Serotonin Dysregulation Correlates with Both Bone and Active Disease In Multiple Myeloma

Abstract Abstract 1920 The biologic mechanisms involved in the pathogenesis of multiple myeloma (MM)-induced osteolytic bone disease are poorly understood. Physiological interactions between the serotoninergic and skeletal systems have been implicated by clinical observations. Brainstem-derived serotonin positively regulates bone mass following binding to 5-HT2C receptors on ventromedial hypothalamic neurons. This is opposed by platelet-derived serotonin that induces bone lysis and osteoclast activation. Moreover, the serotonin transporter SLCA6A4 is universally present among the malignant B cell clones. We examined serotonin dysregulation in two sample types from MM patients: peripheral blood and bone marrow. In the blood we measured by ELISA the ratio of serotonin in serum compared to platelets in patients with MM (n=10), MGUS (n=10) or healthy controls (n=5). We found higher levels of serotonin in platelets for the MM patients compared to the MGUS & healthy controls (p=0.017). Concomitantly there was less serotonin in the serum of MM patients compared to compared to the MGUS & healthy (p=0.002). This implies an imbalance in the compartmentalization of serotonin associated with presence of MM bone disease. Our multiplexed protein kinase signal pathway mapping technology, reverse phase protein microarrays (RPMA), was applied to bone marrow samples for quantifying post-translational modifications (e.g. phosphorylation, cleavage, acetylation) and/or total cell signaling kinase levels. Using reverse-phase protein microarray (RPMA) we retrospectively measured bone remodeling signal pathway perturbations in 15 bone marrow core biopsies from patients diagnosed with MM, at different clinical stages, and correlated this with the presence of MM-related bone disease (documented by scan or MRI). Bone marrow core biopsies exhibited significant elevation of cellular Serotonin, RANK, MMP-11, TNFα, TNF-R1, and Ezrin Tyr353 in MM with active bone disease (n=9) compared to patients without bone disease (n=6) (respectively p=0.031, p=0.038, p=0.0082, p=0.0221, p=0.01, p=0.028). To further evaluate the serotonin dysregulation in bone marrow cells, we measured serotonin bound to plasma cells (CD138+) compared to CD138- cells in bone marrow aspirates (n=21). Bone marrow aspirate were collected from patients undergoing standard of care hematological work up for multiple myeloma at any stage or treatment course. Patients with symptomatic myeloma (defined as presence of at least one of CRAB symptom) had lower serotonin levels in the CD138- bone microenvironment cells compared to non-symptomatic patients (p=0.0235). Plasma cells (CD138+) exhibited larger amounts of serotonin compared CD138- bone microenvironment cells (p=0.016). Taken together, our data show a dysregulation of serotonin in MM suggesting an altered distribution of serotonin in blood and bone marrow. This provides potential insights into diagnosis, prognosis, and/or treatment. Disclosures: No relevant conflicts of interest to declare.

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Osteoclast Gene Expression Profiling in Multiple Myeloma

Blood ◽

10.1182/blood.v112.11.2735.2735 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2735-2735

Author(s):

Jerome Moreaux ◽

Dirk Hose ◽

Thierry Rème ◽

Philippe Moine ◽

Karène Mahtouk ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Bone Resorption ◽

