Bone Marrow Plasma Samples Research Articles

Serum protein N-glycosylation changes in multiple myeloma

BackgroundMultiple myeloma is characterized by clonal proliferation of malignant plasma cells in the bone marrow that produce monoclonal immunoglobulins. N-glycosylation changes of these monoclonal immunoglobulins have been reported in multiple myeloma, but previous studies only detected limited serum N-glycan features. MethodsHere, a more detailed study of the human serum N-glycome of 91 multiple myeloma patients and 51 controls was performed. We additionally analyzed sequential samples from patients (n = 7) which were obtained at different time points during disease development as well as 16 paired blood serum and bone marrow plasma samples. N-glycans were enzymatically released and measured by mass spectrometry after linkage specific derivatization of sialic acids. ResultsA decrease in both α2,3- and α2,6-sialylation, galactosylation and an increase in fucosylation within complex-type N-glycans were found in multiple myeloma patients compared to controls, as well as a decrease in difucosylation of diantennary glycans. The observed glycosylation changes were present in all ISS stages, including the “low-risk” ISS I. In individual patients, difucosylation of diantennary glycans decreased with development of the disease. Protein N-glycosylation features from blood and bone marrow showed strong correlation. Moreover, associations of monoclonal immunoglobulin (M-protein) and albumin levels with glycan traits were discovered in multiple myeloma patients. Conclusions & general significanceIn conclusion, serum protein N-glycosylation analysis could successfully distinguish multiple myeloma from healthy controls. Further studies are needed to assess the potential roles of glycan trait changes and the associations of glycans with clinical parameters in multiple myeloma early detection and prognosis.

Plasma Soluble HLA-Bound Peptides Derived from Acute Myeloid Leukemia Patients during Induction May Predict Individual Response to Therapy

Introduction: Human leukocyte antigen (HLA) molecules are membrane-bound transporters carrying peptides from the cytoplasm to the cell surface where they become exposed to T lymphocytes. Soluble HLA molecules (sHLA) bound to their specific peptides are present in human plasma. Patient-based evidence that most of these peptides are derived from cancerous cells may lead to the identification of cancer-related sHLA peptidomes in the disease state. Leukemic cells in the bone marrow (BM) are usually composed of several malignant subclones even within the same patient. These leukemic subclones may vary in their sensitivity to chemotherapy. During the first days of chemotherapy, a dynamic change in the blast mass and subclonal cell composition is observed. We have thus hypothesized that the repertoire and amount of peptides bound to sHLA molecules derived from the BM plasma of acute myeloid leukemia (AML) patients will be changing during induction. Our study has aimed at identifying peptides derived from resistant subclones, assuming that they could represent potential immunogenic targets specific to these leukemic cells. Particular focus has been made on the peptides common among multiple AML patients.Methods: Mononuclear cells and plasma fractions obtained from AML patients at diagnosis (day 1), during induction chemotherapy (day 5) and on day 14 of induction were studied. HLA molecules loaded with peptides were purified using pretreated pan HLA-A, B and C monoclonal antibody W6/32 TopTip column, and sHLA class I molecules with their bound peptides were eluted with tri-fluoracetic acid. The eluted fraction containing sHLA was separated from its peptides on C18 Micro TipColumn with acetonitrile. The peptide fraction was then examined using liquid chromatography coupled with mass spectrometry (LC-MS/MS). Based on the LC-MS analysis, we excluded peptides longer than 15 amino-acids and shorter than 8 amino-acids, peptides known as contaminators and peptides highly expressed by healthy donors. Immunogenicity of the identified candidate peptides was evaluated by ɣ-IFN Elispot assay.Results: The total amount of sHLA-bound peptides in sequential BM plasma samples of 11 patients was significantly decreased at nadir (day 14) compared to diagnosis or day 5 samples (3 and 1.33 fold, respectively). About 17 percent (16.9%) of the peptides identified on day 5 of induction were not present at diagnosis. These peptides originate from the proteins known to be involved in cell proliferation, migration, death, cycle control, metastasis promotion, DNA damage, or mitochondria processes, and could thus reflect chemotherapy-induced effects. On day 14, 75% of identified peptides were similar to those observed on days 1 and 5, representing cancer-derived peptides expressed by leukemic cells that survived the first days of therapy.Analysis of whole peptidome sequences was performed in the plasma specimens obtained from 8 healthy donors (HLA A02) and collected at the above 3 time points from 11 patients (4 HLA A02, 5 HLA A01, 2 HLA A68). Three major peptides were found to be common across the tested patient samples. The peptide originating from GUCY1B3 protein was solely detected in 10 patients and in none of the healthy donors; one peptide derived from ETS1 protein and one peptide cut from STAG1 protein were identified in 7 and 6 patient samples, respectively, as well as in one healthy donor. Immunogenicity of the 3 peptides was examined against T lymphocytes derived from 5 AML patients (HLA 02) in remission. High T cell reactivity was confirmed in the peptide derived from GUCY1B3 protein. Additionally, similarities between membranal HLA (mHLA) and plasma sHLA peptidomes derived from the same patient were observed (87.7%) (n=3, HLA A01).Conclusions: The requirement of a large number of cells, limiting the application of membranal HLA peptidome analysis could be overcome using sHLA-bound peptides from the BM plasma. Plasma sHLA-originating peptides, detectable by a simple blood test, may serve as potential biomarkers of response to treatment or as targets for immunotherapy. Such peptides derived from resistant subclones during induction chemotherapy could be used to stimulate T cell clones in a peptide-restricted HLA manner and thus act as a personalized immunotherapy adjuvant in AML. DisclosuresOfran:Novartis: Other: Served on a Novartis advisory board.

