Kline, Michael

Abstract Background: Thalidomide and dexamethasone combination has been shown to be an effective therapy for multiple myeloma (MM). In newly diagnosed patients with MM, the combination results in overall response rates of over 70% of patients. However, a considerable number of patients do not respond and will need to proceed to other therapies. The ability to identify patients who are unlikely to respond to particular therapies will allow tailoring of therapeutic approaches and in turn will reduce unnecessary toxicity and morbidity from lack of rapid disease control. With the availability of new technology such as gene expression profiling (GEP), this has become a possibility. We employed this approach to identify genes that can reliably predict lack of response to the thalidomide dexamethasone combination. Methods: Patient samples from the Eastern Co-operative Oncology Group clinical trial (E1A00), that compared thalidomide-dexamethasone combination with dexamethasone, and patient samples from the phase II Mayo Clinic study of thalidomide and dexamethasone, both for newly diagnosed MM, were used for this study. Thirty newly diagnosed patients who were evaluable for response were included in the analysis. GEP was performed using the Affymetrix U133A microarray platform as per manufacturer’s recommendation. The Affymetrix output (CEL files) was imported into Genespring 7.2 (Agilent Technologies) microarray analysis software, normalized across chips using GCRMA followed by per gene normalization to median. For the purposes of this study, responses were defined as reduction in the serum paraprotein (or the urinary M protein in the absence of a serum M protein) of >=50% (PR), 25 to 49% (MR), increase of >=50% (PD) and stable disease for the remaining. Results: Five of the 30 patients had no response to the thalidomide Dexamethasone therapy. Using the class prediction tool available in Genespring (Support Vector Machines), we identified 25 genes that reliable predicted non responders (PD, NC) from the responders (MR, PR, and CR). See table for a list of predictive genes with identified genes. Conclusion: Using a combination of two datasets we have identified a set of genes that can be used to reliably predict lack of response to thalidomide and dexamethasone in patients with newly diagnosed MM. We think that this represents a step towards creation of custom microarrays spotted with genes that are capable of predicting lack of response (or response) for the purpose of tailoring therapy to the patient. Gene Predictive strength hypothetical protein FLJ20719 1.655 Tubulin alpha 6 1.653 SEC3-like 1 (S. cerevisiae) 1.649 Ribosomal protein L18 1.546 Ribosomal protein L4 1.432 Tubulin alpha 6 1.409 cleavage stimulation factor 1.393 Tubulin, alpha, ubiquitous 1.393 CD99 antigen 1.38 hmel2 1.346 Leptin receptor 1.291 Integrin-linked kinaseSMC4 structural maintenance of chromosomes 4-like 1 1.269 Cornichon homolog 1.255 Williams-Beuren syndrome chromosome region 5 1.217 Ubiquitin specific protease 1 1.2 Sarcoma antigen NY-SAR-91 1.2

Abstract Background: Hypusine is a unique amino acid present in all eukaryotes but found in only one highly conserved protein, eukaryotic translation initiation factor 5A. It is formed during a two-step post-translational modification of eIF5A. eIF5A has been identified as a critical factor for cell proliferation in yeast, as a modulator of p53, and recent studies indicate that the unhypusinated form of eIF5A is a pro-apoptotic protein. Because myeloma remains incurable, we hypothesized that induction of apoptosis by modulation of eIF5A may be a useful therapeutic approach. In this study we utilized PEI nanoparticles to deliver plasmid DNA and siRNA to myeloma tumor cells in vitro and in vivo. Methods: An siRNA was used to suppress levels of endogenous hypusinated eIF5A in tumors while an RNAi-resistant plasmid expressing a mutant of eIF5A (eIF5AK50R), that cannot be hypusinated, was used to increase levels of unhypusinated eIF5A which is proapoptotic. In vitro, KAS-6/1 myeloma cells were incubated with control siRNA/control plasmid, eIF5A siRNA alone, or eIF5A siRNA/eIF5AK50R for 72 hours in the presence or absence of IL-6. Apoptosis was quantitated using flow cytometry to detect annexin V/propidium iodide stained cells; results were corrected for percent apoptosis of the mock control to enable comparison between experiments. In vivo, SCID mice (3/group) were injected with 10 million KAS-6/1 cells SQ in the right flank and