ALBERT

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: 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.

Description: This report of a phase 2 trial of thalidomide (THAL) (200 mg/d; 200 mg increment every 2 weeks to 800 mg) for 169 patients with advanced myeloma (MM) (abnormal cytogenetics (CG), 67%; prior autotransplant, 76%) extends earlier results in 84 patients. A 25% myeloma protein reduction was obtained in 37% of patients (50% reduction in 30% of patients; near-complete or complete remission in 14%) and was more frequent with low plasma cell labeling index (PCLI) (below 0.5%) and normal CG. Two-year event-free and overall survival rates were 20% ± 6% and 48% ± 6%, respectively, and these were superior with normal CG, PCLI of less than 0.5%, and β2-microglobulin of 3 mg/L. Response rates were higher and survival was longer especially in high-risk patients given more than 42 g THAL in 3 months (median cumulative dose) (landmark analysis); this supports a THAL dose-response effect in advanced MM.

Description: MGUS is a pre-malignant state, with a 1% annual risk of progression to overt myeloma (Kyle, NEJM346:564, 2004). Progression of MGUS to myeloma is preceded and can be predicted by increased rates of bone turnover (Betaille, JCI88:62, 1991). Progress to overt myeloma is associated with lytic bone disease in about 80% of patients. A simple, reliable test that identifies MGUS patients at risk of progression to symptomatic myeloma would elucidate biological mechanisms that govern the process and identify targets for early intervention. The intimate association of progression with changes in bone biology suggested that early recognition of changes in bone turnover would serve such a purpose. Therefore, we sought to identify protein biomarkers of bone disease in the sera of patients with myeloma that can be used as early predictors of progression. Given the limited reliability of individual markers of bone turnover to identify changes in bone metabolism, we elected to adopt a global proteomics approach. Sera from 62 untreated myeloma patients were collected and stored at −80°C for future analysis. 35 serum samples were from myeloma patients with 1–26 lytic bone lesions as identified on X-ray skeletal surveys and 27 serum samples were from patients without lytic lesions. The sera were analyzed by surface-enhanced laser desorption and ionization-time of flight mass spectroscopy (SELDI-TOF MS) using Ciphergen’s ProteinChip Biology System II (PBS II), to identify protein patterns associated with lytic bone disease. Each sample was applied in 4 replicates to randomly assigned spots on 12 IMAC30 ProteinChips placed in a bioprocessor and activated with Cu++ ions using a Biomek2000 robot. The chips were read on a PBS II reader. The mass spectra of proteins, generated using an average of 66 laser shots, were calibrated using peptide and protein standards and normalized to total ion current using CiphergenExpress 2.0 software. To develop a classification model that separates non-treated patients with focal lesions from those without focal lesions, we identified among the low molecular weight (1500–25000 kDa) proteins a set of 17 protein peaks that were differentially expressed between the two groups at a significance level of p

Description: Families with 3 different syndromes characterized by autosomal dominant inheritance of low platelet count and giant platelets were studied. Fechtner syndrome is an autosomal-dominant variant of Alport syndrome manifested by nephritis, sensorineural hearing loss, and cataract formation in addition to macrothrombocytopenia and polymorphonuclear inclusion bodies. Sebastian platelet syndrome is an autosomal-dominant macrothrombocytopenia combined with neutrophil inclusions that differ from those found in May-Hegglin syndrome or Chediak-Higashi syndrome or the Dohle bodies described in patients with sepsis. These inclusions are, however, similar to those described in Fechtner syndrome. Other features of Alport syndrome, though, including deafness, cataracts, and nephritis, are absent in Sebastian platelet syndrome. Epstein syndrome is characterized by macrothrombocytopenia without neutrophil inclusions, in addition to the classical Alport manifestations—deafness, cataracts, and nephritis—and it is also inherited in an autosomal-dominant mode. We mapped the disease-causing gene to the long arm of chromosome 22 in an Italian family with Fechtner syndrome, 2 German families with the Sebastian platelet syndrome, and an American family with the Epstein syndrome. Four markers on chromosome 22q yielded an LOD score greater than 2.76. A maximal 2-point LOD score of 3.41 was obtained with the marker D22S683 at a recombination fraction of 0.00. Recombination analysis placed the disease-causing gene in a 3.37-Mb interval between the markers D22S284 and D22S693. The disease-causing gene interval in these 3 syndromes is similar to the interval described recently in an Israeli family with a slightly different Fechtner syndrome than the one described here. Recombination analysis of these 3 syndromes refines the interval containing the disease-causing gene from 5.5 Mb to 3.37 Mb. The clinical likeness and the similar interval containing the disease-causing gene suggest that the 3 different syndromes may arise from a similar genetic defect.