Assessment of ASC specks as a putative biomarker of pyroptosis in myelodysplastic syndromes: an observational cohort study

NLRP3 inflammasome-directed pyroptotic cell death drives ineffective haemopoiesis in myelodysplastic syndromes. During inflammasome assembly, the apoptosis-associated speck-like protein containing a CARD (PYCARD, commonly known as ASC) adaptor protein polymerises into large, filamentous clusters termed ASC specks that are released upon cytolysis. Specks are resistant to proteolytic degradation because of their prion-like structure, and therefore might serve as a biomarker for pyroptotic cell death in myelodysplastic syndromes. This observational cohort study was done at the H Lee Moffitt Cancer Center (Tampa, FL, USA). Patients with myelodysplastic syndromes, healthy controls, and patients with non-myelodysplastic syndrome haematological cancers or type 2 diabetes were recruited. We used confocal and electron microscopy to visualise, and flow cytometry to quantify, ASC specks in peripheral blood and bone marrow plasma samples. Speck percentages were compared by t test or ANOVA, correlations were assessed by Spearman's rank correlation coefficient, and biomarker efficiency was assessed by receiver operating characteristics and area under the curve (AUC) analysis. Between Jan 1, 2005, and Jan 12, 2017, we obtained samples from 177 patients with myelodysplastic syndromes and 29 healthy controls for the discovery cohort, and 113 patients with myelodysplastic syndromes and 31 healthy controls for the validation cohort. We also obtained samples from 22 patients with del(5q) myelodysplastic syndromes, 230 patients with non-myelodysplastic syndrome haematological cancers and 23 patients with type 2 diabetes. After adjustment for glucose concentration, the log10-transformed mean percentage of peripheral blood plasma-derived ASC specks was significantly higher in the 177 patients with myelodysplastic syndromes versus the 29 age-matched, healthy donors (-0·41 [SD 0·49] vs -0·67 [0·59], p=0·034). The percentages of ASC specks in samples from patients with myelodysplastic syndromes were significantly greater than those in samples from individuals with every other haematological cancer studied (all p<0·05) except myelofibrosis (p=0·19). The findings were confirmed in the independent validation cohort (p<0·0001). Peripheral blood plasma danger-associated molecular pattern protein S100-A8 and protein S100-A9 concentrations from 144 patients with myelodysplastic syndromes from the discovery cohort directly correlated with ASC speck percentage (r=0·4, p<0·0001 for S100-A8 and r=0·2, p=0·017 for S100-A9). Patients with at least two somatic gene mutations had a significantly greater mean percentage of peripheral blood plasma ASC specks than patients with one or no mutation (-0·22 [SD 0·63] vs -0·53 [0·44], p=0·008). The percentage of plasma ASC specks was a robust marker for pyroptosis in myelodysplastic syndromes (AUC=0·888), in which a cutoff of 0·80 maximised sensitivity at 0·84 (95% CI 0·65-0·91) and specificity at 0·87 (0·58-0·97). Our results underscore the pathobiological relevance of ASC specks and suggest that ASC specks are a sensitive and specific candidate plasma biomarker that provides an index of medullary pyroptotic cell death and ineffective haemopoiesis in patients with myelodysplastic syndromes. T32 Training Grant (NIH/NCI 5T32 CA115308-08), Edward P Evans Foundation, The Taub Foundation Grants Program, the Flow Cytometry, Analytic Microscopy, and Tissue Core Facilities at the H Lee Moffitt Cancer Center and Research Institute, a National Cancer Institute-designated Comprehensive Cancer Center (P30-CA076292).

Microvesicles released from multiple myeloma cells are equipped with ectoenzymes belonging to canonical and non-canonical adenosinergic pathways and produce adenosine from ATP and NAD+

ABSTRACTMultiple myeloma (MM) derives from malignant transformation of plasma cells (PC), which accumulate in the bone marrow (BM), where microenvironment supports tumor growth and inhibits anti-tumor immune responses. Adenosine (ADO), an immunosuppressive molecule, is produced within MM patients' BM by adenosinergic ectoenzymes, starting from ATP (CD39/CD73) or NAD+ [CD38/CD203a(PC-1)/CD73]. These ectoenzymes form a discontinuous network expressed by different BM cells. We investigated the expression and function of ectoenzymes on microvesicles (MVs) isolated from BM plasma samples of patients with MM, using asymptomatic forms of monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM) as controls.The percentage of MVs expressing ectoenzymes at high levels was higher when derived from MM patients than controls. BM CD138+ PC from MM patients expressed high levels of all ectoenzymes. Paired MVs samples confirmed a higher percentage of MVs with high ectoenzymes expression in MM patients than controls. Pooled MVs from MM patients or controls were tested for ADO production. The catabolism of ATP, NAD+, ADPR and AMP to ADO was higher in MVs from MM patients than in those from controls.In conclusion, our results confirmed the hypothesis that MVs in MM niche are main contributor of ADO production. The ability of MVs to reach biological fluids strongly support the view that MVs may assume diagnostic and pathogenetic roles.

Soluble HLA-G and HLA-E Levels in Bone Marrow Plasma Samples Are Related to Disease Stage in Neuroblastoma Patients

The role of nonclassical HLA-class Ib molecules HLA-G and HLA-E in the progression of Neuroblastoma (NB), the most common pediatric extracranial solid tumor, has been characterized in the last years. Since BM infiltration by NB cells is an adverse prognostic factor, we have here analyzed for the first time the concentration of soluble (s)HLA-G and HLA-E in bone marrow (BM) plasma samples from NB patients at diagnosis and healthy donors. sHLA-G and sHLA-E are present in BM plasma samples, and their levels were similar between NB patients and controls, thus suggesting that these molecules are physiologically released by resident or stromal BM cell populations. This hypothesis was supported by the finding that sHLA-G and sHLA-E levels did not correlate with BM infiltration and other adverse prognostic factors (MYCN amplification and age at diagnosis). In contrast, BM plasma levels of both molecules were higher in patients with metastatic disease than in patients with localized NB, thus suggesting that concentration of these molecules might be correlated with disease progression. The prognostic role of sHLA-G and sHLA-E concentration in the BM plasma for NB patients will be evaluated in future studies, by analyzing the clinical outcome of the same NB patients at follow-up.