approximately 6 weeks later injected intratumorally twice weekly as follows: Group 1 control mice received 20 μg empty vector and 10 μg control siRNA. Group 2 received 20 μg eIF5AK50R plasmid and 10 μg control siRNA. Group 3 received 20 μg eIF5AK50R plasmid and 10 μg eIF5A siRNA. Tumor volumes were quantitated in mm3 using a2b/2 where a is the smaller dimension. Results: The in vitro results showed the following % apoptosis: Absence of IL-6 - Control: 6.5%, eIF5A siRNA/eIF5AK50R 30.3%. Presence of IL-6–Control:8.2%; eIF5A siRNA alone 36.5%; eIF5A siRNA/eIF5AK50R 45.2%. Western analysis demonstrated that the siRNA successfully inhibited the production of endogenous eIF5A. In vivo, after 24 days of monitoring tumor volume: group 1 mice showed that the KAS-6/1 cells grew from a mean tumor volume of 7±1 up to 102±16 mm3; tumors in group 2 mice remained relatively stable in size from 21±9 to 26±10 mm3. Group 3 mice showed a mean 92% tumor regression from 99±21 down to 7±1 mm3. Conclusions: Delivery of eIF5A siRNA and eIF5AK50R plasmid by PEI nanoparticles results in significant anti-tumoral reponses in vitro and in vivo. These studies may provide a novel therapy for myeloma patients with end stage disease that have failed standard treatments.

Abstract We have evaluated the efficacy of a novel hydroxamic acid-derived histone deacetylase (HDAC) inhibitor, ITF2357, to promote cell death in multiple myeloma (MM) cells. HDAC inhibitors, which promote histone hyperacetylation and increase gene expression, have been evaluated as candidate agents for combating malignancies because they impact the expression of genes related to proliferation, differentiation, and survival. Exposure of MM cell lines to 1 micromolar ITF2357 led to dramatically increased levels of histone acetylation at 4 hours and 8 hours by Western analysis. Sub-micromolar concentrations of ITF2357 promoted time- and concentration-dependent cell death in MM cell lines. Using 500 nM ITF2357, a concentration potentially achievable in vivo, viability of KAS-6/1 IL-6 dependent myeloma cells was reduced to 28% of control at 24 hrs and 2% of control at 48 hours (Figure 1). In contrast, viability of normal PBMCs was 100% at 24 hours and 80% at 48 hours (Figure 2). U266 and 8226 myeloma cells were found to be sensitive to ITF-2357 in a similar fashion with U266 being least sensitive. Cell death proceeded via apoptosis as measured using Annexin V/propidium iodide staining. ITF 2357 was superior to suberoylanilide hydroxamic acid (SAHA) at inhibition of stromal cell IL-6 production. IL-1beta (10 pg/ml) was used to stimulate bone marrow stromal cell IL-6 production (105 ng/ml) after 48 hours. Concentration of ITF2357:Stromal Cell IL-6 production after 48 hours were as follows - 10 nM: 78 ng/ml; 100 nM: 79 ng/ml; 1000 nM; 32 ng/ml. SAHA at similar concentrations showed no significant decrease in stromal cell IL-6 production compared with the no drug control. In summary, ITF2357 induces significant myeloma cell apoptosis and can inhibit stromal cell IL-6 production. It represents an attractive therapeutic candidate for MM clinical trials. Figure Figure Figure Figure

Abstract Introduction: Features of multiple myeloma (MM) include a proliferative clonal plasma cell population, bone resorption, and neovascularization. Cytokines and chemokines represent two families of molecules that are capable of propagating and enhancing these disease features. In this study, we have utilized antibody array technology to assess the contributions of cytokines and chemokines to the progression of disease, and evaluated the contribution of stromal cells (SCs) in their production. Methods: Wild-type and IL-1beta transduced KAS 6/1 myeloma cell lines or bone marrow cells isolated from patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering myeloma (SMM), and MM were cultured for 48 hrs. Culture supernatants were either analyzed directly, or co-cultured with normal SCs for an additional 48 hrs +/− IL-1 inhibitors, after which the supernatants were removed and analyzed using antibody arrays. SCs alone were also cultured with recombinant IL-1beta +/− IL-1 receptor antagonist (IL-1Ra) to define IL-1 dependent effects. IL-6 and IL-8 ELISAs were utilized to quantify IL-6 and IL-8 levels, and validate antibody array findings. Results: Antibody array analysis of IL-1 effects on stromal cell cultures using recombinant IL-1beta and supernatants from an IL-1beta transduced myeloma cell line demonstrated that stimulation of IL-6, MCP-1 and