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Multiple myeloma (MM) is an incurable bone marrow cancer characterized by the development of osteolytic lesions due to the myeloma-induced increase in osteoclastogenesis and decrease in osteoblastic activity. The standard treatment of MM often involves proteasome inhibitors (PIs), which can also have a beneficial off-target bone anabolic effect. However, long-term treatment with PIs is unadvised due to their high side-effect burden and inconvenient route of administration. Ixazomib is a new-generation, oral PI that is generally well tolerated; however, its bone effect remains unknown. Here, we describe the 3-month results of a single-center phase II clinical trial investigating the effect of ixazomib treatment on bone formation and bone microstructure. Thirty patients with MM in stable disease not receiving antimyeloma treatment for ≥3 months and presenting ≥2 osteolytic lesions received monthly ixazomib treatment cycles. Serum and plasma samples were collected at baseline and monthly thereafter. Sodium 18F-Fluoride positron emission tomography (NaF-PET) whole-body scans and trephine iliac crest bone biopsies were collected before and after three treatment cycles. The serum levels of bone remodeling biomarkers suggested an early ixazomib-induced decrease in bone resorption. NaF-PET scans indicated unchanged bone formation ratios; however, histological analyses of bone biopsies revealed a significant increase in bone volume per total volume after treatment. Further analyses of bone biopsies showed unchanged osteoclast number and COLL1A1High-expressing osteoblasts on bone surfaces. Next, we analyzed the superficial bone structural units (BSUs), which represent each recent microscopic bone remodeling event. Osteopontin staining revealed that following treatment, significantly more BSUs were enlarged (>200,000 μm2), and the distribution frequency of their shape was significantly different from baseline. Overall, our data suggest that ixazomib induces overflow remodeling-based bone formation by decreasing the level of bone resorption and promoting longer bone formation events, making it a potentially valuable candidate for future maintenance treatment.

OBJECTIVES: The Danish Myeloma Study Group initiated a randomized, placebo-controlled, double-blinded phase II study to investigate the efficacy of adding clarithromycin to cyclophosphamide-bortezomib-dexamethasone (VCD) induction therapy in transplant eligible, newly diagnosed multiple myeloma patients. The study was prematurely terminated due to severe complications, and no effect of adding clarithromycin was found. The aim of this study was to compare health-related quality of life (HRQoL) between the two groups and to explore the coherence hereof with adverse event (AE) registration by clinicians.METHODS: Patients completed three validated HRQoL questionnaires at inclusion, before cyclophosphamide priming, and two months after high-dose therapy (HDT). The mean score difference was interpreted by clinically relevant differences between groups. Spearman correlation analysis was used to compare patient-reported toxicities with AEs.RESULTS: Of 58 included patients, 55 participated in the HRQoL reporting. Before cyclophosphamide priming, patients in the clarithromycin group reported clinically relevant reduced HRQoL for eleven domains with persistent reduction in four domains two months after HDT. Poor correlation between patient-reported toxicities and clinician-reported AEs was observed.CONCLUSIONS: Despite the premature study termination, our data demonstrate impaired HRQoL when clarithromycin was added to the VCD regimen. We found clear underreporting of toxicities by clinicians. ClinicalTrials. gov number NCT02573935. This article is protected by copyright. All rights reserved.

Lytic bone disease (LBD) in multiple myeloma (MM) is caused by osteoclast hyperactivation and osteoblast inhibition. Based on in vitro studies, the hepatocyte growth factor (HGF) pathway is thought to be central in osteoblast inhibition. We evaluated the gene expression of the HGF pathway in vivo using bone marrow biopsies (BMBs) of patients with MM and monoclonal gammopathy of undetermined significance (MGUS), and healthy volunteers (HV). BMBs (N = 110) obtained at diagnosis were snap-frozen and used to evaluate gene expression by quantitative reverse transcription polymerase chain reaction. LBD was evaluated using standard radiographs. Enzyme-linked immunosorbent assay (ELISA) was performed on matched bone marrow plasma and immunohistochemistry on matched formalin-fixed paraffin-embedded biopsies. Gene expression of HGF, SDC1, and MET in BMBs were significantly altered in MM versus HV and MGUS, and HGF and MET correlated with the extent of LBD. A significant correlation between gene and protein expression levels was observed for SDC1 (Syndecan-1) and HGF. The HGF bone marrow plasma level was significantly lower in MM patients with no/limited versus advanced LBD. Our novel approach using snap-frozen BMBs seems generally applicable because it allows evaluation of gene expression independent of the extent of MM plasma-cell infiltration. Our study highlights the importance of the HGF pathway in MM LBD.

Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and 125I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5-3.8 fold) and decrease in intact IGFBP-3 (0.6-0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration.

Multiple myeloma (MM) is a common malignant hematological disease displaying considerable heterogeneity. Historical data indicate a prognostic significance of plasmablastic morphology, proliferation, and adverse cytogenetics, but there is little knowledge on the degree of interdependency of these parameters. The aim of this study was to study the degree of overlap between these variables. In a consecutive population-based cohort of 194 untreated MM patients, morphology, and proliferation index, using immunohistochemical double staining for Ki-67 and CD138, was analyzed. In addition, cytogenetic changes were studied by karyotyping and fluorescence in situ hybridization (FISH). Plasmablastic morphology correlated with unfavorable clinical features, high proliferation index, high percentage of plasma cell infiltration in the bone marrow, abnormal karyotype, and del(13q) detected by karyotyping, which indicates that plasmablastic morphology reflects advanced and highly proliferative disease. However, plasmablastic morphology did not correlate with established adverse prognostic cytogenetics identified by FISH, for example, t(4;14), t(14;16) and del(17p).