Description: Syndecan-1 (CD138) is a heparan sulfate-bearing proteoglycan present on the surface of myeloma cells where it mediates myeloma cell-cell and cell-extracellular matrix adhesion. In this study, we examined myeloma cell lines for cell membrane localization of syndecan-1. On some cells we note a striking localization of syndecan-1 to a single small membrane protrusion, with the remainder of the cell surface being mostly negative for syndecan-1. Examination of cell morphology reveals that a proportion of cells from myeloma cell lines, as well as primary myeloma cells, are polarized, with a uropod on one end and lamellipodia on the other end. On these polarized cells, syndecan-1 is specifically targeted to the uropod, but in contrast, on nonpolarized cells syndecan-1 is evenly distributed over the entire cell surface. In addition to syndecan-1, several other cell surface molecules localize specifically to the uropod, including CD44 and CD54. Functional assays reveal that myeloma cell lines with a high proportion of polarized cells have a much higher migratory potential than cell lines with few polarized cells. Moreover, the uropod is the cell pole preferentially involved in aggregation of myeloma cells and in adhesion of myeloma cells to osteoblast-like cells. When polarized myeloma cells are incubated with heparin-binding proteins, like hepatocyte growth factor or osteoprotegerin, they concentrate in the uropod. These data indicate that syndecan-1 is targeted to the uropod of polarized myeloma cells and that this targeting plays a role in promoting cell-cell adhesion and may also regulate the biological activity of heparin-binding cytokines.

Description: High-dose therapy (HDT) has increased complete remission (CR) rates and survival in multiple myeloma (MM). We now report on continuous CR (CCR) and associated prognostic factors in 1000 consecutive patients receiving melphalan-based tandem HDT. Five-year CCR was 52% among 112 CR patients without chromosome 13 (▵13) abnormalities and with beta-2-microglobulin ≤ 2.5 mg/L, C-reactive protein ≤ 4 mg/L, and pre-HDT standard chemotherapy ≤ 12 months. Of all 390 CR patients without ▵13 abnormalities, 35% enjoyed 5-year CCR but none of 54 with ▵13 abnormalities. ▵13 abnormalities, present in overall 16%, reduced 5-year event-free survival from 20% to 0% and overall survival from 44% to 16% (both P 

Description: Families with 3 different syndromes characterized by autosomal dominant inheritance of low platelet count and giant platelets were studied. Fechtner syndrome is an autosomal-dominant variant of Alport syndrome manifested by nephritis, sensorineural hearing loss, and cataract formation in addition to macrothrombocytopenia and polymorphonuclear inclusion bodies. Sebastian platelet syndrome is an autosomal-dominant macrothrombocytopenia combined with neutrophil inclusions that differ from those found in May-Hegglin syndrome or Chediak-Higashi syndrome or the Dohle bodies described in patients with sepsis. These inclusions are, however, similar to those described in Fechtner syndrome. Other features of Alport syndrome, though, including deafness, cataracts, and nephritis, are absent in Sebastian platelet syndrome. Epstein syndrome is characterized by macrothrombocytopenia without neutrophil inclusions, in addition to the classical Alport manifestations—deafness, cataracts, and nephritis—and it is also inherited in an autosomal-dominant mode. We mapped the disease-causing gene to the long arm of chromosome 22 in an Italian family with Fechtner syndrome, 2 German families with the Sebastian platelet syndrome, and an American family with the Epstein syndrome. Four markers on chromosome 22q yielded an LOD score greater than 2.76. A maximal 2-point LOD score of 3.41 was obtained with the marker D22S683 at a recombination fraction of 0.00. Recombination analysis placed the disease-causing gene in a 3.37-Mb interval between the markers D22S284 and D22S693. The disease-causing gene interval in these 3 syndromes is similar to the interval described recently in an Israeli family with a slightly different Fechtner syndrome than the one described here. Recombination analysis of these 3 syndromes refines the interval containing the disease-causing gene from 5.5 Mb to 3.37 Mb. The clinical likeness and the similar interval containing the disease-causing gene suggest that the 3 different syndromes may arise from a similar genetic defect.