IL-8 were induced in an IL-1 and stromal cell dependent manner. Although levels of TIMP-2 varied in these cultures, they appeared unrelated to an IL-1 effect. Studies utilizing supernatants from patient bone marrow cells co-cultured with SCs resulted in levels of IL-6, MCP-1, and IL-8 higher than those seen with patient supernatants alone or SC cultures alone. More interestingly, the IL-8 levels appeared to correlate with diagnosis; MGUS samples generated low levels and MM samples stimulated high levels. Furthermore, this stimulation was reduced by the addition of IL-1 inhibitors, demonstrating a dependence on IL-1. To confirm the relationship between diagnosis and IL-8 production, the levels of IL-8 produced by the bone marrow supernatants were quantified directly by ELISA. Correlating with the antibody array data, background production of IL-8 from the cultures of patient cells alone was lower than the corresponding co-culture value. Supernatants from MM patients and a subset of SMM patients stimulated high levels of SC IL-8 secretion in contrast to bone marrow cell supernatants from MGUS patients and most SMM patients. This activity was inhibited by IL-1 inhibitors (see Figure). The IL-8 levels closely parallel the IL-1beta induced IL-6 levels in the same samples. Conclusion: These data indicate that the concentration of IL-8 may be relevant to the pathogenesis of MM. IL-8 production is largely dependent on SCs, and production appears to be at least partially dependent on IL-1 function. IL-8 is a chemokine with activities including chemotaxis of neutrophils, increased vascular permeability and angiogenesis. IL-8 expression has been implicated in multiple tumor types and may play an important role in the stimulation of angiogenesis during the progression from MGUS to active MM. Figure Figure

Abstract Background: Epigallocatechin-3-gallate (EGCG) is a polyphenol extracted from green tea that has been studied for cancer prevention and treatment. Green tea and its extracts have been an integral part of traditional medicines from Asia and are thought to have considerable medicinal value. EGCG has been studied in many tumor types and in vitro studies have been encouraging. It is currently in Phase I/II clinical trial in the treatment of chronic lymphocytic leukemia. Here we examined the activity of EGCG in the setting of myeloma and its potential mechanisms of action. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of EGCG was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Fresh myeloma cells were obtained from bone marrow aspirates and subjected to ACK lysis to remove red cells. These cells were cultured in RPMI1640 with or with out EGCG and apoptotic rates were determined by Apo 2.7 expression on gated plasma cells. Flow cytometry was used for determining the phosphorylation state of various signaling proteins. Anti-angiogenic activity of EGCG was measured using a human in vitro angiogenesis kit (Angiokit). Results: EGCG treatment of myeloma cell lines resulted in dose- and time-dependent cytotoxicity to the cells. EGCG was cytotoxic to several myeloma cell lines, including IL-6 dependent cell lines KAS-6/1 and ANBL-6 as well as drug resistant cell lines MM1.R, Dox40 and LR5. The effect was maintained when the cells were grown in co-culture with marrow stromal cells, indicating the ability to overcome microenvironment mediated resistance. EGCG induced apoptosis of myeloma cell lines, apoptotic cells being measured as Annexin (+)/PI (−) cells by flow cytometry. It induced a dose-dependent apoptosis of freshly isolated myeloma cells with increased effect seen on the CD45+ myeloma cells compared to CD45- cells, reflecting increased activity on the proliferative fraction. Down regulation of Akt phosphorylation was seen following treatment of myeloma cells with EGCG. Using the Angiokit, we were able to demonstrate inhibition of tubule formation by EGCG at concentrations lower than those cytotoxic to myeloma cells. It was also able to overcome the angiogenic activity of VEGF. Conclusion: We demonstrate potent anti-myeloma activity of EGCG in vitro. In vivo studies in animal models of myeloma are ongoing. We demonstrate, as in other cell lines, inhibition of Akt pathways as a potential mechanism of action of EGCG. Ongoing studies are exploring other mechanisms. Most importantly, we demonstrate anti-angiogenic activity of this compound in the setting of myeloma, a finding in concordance with the inhibition of VEGF activity seen with other tumor systems.