Bone Disease ◽

Plasma Cells ◽

Osteoclastic Bone Resorption ◽

Myeloma Cells ◽

Myeloma Bone Disease ◽

Osteolytic Bone Disease

Abstract Multiple myeloma (MM) is a fatal hematologic malignancy associated with clonal expansion of malignant plasma cells within the bone marrow and the development of a destructive osteolytic bone disease. The principal cellular mechanisms involved in the development of myeloma bone disease are an increase in osteoclastic bone resorption, and a reduction in bone formation. Myeloma cells (MMC) are found in close association with sites of active bone resorption, and the interactions between myeloma cells and other cells within the specialized bone marrow microenvironment are essential, both for tumor growth and the development of myeloma bone disease. In order to investigate the gene expression profile (GEP) of osteoclastic cells, we compare GEP of osteoclastic cells (7 samples) with normal B cells (7 samples), normal bone marrow plasma cells (7 samples), bone marrow stromal cells (5 samples), bone marrow CD3 cells (5 samples), CD14 cells (7 samples), CD15 cells (7 samples), CD34 cells (7 samples) and primary MMC (123 samples). Using SAM analysis, a set of 552 genes was overexpressed in osteoclasts compared to others cell subpopulations with a FDR ≤ 1% and a ratio ≥ 2. Osteoclasts specifically overexpressed genes coding for chemokines (CCL2, CCL7, CCL8, CCL13, CCL18, CXCL5 and CCL23) and MMC growth factors (IGF-1, APRIL and IL-10). Anti- IGF-1 receptor and TACI-Fc inhibit MMC growth induced by osteoclasts. Among the chemokines overexpressed by osteoclasts, the majority of them have a common receptor: CCR2 expressed by MMC. Anti-CCR2 MoAb inhibits migration of the CCR2+ HMCL in response to osteoclasts. Expression data of purified MMC were analyzed by supervised clustering of group with higher (CCR2high) versus lower (CCR2low) CCR2 expression level. Patients in the CCR2high group are characterized by a higher bone disease. A set of 176 genes was differentially expressed between CCR2high and CCR2low MMC. CCR2high displayed a gene signature linked to the dependency of MMC on the interactions with the BM osteoclastic subpopulation and the osteoclastic bone resorption. Taken together, our findings suggest addition of chemokine antagonists to current treatment regimens for MM should result in better therapeutic responses because of the loss of both the protective effect of the bone marrow environment on the MMC and the osteoclastic cells activity.

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Bone disease as the first manifestation of systemic AL-amyloidosis

Terapevticheskii arkhiv ◽

10.26442/00403660.2020.07.000775 ◽

2020 ◽

Vol 92 (7) ◽

pp. 85-89

Author(s):

L. P. Mendeleeva ◽

I. G. Rekhtina ◽

A. M. Kovrigina ◽

I. E. Kostina ◽

V. A. Khyshova ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Disease ◽

Plasma Cells ◽

Interventricular Septum ◽

Bone Destruction ◽

Amyloid Deposition ◽

Bone Lesions ◽

Al Amyloidosis ◽

Linear Type

Our case demonstrates severe bone disease in primary AL-amyloidosis without concomitant multiple myeloma. A 30-year-old man had spontaneous vertebral fracture Th8. A computed tomography scan suggested multiple foci of lesions in all the bones. In bone marrow and resected rib werent detected any tumor cells. After 15 years from the beginning of the disease, nephrotic syndrome developed. Based on the kidney biopsy, AL-amyloidosis was confirmed. Amyloid was also detected in the bowel and bone marrow. On the indirect signs (thickening of the interventricular septum 16 mm and increased NT-proBNP 2200 pg/ml), a cardial involvement was confirmed. In the bone marrow (from three sites) was found 2.85% clonal plasma cells with immunophenotype СD138+, СD38dim, СD19-, СD117+, СD81-, СD27-, СD56-. FISH method revealed polysomy 5,9,15 in 3% of the nuclei. Serum free light chain Kappa 575 mg/l (/44.9) was detected. Multiple foci of destruction with increased metabolic activity (SUVmax 3.6) were visualized on PET-CT, and an surgical intervention biopsy was performed from two foci. The number of plasma cells from the destruction foci was 2.5%, and massive amyloid deposition was detected. On CT scan foci of lesions differed from bone lesions at multiple myeloma. Bone fragments of point and linear type (button sequestration) were visualized in most of the destruction foci. The content of the lesion was low density. There was no extraossal spread from large zones of destruction. There was also spontaneous scarring of the some lesions (without therapy). Thus, the diagnosis of multiple myeloma was excluded on the basis based on x-ray signs, of the duration of osteodestructive syndrome (15 years), the absence of plasma infiltration in the bone marrow, including from foci of bone destruction by open biopsy. This observation proves the possibility of damage to the skeleton due to amyloid deposition and justifies the need to include AL-amyloidosis in the spectrum of differential diagnosis of diseases that occur with osteodestructive syndrome.

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Role of microRNAs in Diagnosis, Prognosis and Management of Multiple Myeloma

International Journal of Molecular Sciences ◽

10.3390/ijms21207539 ◽

2020 ◽

Vol 21 (20) ◽

pp. 7539

Author(s):

Amro M. Soliman ◽

Teoh Seong Lin ◽

Pasuk Mahakkanukrauh ◽

Srijit Das

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Growth ◽

Malignant Transformation ◽

Bone Disease ◽

Clinical Management ◽

Potential Role ◽

Plasma Cells ◽

The Usa

Multiple myeloma (MM) is a cancerous bone disease characterized by malignant transformation of plasma cells in the bone marrow. MM is considered to be the second most common blood malignancy, with 20,000 new cases reported every year in the USA. Extensive research is currently enduring to validate diagnostic and therapeutic means to manage MM. microRNAs (miRNAs) were shown to be dysregulated in MM cases and to have a potential role in either progression or suppression of MM. Therefore, researchers investigated miRNAs levels in MM plasma cells and created tools to test their impact on tumor growth. In the present review, we discuss the most recently discovered miRNAs and their regulation in MM. Furthermore, we emphasized utilizing miRNAs as potential targets in the diagnosis, prognosis and treatment of MM, which can be useful for future clinical management.