Effects of in-vivo G-CSF stimulation on cytokine levels in bone marrow plasma samples from healthy donors

Effects of in-vivo G-CSF stimulation on cytokine levels in bone marrow plasma samples from healthy donors

Differential cytokine expression profile of healthy human bone marrow plasma samples from the endosteal and vascular region

Differential cytokine expression profile of healthy human bone marrow plasma samples from the endosteal and vascular region

Assessment of human bone marrow plasma samples suggests a different role for mesenchymal stromal cells in distinct areas mediated by PDGF-BB/PDGF-R

P1144 ASSESSMENT OF HUMAN BONE MARROW PLASMA SAMPLES SUGGESTS A DIFFERENT ROLE FOR MESENCHYMAL STROMAL CELLS IN DISTINCT AREAS MEDIATED BY PDGF-BB/PDGF-R Fatima Aerts Kaya, Ilgin Cagnan, Gozde Aydin, Sevil Kose, Asli Semerci, Baris Kuskonmaz, and Duygu Uckan Center for Stem Cell Research and Development, Hacettepe University, Ankara, Turkey; Pediatric Hematology, BMT unit, Hacettepe University Childrens Hospital, Ankara, Turkey

Decorin is down‐regulated in multiple myeloma and MGUS bone marrow plasma and inhibits HGF‐induced myeloma plasma cell viability and migration

Decorin is a stromal-produced small leucine-rich proteoglycan known to attenuate tumour pro-survival, migration, proliferation and angiogenic signalling pathways. Recent studies have shown that decorin interacts with the hepatocyte growth factor (HGF) receptor c-Met, a potential key pathway in multiple myeloma (MM). Decorin levels in paired peripheral blood and bone marrow plasma samples from healthy volunteers (HV) (n=23), and patients with monoclonal gammopathy of undetermined significance (MGUS) (n=41) and MM (n=19) were determined by ELISA. Further, the ability of decorin to inhibit HGF-induced effects on MM cell lines were analysed in vitro using cell viability and Transwell migration assays. We found that decorin concentrations were significantly higher (P<0.05) in bone marrow (BM) plasma from HVs (median 35.2ng/mL; range, 15.3-99.1) compared with MGUS (median 22.5ng/mL; range, 11.1-59.5) and patients with MM (median 21.5ng/mL; range, 10.6-35.9). Decorin levels were higher in BM plasma than in peripheral blood in all groups, with a BM/PB ratio of 3.9, 3.4 and 2.5 for HV, MGUS and MM, respectively. A positive correlation (Spearman's ρ=0.51, P<0.05) was found between simultaneously measured levels of HGF and decorin in BM plasma in HVs, but not in MGUS or MM samples. Functionally, decorin inhibited HGF-induced migration and viability of INA-6 and ANBL-6 MM cell lines, independent of c-Met down-regulation. Our results show that decorin is down-regulated in MGUS and MM bone marrow plasma and that it inhibits HGF-induced viability and migration of myeloma cell lines in vitro.

Detection of Multiple Myeloma Cells in Peripheral Blood Using High-Throughput Sequencing Assay

AbstractAbstract 321 Background:Multiple myeloma (MM) is characterized by the presence of monoclonal protein (M-protein) in serum and/or urine, clonal plasma cell accumulation in bone marrow (BM), and related organ or tissue impairment. MM patients are monitored during therapy and posttherapy using immunoglobulin, M-protein and free light chain assays. Assessing pathological myeloma cells using flow cytometry and RT-PCR has been shown to have superior prognostic value. However, the sensitivity of these techniques has generally limited their use to assessment of BM. In order to determine whether myeloma minimal residual disease could be detected in peripheral blood (PB), we assessed a cohort of MM patients using a sequencing based platform, LymphoSIGHT, with a sensitivity of 1 cancer cell per million leukocytes. Methods:We obtained from 4 sources (UCSF, NYU, Washington Univ, commercial source) pairs of BM and PB samples from 60 MM patients at different points of disease. BM samples were used to identify the clonal MM sequence and detection of that sequence in the PB was assessed. For some patients BM/PB sample pairs were obtained from >1 time point resulting in a total of 78 pairs. In addition blood and bone marrow plasma samples were available for 44 and 6 patients, respectively, to assess presence of the myeloma clonotype in cell free DNA. Altogether there were 206 samples. BM samples were either available as BM mononuclear cells (BMMC) or bead enriched CD138+ cells. Using universal primer sets, we amplified IgH@ variable (V), diversity (D), and joining (J) gene segments from genomic DNA and/or RNA samples, the incomplete IgH rearrangement (DJ), and IgK from genomic DNA. Amplified products were sequenced to obtain >1 million reads and analyzed using standardized algorithms for clonotype determination. Myeloma-specific IgH, IgK, and DJ clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in all PBMC (DNA and RNA), BM plasma (DNA), and PB plasma (DNA) samples using the same IgH and in some samples using the IgK sequencing assays. A quantitative and standardized measure of clone level among all leukocytes in each PB or BM sample was determined using internal reference DNA. Here we describe data on 46/60 patients; data from all 60 patients will be presented. Results:In BM samples, we detected the myeloma clonal rearrangement of at least one receptor (“calibrating receptor”) in 34/46 (74%) of MM patients (Table 1). The calibration rate varied by receptor, with 30/46 (65%) patients calibrating with IgH, 14/43 (33%) with IgH DJ, and 22/43 (51%) with IgK (Table 1). Identification of myeloma-specific clonal rearrangement is based on presence at high frequency and may not occur in samples from patients with low disease load (e.g., post-treatment). Of the 12 non-calibrating patients, only 3 had high disease load. The myeloma clonotype that was identified in the BM was also detected in PBMC in 22/30 (73%) and 28/30 (93%) patients with the DNA and RNA IgH analysis, respectively (Table 2). IgK DNA analysis showed the presence of the myeloma clonotype in 9/10 PBMC samples, all of which were concordant with IgH results. The myeloma clonotype that was identified in the BM was also detected in the cell-free BM and PB samples in 5/5 and 7/11 patients, respectively, using the IgH DNA assay. The evaluation of blood plasma and PBMC were at times complementary in detecting the myeloma. Conclusions:Results from the application of a high-throughput sequencing method for detection of myeloma-specific clonotypes in 46 MM patients are shown. A clonal rearrangement was detected in 74% of MM BM samples. Importantly, 93% of peripheral blood samples from 30 patients showed evidence of circulating myeloma in PBMC. Analysis of BM and PB samples from 14 additional MM patients as well as association of the level of myeloma in PBMC and BM with clinical measures is ongoing.Table 1Calibration rates for IgH and IgK receptors in MM patients.Sample sourceIgH (complete)IgKIgH (incomplete)Any receptorUCSF8/126/123/129/12NYU15/2113/2110/2117/21Commercial source7/133/101/108/13Total30/4622/4314/4334/46Table 2Detection of circulating myeloma material in MM patients.Sample sourceIgH clone present in blood DNAIgH clone present in blood RNAIgK clone present in blood DNAUCSF4/87/82/3NYU13/1515/157/7Commercial source5/76/7N/ATotal22/3028/309/10 Disclosures:Faham:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Klinger:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Expression of Factors in the Hepatocyte Growth Factor (HGF) Pathway in Whole Bone Marrow Biopsies in Association to the Osteolytic Bone Disease of Multiple Myeloma

