to poor prognosis.

Description: cAMP-mediated signaling potentiates glucocorticoid-mediated apoptosis in lymphoid cells, but an effective means by which to take advantage of this observation in the treatment of lymphoid malignancies has not been identified. The PDE4 enzyme family regulates the catabolism of cAMP to AMP in a wide range of tissues. PDE4 inhibitors have recently been submitted for approval for use in asthma and COPD. In leukemic samples from 11 B-CLL patients, rolipram and RO20-1724, two structurally unrelated PDE4 inhibitors, synergized with either hydrocortisone or dexamethasone in inducing B-CLL but not T cell apoptosis. Dose titration studies demonstrated that addition of a PDE4 inhibitor augmented B-CLL apoptosis even when maximally effective doses of either glucocorticoid were utilized. In five patients so analyzed, 10 uM rolipram augmented the induction of apoptosis by 100 uM hydrocortisone by 40 +/− 18%. Using transient transfection of a GRE-luciferase construct with an Amaxa nucleofector technique, we determined that treatment with PDE4 inhibitors augmented glucocorticoid receptor (GR)-mediated GRE transactivation in primary B-CLL cells. Strikingly, inhibition of PKA with the cAMP antagonist Rp-8Br-cAMPS inhibited glucocorticoid-induced apoptosis by 86 +/− 14% in 6 patients so tested and GRE transactivation by 83% in 8 patients so tested. Similarly, treatment with Ht31 peptide, a 23 residue peptide derived from an AKAP that binds with 4.0 nM dissociation constant to PKA RII subunits, also reduced hydrocortisone-induced transactivation. These studies suggest that PKA activity is required for both the ability of glucocorticoids to induce apoptosis and GRE transactivation in B-CLL cells. CCRF-CEM cells, a well-studied model of glucocorticoid and cAMP-induced apoptosis, differed from B-CLL cells in that stimulation of adenylyl cyclase with the diterpene forskolin was required to increase both glucocorticoid-mediated apoptosis and GRE activation, while PDE4 inhibition had no effect. We isolated both dexamethasone-sensitive and dexamethasone-resistant CCRF-CEM clones for these studies and demonstrated that forskolin induced glucocorticoid sensitivity even in the initially dexamethasone resistant clone. 1,9 dideoxyforskolin, a forskolin analogue that does not activate adenylyl cyclase, failed to augment glucocorticoid sensitivity in CCRF-CEM cells. Given the marked discrepancy in the sensitivity of B-CLL cells and CCRF-CEM cells to PDE4 inhibitor-induced augmentation of glucocorticoid apoptosis and GRE transactivation, we next examined the cAMP response and PDE4 isoforms in these two cell types. Inhibition of PDE4 induced cAMP elevation in B-CLL but not CCRF-CEM cells, while forskolin augmented cAMP levels in CCRF-CEM but not B-CLL cells. While rolipram but not forskolin treatment up-regulated 63 and 68 kDa forms of PDE4B (most likely PDE4B2) in B-CLL, forskolin but not rolipram treatment up-regulated 67 and 72 kDa forms of PDE4D (most likely PDE4D1/D2) in CCRF-CEM cells. These studies suggest that PKA is required for and enhances glucocorticoid-induced apoptosis in B-CLL by modulating GR signal transduction and that inhibition of PDE4 in the absence of exogenous adenylyl cyclase activation is a clinically tenable means by which to achieve such PKA activation. Clinical trials that examine whether PDE4 inhibitors enhance the efficacy of glucocorticoid-containing chemotherapy regimens in B-CLL and other lymphoid malignancies are indicated.

Description: We have performed 2,000+ Fluorodeoxyglucose PET (PET) scans for multiple myeloma (MM) staging and restaging at our facility since October 2001. While the usefulness of the PET scan for MM is reported by us and others elsewhere, we have reviewed our list of “incidental” but important findings, some of which are unique or occur more commonly with MM patients and some of which are common to many patients, the more common of which we present below: Occult infection occurs very commonly in patients with MM due to direct tumor effect on the immune system and to medication (especially high dose dexamethasone). Of the 2000+ PET scans for MM done at our facility, 300+ infections (about one half occult) have been detected by PET. These most commonly involve central lines (septic thrombophlebitis), diskitis, lung (either bacterial or fungal), and periodontal abscesses (a source of bacteremia in these patients), though non-catheter related spontaneous septic thrombophlebitis also occurs. While related to MM, extramedullary disease from MM (EMD) is seen more commonly with PET than MRI since PET has a wider field of view. In our series of 172 patients with both baseline PET and MRI, PET detected EMD in 11/11 patients versus MRI detection rate of 7/11. On follow-up, PET detected three times more EMD (29/31 sites) than MRI (9/31sites). Detection of EMD by PET at baseline is a profoundly negative prognostic factor in our series (12 month EFS 20% for EMD+ vs. 47% for EMD−, p = 0.002, and OS 42% for EMD+ vs. 70% for EMD−, p=0.005, n=48). Following tumor response in hypo- or non-secretory disease is difficult by standard prognostic factors (SPFs). FDG PET results were actively used for clinical management in 10 of 11 non-secretory patients at our facility. A major pitfall of PET scanning for MM comes from the use of steroids. Treatment with chemotherapy in general and steroids (i.e. prednisone or dexamethasone) in particular can result in a false negative PET scan by producing a profound but transient suppression of tumor metabolism. In addition, the hyperglycemic effect of the steroids produces competitive inhibition of FDG (a glucose analog) uptake, also causing suppression of FDG tumor uptake that can lead to an underestimation of disease. Second primary malignancies are frequently seen the MM age group. In our population, we have found unsuspected breast cancer, breast lymphoma, thyroid cancer, melanoma, colon cancer and lung cancer. In addition, we have found several functioning thyroid adenomas and premalignant colon polyps. Being a whole body imaging device for metabolism, FDG PET is a powerful though nonspecific tool for imaging that can not only help stage and restage the malignancy under question but which can yield a plethora of additional clinically useful information if used properly and with its limitations understood.