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Targeting Wnt Signaling in Myeloma In Vivo; Differential Effects on Tumor Burden and Myeloma Bone Disease.

Blood ◽

10.1182/blood.v110.11.812.812 ◽

2007 ◽

Vol 110 (11) ◽

pp. 812-812

Author(s):

Claire M. Edwards ◽

James R. Edwards ◽

Seint T. Lwin ◽

Gregory R. Mundy

Keyword(s):

Bone Marrow ◽

Wnt Signaling ◽

Bone Disease ◽

Tumor Burden ◽

Bone Microenvironment ◽

Myeloma Cells ◽

Myeloma Bone Disease ◽

Osteolytic Bone Disease

Abstract Multiple myeloma is characterized by uncontrolled proliferation of myeloma cells within the bone marrow and the development of a severe osteolytic bone disease. In addition to a well characterized increase in osteoclastic bone resorption, myeloma bone disease is associated with a reduction in bone formation. Osteoblast differentiation and bone formation are regulated in vivo by canonical Wnt signaling and activation of β-catenin. Therefore increasing Wnt signaling in the bone microenvironment in multiple myeloma may prevent the development of myeloma bone disease. In support of this, we have previously demonstrated that activation of Wnt signaling with lithium chloride (LiCl) in the 5TGM1 murine model of myeloma reduces tumor burden and osteolytic bone disease. However, we also found that LiCl treatment increased subcutaneous (s.c.) tumor growth. This suggests that the reduction in tumor burden within the bone microenvironment may be an indirect effect mediated through the effects of LiCl to prevent myeloma bone disease. The aim of the current study was to determine the effect of specific molecular blockade of Wnt signaling in myeloma cells in vivo. 5TGM1-GFP myeloma cells were transfected by electroporation with either myc-tagged dominant negative TCF4 (DNTCF4) or pcDNA. Following stable selection by culture in G418, expression of DNTCF4 was confirmed by western blot for myc. No difference was found in the growth rates of 5TGM1-pcDNA or 5TGM1-DNTCF4 in vitro. Treatment with LiCl or Wnt3A had no significant effect on cell viability in vitro, but significantly increased β-catenin activity, as measured by TOPFLASH activity in 5TGM1-pcDNA cells. This increase was not observed in 5TGM1-DNTCF4, confirming that expression of DNTCF4 blocked Wnt signaling induced by LiCl in 5TGM1 myeloma cells. C57Bl/KaLwRij mice were inoculated with 5TGM1-pcDNA or 5TGM1-DNTCF4 cells by either intravenous (i.v.) or s.c. injection. Mice were treated from time of tumor cell inoculation with 200mg/kg/day LiCl or vehicle control (d.H20) by oral gavage for 28 days. I.v. inoculation of myeloma cells resulted in a significant increase in serum IgG2bκ concentrations and the proportion of GFP-positive cells in the bone marrow. A significant reduction in trabecular bone volume was also observed. MicroCT analysis of the tibia demonstrated that LiCl significantly increased trabecular bone volume in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. LiCl significantly decreased serum IgG2bκ concentrations in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice, with a greater effect in 5TGM1-DNTCF4 myeloma-bearing mice. FACS analysis of GFP-positive cells demonstrated that LiCl significantly reduced tumor burden in the bone marrow in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. However, following s.c inoculation, LiCl significantly increased s.c. tumor volume of 5TGM1-pcDNA tumors, but had no effect on 5TGM1-DNTCF4 s.c. tumor volume. Taken together these results demonstrate that the effect of increasing Wnt signaling in myeloma is dependent upon the microenvironment. By specific inhibition of β-catenin activity in myeloma cells combined with systemic stimulation of the Wnt signaling pathway, our results suggest that increasing Wnt signaling in myeloma in vivo has dual effects; firstly to enhance myeloma growth directly, and secondly to enhance osteoblast differentiation and thus indirectly reduce tumor burden in bone, highlighting the importance of the bone marrow microenvironment in regulating myeloma growth and survival.