OBJECTIVES: Syndecan-1 (SDC1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and interleukin-6 (IL6) are expressed by malignant plasma cells and cells in the bone marrow microenvironment and may be involved in the angiogenic process in multiple myeloma (MM).METHODS: In this study, we examined the association between bone marrow angiogenesis estimated as micro-vessel density (MVD) and gene expression of SDC1, HGF, VEGF and IL6 in whole bone marrow biopsies from healthy volunteers (n = 10), patients with monoclonal gammopathy of undetermined significance (MGUS) (n = 35) and MM (n = 65).RESULTS: MVD was significantly higher in patients with MM than MGUS (P = 0.03) and was positively correlated with plasma cell percentage (P = 0.002). SDC1 gene expression increased with increasing MVD in patients with MGUS and MM (P < 0.001). A positive correlation between bone marrow plasma cell percentage and SDC1 gene expression was detected in patients with MM (P < 0.001). Importantly, after adjustment for plasma cell percentage, the association between MVD and SDC1 gene expression remained significant (P = 0.026). No association between bone marrow angiogenesis and gene expression of HGF, VEGF and IL6 was seen.CONCLUSION: Our study indicates that SDC1 expressed by the bone marrow microenvironment is involved in angiogenesis in MM.

Multiple myeloma (MM) lytic bone disease (LBD) is caused by osteoclast activation and osteoblast inhibition. RANK/RANKL/OPG play central roles in osteoclast activation and Wnt inhibitor DKK1 in osteoblast inhibition. The role of other Wnt inhibitors is less clear. We evaluated gene expression of osteoclast regulators (RANK, RANKL, OPG, TRAIL, MIP1A), Wnt inhibitors (DKK1, SFRP2, SFRP3, sclerostin, WIF1) and osteoblast transcription factors (RUNX2, osterix) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) in the bone marrow (BM) microenvironment using snap-frozen BM biopsies, thereby achieving minimal post-sampling manipulation, and gene expression profiling (GEP) data, reflecting the in vivo situation. We analyzed 110 biopsies from newly diagnosed patients with MM and monoclonal gammopathy of unknown significance (MGUS) and healthy volunteers. LBD was evaluated using standard radiographs and the bone resorption marker CTX-1. Protein levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Among Wnt inhibitors, only SFRP3 and DKK1 were significantly overexpressed in advanced LBD, correlating with protein levels. SFRP3 correlated with CTX-1. Our findings support osteoblast inhibition as the driving force behind MM LBD.

Objectives 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). Methods 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. Results We found that decorin concentrations were significantly higher (P

Osteolytic bone disease (OBD) in multiple myeloma (MM) is not fully understood. Microarray analysis of isolated plasma cells from patients with MM revealed overexpression of the osteoblast-regulati...

Syndecan-1 is a heparan sulfate proteoglycan expressed by both normal and multiple myeloma (MM) plasma cells. Matsui et al. [1][1] reported the identification of a potential MM “stem cell” resembling less differentiated post germinal center B cells. These cells lacked expression of CD138 but

Abstract 2922 Osteolytic 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 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 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. Disclosures: No relevant conflicts of interest to declare.

Udgivelsesdato: 2009-Oct The morphological and contractile changes of muscles below the level of the lesion after spinal cord injury (SCI) are dramatic. In humans with SCI, a fiber-type transformation away from type I begins 4-7 months post-SCI and reaches a new steady state with predominantly fast glycolytic IIX fibers years after the injury. There is a progressive drop in the proportion of slow myosin heavy chain (MHC) isoform fibers and a rise in the proportion of fibers that coexpress both the fast and slow MHC isoforms. The oxidative enzymatic activity starts to decline after the first few months post-SCI. Muscles from individuals with chronic SCI show less resistance to fatigue, and the speed-related contractile properties change, becoming faster. These findings are also present in animals. Future studies should longitudinally examine changes in muscles from early SCI until steady state is reached in order to determine optimal training protocols for maintaining skeletal muscle after paralysis.

Udgivelsesdato: 2009-null We report here a case of pyoderma gangrenosum in a patient with myelodysplastic syndrome developing into myeloid sarcoma as a sign of transformation to acute leukaemia. The patient was treated successfully with intensive chemotherapy and achieved complete remission, and her otherwise expanding ulcers started to heal. This is the first reported case of secondary blastic infiltration in pyoderma gangrenosum, and it underlines the importance of performing re-biopsy of non-healing ulcers, especially in patients with an underlying haematological disease.

Two cases of epithelial inclusion cyst as a late complication of childhood female circumcision in patients aged 39 and 27 years are reported. Symptoms were interference with sexual intercourse and discomfort during sitting. Surgical treatment of the condition is known to be effective with few complications and gave correct aesthetic and functional results. Histology confirmed the diagnosis of epithelial inclusion cyst.

Abstract 3977 Osteolytic 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 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 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. Figure 1A. HGF Gene Expression levels in whole snap-frozen BM biopsies. Figure 1B. HGF protein levels in BM plasma (pg/mL). 1 = HV, 2 = MGUS, 3 = no/low OBD MM, 4 = advanced OBD MM. Figure 1A. HGF Gene Expression levels in whole snap-frozen BM biopsies. Figure 1B. HGF protein levels in BM plasma (pg/mL). 1 = HV, 2 = MGUS, 3 = no/low OBD MM, 4 = advanced OBD MM. Disclosures: No relevant conflicts of interest to declare.