AbstractAbstract 3977Osteolytic bone disease (OBD) in multiple myeloma (MM) is known to be caused by a combination of osteoclast hyperactivation and osteoblast inhibition. One of the pathways known to be involved in osteoblast inhibition from in vitro studies is the HGF pathway consisting of HGF, its receptor MET, the co-receptor Syndecan-1 (SDC-1), the partial MET antagonist Decorin and HGF activator responsible for HGF processing to its active form. So far, gene expression studies in MM have been performed on isolated MM plasma cells or bone marrow (BM) aspirates, which are not completely representative of the cell composition in the BM micro-environment. We used a novel strategy, whereby gene expression of factors associated with the HGF pathway was evaluated in snap-frozen BM biopsies, and moreover we determined the protein levels in matched BM plasma samples.An additional BM core biopsy obtained during the diagnostic procedure of MM patients was snap-frozen. Biopsies were cut, homogenized and RNA was purified and analyzed by qRT-PCR using low density arrays (Applied Biosystems). The relative quantitative gene expression was calculated using 3 internal reference genes (ABL, GAPDH and GUS). OBD was evaluated using standard radiographs. All patients were untreated and did not receive medicine that could influence bone remodeling. We examined 10 healthy volunteers (HV), 35 monoclonal gammopathy of unknown significance (MGUS) and 65 untreated MM patients, which according to radiographic findings were divided into NO/LOW and advanced OBD, i.e. OBD in ≥2 regions.ELISA was performed on a total of 31 matched BM plasma samples of HV, MGUS and MM obtained at the same time point as the biopsies. In addition, extra samples without gene data (N=52) were analyzed. Commercial kits for SDC-1 (Diaclone), HGF (RnD, Quantikine) and Decorin (RnD, Duoset) were run in duplicates according to manufacturer's instructions.Gene expression of HGF, SDC1, and MET were significantly different comparing HV, MGUS, no/low and advanced OBD (p<0.05) (For HGF, see figure 1). Decorin was not associated to OBD. HGF activator was not expressed in any of our samples, but only in the positive control.A significant correlation between gene and protein expression levels measured by ELISA was found for SDC-1 (Spearman's rho= 0.463, p=0.0058) and HGF (Spearman's rho=0.45, p=0.01). No correlation was found between Decorin gene levels and BM plasma levels (Spearman's rho =-0.24, p=0.22).The protein level of SDC-1 and HGF in BM plasma were both upregulated in MM and associated significantly to OBD level (p<0.05), while Decorin were significantly downregulated in MGUS and MM samples compared to HVs (p<0.05). A significant difference in HGF BM plasma levels were found between patients with no/limited OBD (median: 1.7ng/mL) and advanced OBD (median: 6.2ng/mL) in BM plasma.In our expression study reflecting the in vivo situation in MM patients, genes in the HGF pathway and proteins were significantly associated to OBD. The use of whole snap-frozen BM biopsies is a novel strategy for evaluation of gene expression in MM making it possible to investigate patients independent of degree of MM plasma cell infiltration. In addition to the dys-regulated gene expression levels alteration of SDC-1 and HGF was also observed at protein level, supporting the gene expression findings, and underscoring the usefulness of BM biopsies for gene expression studies in MM. Furthermore, our study for the first time shows up regulation of HGF in association with OBD at both gene and protein level in a large clinical material. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Expression of Wnt-Inhibitors and SDF-1 in Whole Bone Marrow Biopsies in Association to the Osteolytic Bone Disease of Multiple Myeloma.