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miRNA in Serum and Bone Marrow Plasma Cells From Multiple Myeloma Patients.

Blood ◽

10.1182/blood.v120.21.2921.2921 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2921-2921

Author(s):

Andrew Stiff ◽

Alberto Rocci ◽

Craig C. Hofmeister ◽

Paola Omedè ◽

Susan Geyer ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Peripheral Blood ◽

Plasma Cells ◽

Immunoglobulin M ◽

Differentially Expressed ◽

Healthy Controls ◽

Circulating Mirnas ◽

Serum Samples ◽

Screening Analysis

Abstract Abstract 2921 Background: Multiple myeloma (MM) is a clonal B-cell malignancy characterized by the aberrant expansion of clonal plasma cells (PCs) within the bone marrow. Malignant PCs produce intact or partial monoclonal immunoglobulin (M protein) and cause organ damage. More than 20,000 new cases of multiple myeloma (MM) are diagnosed every year in the US with approximately 10,700 deaths occurring. The pathogenesis of MM is still largely unclear, but several reports suggest that interaction of tumor cells with the bone marrow microenvironment and microRNAs (miRNAs) deregulation may play a role in the etiology and progression of MM. miRNAs are small non-coding RNAs capable of regulating protein expression by binding to mRNA, and have been implicated in the development of MM. First identified inside cells, miRNAs can also be detected in body fluids, including serum and plasma, and may be a valid biomarker. Few studies have investigated the agreement between circulating miRNAs and intracellular myeloma PC miRNAs at diagnosis. Methods: Using Nano-String nCounter technology we first performed a screening analysis on serum samples obtained from MM patients and healthy controls. We identified a candidate set of miRNAs differentially expressed in the serum of MM patients. The levels of these miRNA markers were validated by RT-PCR in both serum and bone marrow PCs from the same cohort. Agreement of the quantitative miRNA marker levels between sample types was evaluated using intraclass correlation coefficients (ICC) (both for normalized and log2 measures). Results: Thirty-nine MM patients (21 male, 18 female) with a median age of 72 years (range: 65 – 83) were included in the analysis. Most were ISS stage I or II (59% vs. 41% ISS stage III) and 39% were high risk according to FISH abnormalities – 21% of patients carried del17p, 24% t(4;14) and 5% t(14;16). Medians and ranges for lab markers were as follows: hemoglobin 10.0 g/dl (7.2 to 15.1), beta2-microglobulin 5.18 ug/ml (1.38 – 12.1), creatinine 0.94 mg/dl (0.65 – 2.49), CRP = 1.6 mg/dl (0.02 – 116.0). Nine age-matched healthy controls were also used for the analysis. After the screening analysis, the following miRNAs were differentially expressed between healthy subjects and MM patients in serum samples: miR-92a, miR-451, miR-19b, miR-21, miR-16, miR-25, miR-30a, and miR-126. There was no significant agreement or correlation between serum and myeloma cell samples using either untransformed as well as log2measures (all p>0.40) (Table 1). Conclusion: Our preliminary results suggest a difference between circulating miRNAs in myeloma patients from controls. This indicates that future studies are needed to better define the role of miRNAs in the peripheral blood as a prognostic and even diagnostic biomarker in myeloma. From our preliminary data it also appears that circulating miRNAs are not simply secreted into the peripheral blood by myeloma PCs as it seems that circulating miRNAs do not reflect those of myeloma PCs. Differential expression could be determined by other cells that can release and or modify their miRNA expression in response to MM. Ongoing studies are examining the origin and function of miRNAs in the peripheral blood. Disclosures: No relevant conflicts of interest to declare.

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Myeloma-Induced Alterations of Glutamine Metabolism Impair Bone Microenvironment Niche in Multiple Myeloma Patients

Blood ◽

10.1182/blood-2018-99-111337 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4481-4481

Author(s):

Denise Toscani ◽

Martina Chiu ◽

Giuseppe Taurino ◽

Emanuela Vicario ◽

Valentina Marchica ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Bone Disease ◽