Abstract Background: The clonal plasma cells in multiple myeloma have relatively low rates of apoptosis. We have previously shown that the apoptotic rates of the plasma cells have prognostic value in these patients. However, several methods are used both for targeting the myeloma cells for study as well as for detection of apoptosis in these cells. It is not clear which method is optimal for studying apoptotic rates of plasma cells from primary patient samples. We examined two different ways of studying the plasma cells, namely CD138 magnetic bead based positive selection of myeloma cells (sorted) and gating on the plasma cells in whole bone marrow (unsorted) using CD38/CD45 expression patterns. Additionally, we compared two different methods of identifying apoptotic plasma cells. Methods: Paired samples processed by two different methods were studied. The bone marrow aspirate samples were initially processed using ACK lysis to remove the red cells. The samples were then studied using flow cytometry (unsorted sample) or subjected to a second step of magnetic bead based separation of myeloma cells based on CD138 expression (sorted sample). Each of the samples was studied using Annexin/PI and Apo 2.7 expression to identify the plasma cells undergoing apoptosis. Plasma cells were identified in the unsorted samples by virtue of their CD38/CD45 expression. Samples were studied at collection and after 48 hours of culture. Results: At collection, the median percentages of cells considered apoptotic by annexin/PI (Annexin pos, PI neg) were higher than that detected by the Apo 2.7 method (Apo 2.7 + cells), for both sorted (6.5% vs. 1.5%) and unsorted cells (26% vs. 10%). However, after 48 hours of culture the percentage of apoptotic cells detected using the Apo 2.7 method were higher than that by Annexin/PI for both sorted (22.5% vs. 14.5%) and unsorted cells (43.5% vs. 10%). When the 48 hr study was compared with the initial study, percent apoptotic cells detected by Apo 2.7 had increased at 48 hrs for both sorted and unsorted cells. However, the percentage of apoptotic cells by annexin/PI had decreased in the unsorted cells at 48 hrs compared to initial study. The sorted samples had an overall increase in apoptosis at 48 hours as measured by annexin/PI. We then looked at the percentage of apoptotic cells detected by either method on initial study and compared the sorted cells and unsorted cells. Using either method, the percentage of apoptotic cells was significantly less (P=0.01; paired t-test) among the sorted cells compared to the unsorted cells. Conclusion: Previous studies have shown that CD138 expression is lost from PCs as they enter apoptosis. Here we show in paired samples that the percentage of apoptotic myeloma cells significantly decreases following CD138 sorting. CD138 sorting should not be used when studying apoptosis of myeloma cells in patient samples. Use of unsorted cells also allows for examination of the effects of interventions on other cell populations as well. When serial studies of apoptosis are done with unsorted samples, we recommend using Apo 2.7 rather than Ann/PI, since the percentage of apoptotic cells by Ann/PI expression actually appear to decrease with time in culture, thus making it difficult to assess effects of intervention such as in drug treatment studies. We hypothesize that this is due to an artificially high level of Annexin positivity at the initial study that might be brought on by non-specific damage of the membranes secondary to cell processing.