AbstractAbstract 2922Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by a combination of osteoclast hyperactivation and osteoblast inhibition. Several studies have shown that the Wnt-inhibitor DKK1 is one of the factors involved in osteoblast inhibition (Tian, NEJM, 2003), while the role of other Wnt-pathway inhibitors, such as secreted frizzled related protein-2 (SFRP2), SFRP3, sclerostin and Wnt inhibitory factor 1 (WIF1) are less studied. In addition stromal derived factor 1 (SDF-1/CXCL12) that interacts with CXCR4 in bone-marrow (BM) is a potential factor in OBD, although conflicting data has been reported. SDF-1has in mesenchymal stromal cells (MSC) been found downregulated in MM patients compared to healthy volunteers (HV) and also downregulated in MM plasma cells vs. normal plasma cells, while SDF-1 serum levels has been correlated to the extent of OBD in MM.Until now, gene expression studies in MM have been performed on isolated MM plasma cells or bone marrow aspirates, which are not completely representative of the cell composition in the BM micro-environment. We used a novel strategy whereby BM biopsies are snap-frozen and subsequently gene expression is analyzed. Using this strategy we have examined the gene-expression of Wnt inhibitors and SDF1. In addition, we have evaluated the protein levels of DKK1 and SDF-1 in matched BM plasma samples.An additional BM core biopsy obtained during the diagnostic procedure of MM patients was snap-frozen. Biopsies were cut, homogenized and RNA was purified and analyzed by qRT-PCR using low density arrays (Applied Biosystems). The relative quantitative gene expression was calculated using 3 internal reference genes (ABL, GAPDH and GUS). OBD was evaluated using standard radiographs. All patients were untreated and did not receive medicine that could influence bone remodeling. We examined 10 healthy volunteers (HV), 35 monoclonal gammopathy of unknown significance (MGUS) and 65 untreated MM patients, which according to radiographic findings were divided into NO/LOW and advanced OBD, i.e. OBD in ≥2 regions.ELISA was performed on a total of 31 matched BM plasma samples of HV, MGUS and MM obtained at the same time point as the biopsies. In addition, extra samples without gene data (N=52) were analyzed. Commercial kits for DKK-1 (RnD, Quantikine) and SDF-1 (RnD, Quantikine) were run in duplicates according to manufacturer's instructions.Gene expression of DKK1 and SFRP3 were significantly (p<0.05) associated with OBD with increasing gene-expression with increasing OBD, while SDF-1 were significantly associated with decreasing gene-expression with increasing OBD. Gene expression of SFRP2, sclerostin and WIF1 did not associate with the extent of OBD. When comparing HV, MGUS and MM independent of OBD, sclerostin showed a tendency to be higher expressed in MM vs. MGUS and HV (p=0.08).A significant correlation between gene expression levels and protein expression levels was found for DKK-1 (Spearman's rho= 0.57, p=0.0006,) while the correlation of gene and protein expression levels of SDF-1 did not reach significance (Spearman's rho =0.31, p=0.074).The protein level of DKK1 in BM plasma was upregulated in MM and associated significantly with the OBD level (p<0.05). The protein level of SDF1 did not correlate to the extent of OBD.Our gene expression study of MM with minimal manipulation of the samples showed that the Wnt-inhibitors DKK1 and SFRP3 were significantly associated to the extent of OBD, while SDF1 gene expression inversely correlated with the extent of OBD. The report of simultaneous measurement of not only DKK1 but also SFRP3 and other Wnt-inhibitors is novel. The use of whole snap-frozen BM biopsies is a new method to evaluate gene expression in MM that makes it possible to include the tumor micro-environment and investigate patients independent of degree of MM plasma cell infiltration, and we were able to reproduce the DKK1 findings by others. The gene expression results were complemented by protein data for DKK1 and SDF-1. Our study shows for the first time the difference between SDF1 gene and protein levels in matched MM patient samples, and the difference measured incites us to further investigate the post-translational regulation of SDF-1, and the possibility of SDF1 protein being less bound to CXCR4 in MM. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Bone Marrow Mesenchymal Stem Cells in Patients with Beta Thalassemia Major: Molecular Analysis with Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy Study as a Novel Method

Bone marrow mesenchymal stem cells (BM-MSCs) are the main cellular components of the bone marrow, providing a supportive cellular microenvironment to maintain healthy hematopoiesis. β-thalassemia major (β-TM) is characterized by anemia that is caused by a genetic defect in hemoglobin synthesis and results in ineffective erythropoiesis (IE). The alterations in the microenvironment in thalassemic bone marrow during IE can cause changes in BM-MSCs. This study aimed to investigate global structural and compositional changes in BM-MSCs in β-TM that may provide a basis in understanding interactions of hematopoietic stem cells (HSCs)-MSCs in such a pathological bone marrow microenvironment. Following characterization of morphological, immunophenotypical, and differentiation properties, the changes in healthy and thalassemic BM-MSCs before and after bone marrow transplantation (BMT) were examined by attenuated total reflection-Fourier transform infrared (ATR-FTIR). The significant increase in lipid, protein, glycogen, and nucleic acid contents in thalassemic BM-MSCs with respect to healthy BM-MSCs was attributed to enhanced cell proliferation and BM activity during IE. The significant decreases in the content of mentioned macromolecules in post-transplant group BM-MSCs versus pre-transplant BM-MSCs was interpreted as restoring effect of BMT therapy on IE and defective BM microenvironment. These alterations were also supported by ELISA results of erythropoietin (EPO) and growth differentiation factor (GDF15) in bone marrow plasma samples as a reflection of IE and by MTT proliferation assay on BM-MSCs. Based on these changes, sampling groups were discriminated by cluster analysis. These results provide information for the studies that concentrate on interactions between HSCs-MSCs in bone marrow.

