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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.

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.

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...

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.