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that results in considerable morbidity and mortality. As it is incurable with the current therapeutic approaches, more effective therapies based on better understanding of the pathobiology of the disease are needed. In MM, malignant plasma cells are characterized by low proliferative and apoptotic rates compared to other malignancies. The tumor suppressor gene p53, responsible for induction of cellular apoptosis in response to genotoxic stimuli, is relatively intact in most cases of myeloma. However, p53 mutations or deletion can occur late in the course of disease. Here we evaluate a novel small molecule inhibitor of the interaction between p53 and its negative regulator, MDM2, in the setting of myeloma. Methods and Results: Mi-63 was cytotoxic to several different myeloma cell lines with a median effect observed at approximately 2.5 μM in cell lines including MM1.S that express wild type p53 and between 10–15 μM in cells with mutated p53 as measured using an MTT cell viability assay. Additionally, Mi63 induced cytotoxicity in myeloma cell lines resistant to conventional agents such as Melphalan (LR50), Doxorubicin (Dox40) and Dexamethasone (MM1.R), indicating non-overlapping mechanisms. To evaluate the ability of the drug to induce cell death in the tumor microenvironment, MM cells were co-cultured with marrow stromal cells or in the presence of VEGF or IL-6, two cytokines known to be important for myeloma growth and survival. Mi63 was cytotoxic to myeloma cells under these conditions as well, at doses similar to those seen with myeloma cells alone. Mi63 was able to inhibit proliferation and induce apoptosis in myeloma cells in a dose- and time-dependent fashion, as demonstrated by flow cytometry using Annexin/PI staining as well as cell cycle studies. Treatment of myeloma cells with Mi63 was associated with early mitochondrial membrane depolarization, inversion of Bax/Bcl-2 ratio, and down regulation of Mcl-1, indicating induction of mitochondrial mechanisms of cell death. Mi63 was also cytotoxic to freshly isolated primary patient myeloma cells, inducing apoptosis in a dose-dependent manner. In the patient cells the drug appears to have a differential effect on the CD45 positive and negative cells. Conclusion: Mi-63 has significant activity in vitro in the setting of myeloma as demonstrated by its effect on myeloma cell lines and primary patient cells. It clearly induces apoptosis in myeloma cells, with higher activity seen in cells with wild type p53. Given the lack of p53 abnormalities in most of the patients with myeloma, this drug alone or in combination is likely to have significant clinical activity. Studies combining this with various DNA damaging drugs are in progress. These studies will eventually form the framework for future clinical studies.

Abstract Background: Isolation of malignant plasma cells from bone marrow of patients with monoclonal gammopathies is critical for studies into the disease biology. The isolation of plasma cells have generally been performed by positive selection using plasma cell markers such as CD138 or by negative selection by removing other marrow cells using a cocktail of antibodies. We have previously demonstrated differences in apoptotic rates in plasma cells with and without this enrichment step. Here we have examined the effect of CD138 magnetic bead selection on the surface phenotype of plasma cells by flow cytometry. Methods: Bone marrow aspirates from patients with myeloma (n=12) were first washed, and then lysed with ACK to eliminate red cells. The samples were split with a portion of the cells further processed for CD138 selection. ACK lysed only whole bone marrow (WBM) and sorted cells were stained with CD38, CD45, CD56, activation markers CD71 and CD69, adhesion markers CD49d, CD11b, and CD66, B cell markers CD19 and CD20, and clonality (kappa and lambda). Gates were drawn around the plasma cells and plasma cell subsets on the basis of CD38/45 expression for both sorted and unsorted samples. In addition, an aliquot of the sorted preparation was examined by immunohistochemistry to calculate the purity of the sorted sample. Results: Significant differences were observed in terms of the percentage of plasma cells expressing the different antigens when the cells were selected using CD138. This difference included a greater than 10% difference in expression between the two preparations as well as a change from positive to negative in several cases. There was a substantial loss in the expression of CD20, CD71 and CD11b on plasma cells following CD138 based sorting (Table). Among the other markers, CD49d remains unchanged and changes are variable for the other markers. In addition, in 8 of the 12 cases, there was a nearly complete loss of the CD45 positive subset with a loss of discrimination between CD45 negative and CD45 positive plasma cell subsets in the remaining CD138 sorted preparations (figure: unsorted left, sorted right). Conclusion: In conclusion, the process of CD138 sorting of plasma cells appears to change important markers on the plasma cells and may even eliminate a key subset from further analysis. This should be kept in mind when isolating plasma cells using CD138 positive selection for analysis such as gene expression profiling. Consideration should be given to negative selection using antibodies against other cell types to deplete them. Marker Unsorted % Sorted % CD56 74 67 CD69 11 2 CD71 23 8 CD49d 95 92 CD11b 33 4 CD66 5 1 CD20 16 6 Figure Figure