Production of TRAIL by Multiple Myeloma Cells: a Potential Prediction Marker for Skeletal-Related Events

AbstractAbstract 2975 Introduction:Skeletal complications including pathologic bone fractures, skeletal related plasmacytoma, bone pain and hypercalcemia are major clinical problems for patients with multiple myeloma (MM). Osteoclastgenesis are reportedly triggered by receptor activator of nuclear factor kappa β ligand (RANKL) and inhibited by osteoprotegerin (OPG). Since OPG is an inhibitor of RANKL, a possible role of TRAIL as an osteoclast inducer is suggested. We have previously reported that TRAIL was produced from myeloma cells and it correlated with skeletal-related events (SRE) at the 51th ASH meeting. In the present study, we analyzed a total of 56 cases with MM and examined their urine deoxypyridinorine (U-DPD) concentration, and the expression of DKK-1, MIP-1a by their MM cells, revealing that TRAIL is a powerful and independent predictor of SRE. Materials and Methods:Myeloma cells were purified from bone marrow samples from 56 MM patients using CD138-immunomagnetic beads. Plasma cells from MGUS patients were also evaluated. All samples were obtained under informed consent according to Helsinki Declaration. TRAIL, DKK1 and MIP1a RNA expression in purified MM cells was analyzed using real time PCR. Serum concentrations of TRAIL from MM patients and healthy donors were determined with ELISA. We also examined concentrations of TRAIL in bone marrow plasma from MM and MGUS patients. MM patients were given Skeletal-Related Event Score (SRE score) according to skeletal complications and therapeutic events such as irradiation and operation. Osteoclasts were generated using peripheral blood mononuclear cells (PBMC), M-CSF, sRANK-L, and the murine pre-osteoclastic cell line RAW264.7. Numbers of viable osteoclasts and RAW264.7 cells were determined upon treatment with rTRAIL. Results:TRAIL expression in myeloma cells from MM patients was 5-fold greater than that in plasma cells from MGUS patients (p<0.01). No correlation was observed between TRAIL expression levels in myeloma cells and serum TRAIL concentrations. Serum TRAIL concentrations in healthy donors were not significantly different from those in MM patients. However, TRAIL expression levels in myeloma cells correlated to TRAIL concentrations in corresponding bone marrow plasma samples (p<0.05). Univariate analysis revealed that there was a significant correlation of TRAIL expression levels with serum calcium levels (p<0.05), U-DPD concentrations (p<0.05) and SRE scores (p<0.01). DKK1 expression levels in myeloma cells had no correlation with serum calcium (p=0.20), U-DPD (p=0.26), or SRE scores (p=0.12), while MIP1a expression levels correlated with serum calcium (p<0.01) and U-DPD (p<0.05) levels, but not with SRE scores (p=0.53). Summary of statistical analysis is shown in table 1. Although PBMC-derived osteoclasts constitutively expressed TRAIL receptors, DR4 and DR5, rTRAIL did not trigger apoptosis in osteoclasts or RAW264.7 cells. Conclusions:We propose that MM patients with high TRAIL expression levels in MM cells tend to have greater chances of skeletal complications. Since serum TRAIL concentrations did not correlate with TRAIL RNA expression in myeloma cells, increase of TRAIL in bone marrow microenvironment should be important to develop bone destruction. TRAIL is an important predictable factor for SREs, serum calcium, and U-DPD. Since DKK-1 suppresses osteoblast differentiation rather than manipulating osteoclastgenesis, DKK1 expression may not be capable enough of reflecting SRE events. At this time, how MM cells survive from TRAIL expression, which is associated with apoptosis, is not known. We hypothesize that MM cells are protected by their adhesion to stromal cells as previously reported (J. Immunol. 180:1545-55, 2008), while osteoclasts may be directly activated by TRAIL. Since direct induction of apoptosis of osteoblast by TRAIL is also reported, taken our findings together, TRAIL may be a key molecule inducing SRE in MM patients. Our findings may introduce a new strategy targeting TRAIL to reduce skeletal events in MM. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Characteristic Cytokine/Chemokine Milieu Is Present in the Bone Marrow Environment of Multiple Myeloma Patients Before and After Allogeneic Stem Cell Transplantation.

Abstract 2795Poster Board II-771 Background:The interaction of Multiple Myeloma (MM) with its bone marrow (BM) microenvironment is important for the homing, survival, and proliferation of the malignant plasma cells. In this study, we aimed at answering the question which cytokines, chemokines, and growth factors are typically found in the BM of untreated MM patients as well as in MM patients after allogeneic stem cell transplantation (alloSCT) and might be involved in the pathogenesis of MM and/or Graft-versus-Host Disease (GvHD). Design and Methods:Applying Multiplex Bead-based Luminex technology and Quantikine ELISAs, we determined the concentrations of 34 cytokines/chemokines in the supernatants of 10 myeloma cell lines as well as in the plasma derived from BM and peripheral blood (PB) samples of 10 newly diagnosed MM patients, 20 MM patients post alloSCT, and 20 healthy donors. Results:Besides factors known to be secreted by myeloma cell lines (IL-1RA, IL-8, MCP-1, MIP-1α, MIP1β, MIP-3α) we observed secretion of other cytokines/chemokines such as EGF, HGF, IL2R, IL-12p40/p70, IL-22, IP-10, MIG, and RANTES. In the BM plasma samples of MM patients, we detected elevated levels of HGF, IL-2R, IL-16, EGF, IL-1RA, IP-10, MCP-1, and MIG (p<0.01 or p<0.05). Most of these factors were significantly increased in the BM compared to PB. In addition to increased levels of the factors mentioned above, the BM plasma of MM patients post alloSCT showed selectively elevated concentrations of IL-4, IL-6, IL-8, IL-12p40/p70, and eotaxin (p<0.01 or p<0.05). Eotaxin levels were particularly high in patients with chronic GvHD. Correlating the BM cytokine/chemokine levels with the clinical characteristics of MM patients, we observed that BM levels of IL-16 (r=0.829, p=0.003) and HGF (r=0.805, p=0.005) correlated positively with myeloma cell infiltration within the BM. In addition, IP-10 (r=0.770, p=0.009), HGF (r=0.645, p=0.044), and IL-6 (r=0.664, p=0.036) correlated significantly with the initial stage of disease. Conclusions:Our study demonstrates that myeloma cells themselves generate a significantly higher number of cytokines/chemokines than previously described. We show that characteristic cytokine/chemokine patterns exact in the BM environment of MM patients before and after alloSCT. Certain factors, such as MIP-1α, MCP-1, HGF, IL-16, IP-10, and eotaxin might not only be developed into diagnostic instruments and/or predictive biomarkers, but might also represent potential targets for future myeloma- or GvHD-specific therapies. Disclosures:No relevant conflicts of interest to declare.

Longitudinal and Functional Analysis of Spontaneous NY-ESO-1-Specific Antibody Responses in Multiple Myeloma Patients.

Abstract 2831Poster Board II-807Its tumor-restricted expression and its high immunogenicity render cancer-testis (CT) antigen NY-ESO-1 an exquisite target for antigen-specific immunotherapies. Spontaneous antibody responses against NY-ESO-1 are typically found in a subset of patients with solid tumors. However, little is known regarding serological immune responses against NY-ESO-1 in patients with hematological malignancies including multiple myeloma (MM). Furthermore, no consequent longitudinal analyses have been performed correlating NY-ESO-1-specific antibody titers with the clinical development of the given malignancy. Finally, nothing is known regarding the functional capabilities of spontaneously occurring anti-NY-ESO-1 antibodies in MM or other malignancies.Here, we performed the first longitudinal and functional investigation of NY-ESO-1-specific antibody responses in MM analyzing 1100 sera and 200 bone marrow plasma samples of 194 MM patients. Sera and BM plasma samples of 100 healthy donors served as controls.Screening sera and bone marrow plasma of our MM patients by Enzyme-linked Immunosorbent Assay (ELISA) using full length recombinant NY-ESO-1 protein, we found that 5/194 patients had high-titered antibody responses against this CT antigen. A quantitative B cell ELISPOT demonstrated NY-ESO-1-specific B cells in the peripheral blood as well as in the bone marrow of the respective MM patients. In a western blot analysis, spontaneous NY-ESO-1-specific immune responses in the patients were found to be highly specific for both native and recombinant protein. Epitope mapping in an ELISA using 18 overlapping NY-ESO-1 20mer peptides showed that antibody responses were restricted to the first 70 amino acids of the full-length protein. NY-ESO-1-specific antibodies consisted mainly of IgG1 and to a lower extent of IgG3 subtypes. No IgG2, IgG4, IgM or IgA antibodies against NY-ESO-1 were detected. Interestingly, antibody affinity increased over the course of the disease suggesting an affinity driven antibody maturation. Accordingly, NY-ESO-1-specific antibodies of MM patients were found to be potent complement activators in a western blot technique. On the other hand, despite the high functional capabilities of NY-ESO-1-specific antibodies, antibody titers increased with each NY-ESO-1-expressing (as indicated by reverse-transcriptase-polymerase-chain-reaction and immunohistochemistry) recurrence of the disease.In conclusion, we demonstrate here the spontaneous occurrence of high-titered NY-ESO-1-specific antibodies in MM patients. One reason for the relatively low frequency of antibody responses against NY-ESO-1 might be that most patients were in early stages of the disease or in remission at the time the analysis was performed. Antibodies were produced by NY-ESO-1-specific B cells detectable in the bone marrow as well as in the peripheral blood of the patients. NY-ESO-1-specific antibodies were evoked by a distinct and immunodominant fragment of NY-ESO-1. Affinity maturation of this response and complement activation by the spontaneously occurring NY-ESO-1-specific IgG1-type antibodies speak in favour of an effective serological immune response. However, positive correlation of antibody titers with tumor burden and recurrence of the disease suggest an inability of antibodies targeting intracellular protein NY-ESO-1 to control the course of the disease, at least in the long run. Antigen-specific immunotherapy might be necessary to shape NY-ESO-1-specific immunity in MM patients and, particularly, to mobilize tumor-specific T cell responses. Disclosures:No relevant conflicts of interest to declare.

Acute Lymphoblastic Leukemia Blast Cells Stimulate the Autocrine Production of CCL2 and IL-8 in Bone Marrow Stromal Cells

The interactions of Acute Lymphoblastic Leukemia (ALL) blasts with bone marrow (BM) stromal cells have a positive impact on leukemia cell survival and resistance to chemotherapy. ALL stimulates BM stromal cells, which reciprocally promotes leukemia cell survival. To identify molecules critically involved in leukemia–microenvironment crosstalk, we performed gene expression profiling analyses of primary BM endothelial cells (BMEC) and BM mesenchymal stem cells (BMMSC) following stimulation by primary ALL cells. Leukemia stimulation of BM stromal cells upregulates the expression of several inflammatory chemokines, including CCL2 and IL-8/CXCL8. Secretion of these molecules was confirmed by ELISA assays of in vitro co-culture experiments and in BM plasma samples from pediatric ALL patients. Most primary ALL samples were found to express mRNA for CCR2 and CXCR1/CXCR2, which are the cognate receptors for CCL2 and IL-8, respectively. Primary ALL cells expressing at least one myeloid marker (CD13, CD15 or CD33) exhibited increased mRNA expression of CCR2 (p = 0.02). Leukemia cells from most patients express CCL2 and IL-8 chemokines (ELISA test) but at lower levels than that of BMEC and BMMSC. In vitro functional studies revealed that the proliferation, survival and migration of primary ALL cells co-cultured with BM stromal cells were not affected by addition of CCL2, IL-8 or of neutralizing antibodies to these chemokines. On the other hand, both chemokines were found to enhance BMEC and BMMSC survival in serum-free medium and to increase their proliferation in serum-starved conditions. Interestingly, CCL2 and IL-8 affected endothelial morphogenesis as shown in Matrigel assays. Since CCL2 and IL-8 have suppressive effects in normal hematopoiesis but do not seem to affect primary ALL cells, it is possible that these chemokines may contribute to the establishment of survival/proliferative selective advantage for ALL cells in the leukemic BM microenvironment. In addition, CCL2 and IL-8 seems indirectly to contribute to ALL cell survival by stimulating the supporting BM stromal cells. Finally, preliminary results showed that standard risk pediatric ALL patients with BM plasma levels below 577pg/ml have better survival rates than those with higher CCL2 levels (p = 0.08). In conclusion, this work suggests a significant role for the chemokines CCL2 and IL-8 in the leukemia/microenvironment crosstalk in human ALL, and suggests that these molecules may represent valuable targets for therapeutic intervention in this cancer. Supported by: CNPq, FAPESP.