Abstract Background: Multiple myeloma remains incurable with current approaches and newer therapies are needed to improve the outcome of these patients. While monoclonal antibody based therapies have been successful in some of the hematological malignancies, such approaches have had limited efficacy in the setting of myeloma. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) (Thymo) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and solid organ transplants. Given the polyclonal nature of this product, with antibodies against different B cell antigens, we evaluated the in vitro and in vivo activity of Thymo in myeloma. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Apoptosis was measured by flow cytometry using Annexin V/PI in cell lines and Apo 2.7 in primary patient plasma cells. Shifts in expression of a variety of different B cell and plasma cell antigens were examined on several different myeloma cell lines following Thymo treatment in order to identify the potential antigenic targets. In vivo activity of thymo was evaluated in a SCID plasmacytoma model injected with RPMI myeloma cell lines. Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1 with LC50 of around 1 mg/mL. Additionally, thymo was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). Thymo induced apoptosis in MM cell lines and in patient derived primary myeloma cells. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Using competitive flow cytometry, we identified CD138, CD38, Cd45, CD126, CD49d (VLA4), as well as CD20 as antigens likely to be targeted by Thymo. Tumor bearing mice injected with Thymo at two different doses (5 mg/kg and 10 mg/kg for five days) had significantly delayed tumor growth compared to non-injected mice, and this translated into a better survival for these mice. Mice receiving 10 mg/kg dose had a slower tumor growth compared to 5 mg/kg dose (Figure). Conclusions: Thymoglobulin has promising in vitro and in vivo activity in the setting of myeloma. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents. Based on these results, we are in the process of initiating a clinical trial combining Thymo with Melphalan. Figure Figure

Abstract Background: Multiple myeloma cells are heterogenous in terms of the expression of CD45 and previous studies have suggested important clinical and biological implications of the CD45 expression. It has been shown that expression of CD45 on myeloma cells has prognostic value and that the CD45 positive cells contain the proliferative fraction of the myeloma cells. We have also demonstrated that the CD45+ population has a higher apoptotic rate compared to CD45− cells. Given the hypothesis that CD45+ cells represent myeloma cells earlier in their development and are more proliferative and more dependent on the marrow microenvironment we studied differences between the two populations in patients with different stages of monoclonal gammopathies and in normal plasma cells. Methods: Fresh bone marrow aspirates were subjected to ACK lysis for removal of RBCs subjected to multicolor flow cytometry to study the expression of different surface antigens on the plasma cells. Plasma cells were identified based on their characteristic CD38/45 expression. The surface expression of CD19 (B cell marker), CD56 (NCAM), CD87, (involved in cellular adhesion, cell motility and angiogenesis), CD126 (IL-6 receptor alpha chain), sVEGF (surface VEGF; surrogate measure of VEGF receptor expression), and CD71 (proliferation marker) were studied and compared between the CD45 + and CD45− cells by gating separately on these populations. Results: Patients with active myeloma (76), smoldering myeloma (32), MGUS/amyloid (44), and normal patients (23) were studied. Not all markers were performed on all patients. Among the patient samples, the expression of CD19, CD87, CD71, CD126, and sVEGF were higher among the CD45+ cells compared to CD45− cells (Table: % of cells with expression). There was no significant difference between the two in terms of the expression of CD56. Among the normal plasma cells, the expression of CD19, CD87, CD126 and sVEGF was greater on the CD45+ cells compared to CD45− plasma cells and CD56 was not different. When the patient cells were compared to normal cells, the expression of CD19 was higher among the normal CD45+ plasma cells and CD56, CD87 and CD126 were less. The expression sVEGF was comparable Conclusion: In this study we demonstrate differences between CD45+ and CD45− plasma cells in patients with monoclonal gammopathies. These findings are important for understanding the disease biology and provide an insight into the functional differences seen between these two populations. The increased expression of CD19 in the CD45+ cells supports the notion that these cells are likely to be at an earlier developmental stage. The comparable CD56 expression is not in accordance with previous studies and needs to be examined in a larger group of patients. Increased expression of sVEGF and CD126 as well as the adhesion molecules is in accordance with increased responsiveness and dependence of the CD45+ cells to VEGF and IL-6 and the increased proliferative rates. These findings indirectly support the hypothesis that CD45 negative cells are less dependent on the marrow microenvironment and the cytokine milieu that it provides.