A critical evaluation of loss of heterozygosity detected in tumor tissues, blood serum and bone marrow plasma from patients with breast cancer

IntroductionThe aim of the study was to perform a comparative analysis of LOH (loss of heterozygosity) in primary tumors as well as peripheral blood and bone marrow (BM) of patients with breast cancer (BCa).MethodsPerforming PCR-based fluorescence microsatellite analysis and using a panel of seven polymorphic microsatellite markers, we compared the profiles of LOH in primary tumors, peripheral blood and BM plasma from patients with primary BCa (n = 40, stage M0) as well as tumor tissues and blood serum from metastatic BCa patients (n = 48, stage M1). During the course of systemic treatment blood samplings from 12 M0 and 16 M1 patients were at least once repeated.ResultsThe overall incidences of LOH in tumor tissues, blood and BM samples were 27.5%, 9.0% and 5.0%, respectively. The marker D3S1255 was the only one in the panel that showed similar frequencies of LOH ranging from 19.0 to 24.5% in tumor, blood and BM samples. Both M0 blood serum and BM plasma samples displayed the same rate of 19.0%, whereas tumor and M1 serum showed a rate of 24.5% and 24.0%, respectively, at this locus. This marker also showed the highest frequency of LOH in serum and BM samples, whereas in tumor samples LOHs at the markers D13S218 (38%) and D17S855 (36%) were more frequent. Statistical analysis of the tumor samples showed that occurrence of LOH at the markers D3S1255 (P < 0.04), D9S171 (P < 0.05) and D17S855 (P < 0.03) correlated with undifferentiated nuclear grade. Additionally, significant associations of the number of LOH recorded at D17S250 with estrogen receptor (P < 0.02), progesterone receptor (P < 0.03) expression and high proliferation score (Ki-67 expression, P = 0.009) were observed. In blood serum samples a relationship between positive lymph node status and LOH at the marker D3S1255 was revealed (M0 stage, P = 0.05; M0+M1 stage, P = 0.004).ConclusionOur study demonstrates heterogeneous profiles and low rates of LOH, particularly on free DNA in BM and blood samples. However, the significant associations of LOH with some risk factors and the demonstrated possibility of monitoring free DNA in patients undergoing systemic therapy suggest that LOH analysis may be developed into a useful diagnostic tool.

Functional CXCR4-Expressing Microparticles and SDF-1 Correlate with Circulating Acute Myelogenous Leukemia Cells

Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of acute myelogenous leukemia (AML). Cellular microparticles, submicron vesicles shed from the plasma membrane of various cells, are also associated with human pathology. In the present study, we investigated the putative relationships between the SDF-1/CXCR4 axis and microparticles in AML. We detected CXCR4-expressing microparticles (CXCR4(+) microparticles) in the peripheral blood and bone marrow plasma samples of normal donors and newly diagnosed adult AML patients. In samples from AML patients, levels of CXCR4(+) microparticles and total SDF-1 were elevated compared with normal individuals. The majority of CXCR4(+) microparticles in AML patients were CD45(+), whereas in normal individuals, they were mostly CD41(+). Importantly, we found a strong correlation between the levels of CXCR4(+) microparticle and WBC count in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, levels of functional, noncleaved SDF-1 were reduced in these patients compared with normal individuals and also strongly correlated with the WBC count. Furthermore, our data indicate NH(2)-terminal truncation of the CXCR4 molecule in the microparticles of AML patients. However, such microparticles were capable of transferring the CXCR4 molecule to AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID/beta2m(null) mice. The CXCR4 antagonist AMD3100 reduced these effects. Our findings suggest that functional CXCR4(+) microparticles and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML variable.

Interleukin 6. A potential autocrine/paracrine factor in Paget's disease of bone.

Pagetic osteoclasts are greatly increased in number and size and have increased numbers of nuclei per cell compared to normal osteoclasts. The mechanisms responsible for enhanced osteoclast formation in Paget's disease are unknown. We have used our recently described model system for pagetic osteoclast formation to evaluate culture media conditioned by these atypical multinucleated cells (MNC) to determine if pagetic osteoclasts produce an autocrine or paracrine factor that enhances osteoclast formation. Conditioned media from long-term bone marrow cultures from patients with Paget's disease stimulated osteoclast-like MNC formation in normal marrow cultures. At least part of this activity could be ascribed to interleukin 6 (IL-6). In contrast, conditioned media from normal marrow cultures contained lower levels of IL-6 and did not stimulate formation of osteoclast-like MNC. 7 of 8 bone marrow plasma samples taken from involved bones and 18 of 27 peripheral blood serum samples from Paget's patients had high levels of IL-6. Normal marrow plasma and peripheral blood serum had no or very low levels of IL-6. These results suggest that IL-6 produced by marrow and/or bone cells in patients with Paget's disease may be an autocrine/paracrine factor for pagetic osteoclasts.