Research Funding ◽

Bone Cells ◽

Bone Microenvironment ◽

Advisory Committees ◽

Osteolytic Lesions ◽

Osteolytic Bone Disease ◽

The Relationship

Abstract Multiple myeloma (MM) cells are characterized by tight dependence on the bone marrow (BM) microenvironment that exerts a permissive role on cell growth and survival. In turn, MM cells markedly modify their microenvironment leading, in particular, to the development of osteolytic bone lesions. Recently, we demonstrated that metabolic alterations is a major feature of MM cells showing that BM plasma of MM patients is characterized by lower levels of Glutamine (Gln) and higher levels of Glutamate (Glu) and ammonium when compared with patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). In the majority of MM patients MM cells are Gln-addicted since they strictly depend on extracellular Gln, do not express Glutamine Synthetase (GS), the enzyme that synthetizes Gln from Glu and ammonium, and are endowed with high levels of the Gln transporter ASCT2. Based on this evidence, we have hypothesized that the peculiar Gln metabolism of MM cells may have a significant impact on the relationship with the bone microenvironment and contribute to the development of osteolytic lesions. We firstly characterized a panel of human MM cell lines (HMCLs) for their GS expression and response to decreasing levels of Gln. The majority of HMCLs, which did not express GS, consumed large amounts of extracellular Gln but secreted nearly half of the amino acid as Glu. Two HMCLs, MM1.S and U266, with a sizable GS expression, were less sensitive to Gln deprivation and secreted less Glu in the extracellular space compared with GS-negative HMCLs. Consistently, the activity of the Glu exchanger x-CT (the product of SLC7A11 gene) was lower in GS-positive than in GS-negative cells. The response to Gln starvation was then studied in mesenchymal stromal cell line (MSC), as well as in osteoblastic (HOBIT) and pre-osteocytic cells (HOB-01). HOBIT and HOB-01 were more sensitive to Gln depletion than MSC. Indeed, while MSC showed a low EC50 for Gln (0.064mM), which is 10-times lower than the physiological blood Gln concentration (around 0.6 mM), the EC50 values of HOBIT and HOB-01 cells were 0.250 mM and 0.297mM, respectively. Furthermore, L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, emphasized the effects of Gln deprivation on all the cell lines tested. Indeed, Gln deprivation enhanced the expression of GS, suggesting that both stromal and osteoblastic cells exploit the enzyme to counteract Gln deprivation. On the basis of these data, we assessed the effects of Gln and Glu on osteogenic differentiation by incubating MSC, either immortalized or primary, with an osteogenic medium containing different concentrations of Gln and Glu. After 2 weeks, compared with cells differentiated in high Gln/high Glu conditions, MSC incubated in the presence of decreased Gln and increased Glu showed lower osteogenic ability, as assessed by real time PCR and ALP staining. Lastly, MSC co-cultured for 72 hours with GS-negative, but not with GS-positive HMCLs, showed reduced viability and increased GS expression. Lastly, to put in a translational perspective these in vitro observations, we analyzed the BM plasma levels of Gln and Glu in a cohort of 41 patients with newly diagnosed MM, including 9 smoldering MM (SMM) and 32 active MM patients (20 of them with osteolytic bone disease, 12 of them without bone disease). All 20 osteolytic MM patients had more than three osteolytic lesions. We found that MM patients had lower Gln levels and higher Glu levels than SMM patients. Moreover, when compared with MM patients without bone disease, MM patients with bone disease showed lower levels of Gln and higher levels of Glu. The results of these analyses are being continuously updated increasing the number of samples tested. Overall, these results indicate that MM cells are able to create a low-Gln/high-Glu bone marrow microenvironment that sustains GS expression in bone cells and impairs their differentiation and viability. Thus, the peculiar metabolic milieu in the MM bone microenvironment affects the relationship between neoplastic and bone cells and may contribute to the development of osteolytic bone disease in MM patients. Disclosures Aversa: Astellas: Honoraria; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

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

Blood ◽

10.1182/blood.v120.21.2922.2922 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2922-2922

Author(s):

Ida Bruun Kristensen ◽

Jacob Haaber ◽

Maria B Lyng ◽

Lise Pedersen ◽

Lars Melholt Rasmussen ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cells ◽

Plasma Samples ◽

Expression Levels ◽

Protein Levels ◽

Osteolytic Bone Disease ◽

The Difference ◽

Wnt Inhibitors

Abstract 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<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. Disclosures: No relevant conflicts of interest to declare.

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Deregulation of the bone marrow stromal compartment occurs early in multiple myeloma and precedes osteolytic bone disease

Bone ◽

10.1016/j.bone.2011.03.619 ◽

2011 ◽

Vol 48 ◽

pp. S253-S254

Author(s):

D. Kassen ◽

N. Rabin ◽

D. Lath ◽

P. Croucher ◽

K. Yong

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Disease ◽

Stromal Compartment ◽

Osteolytic Bone Disease

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Cellular Mechanisms of Multiple Myeloma Bone Disease

Clinical and Developmental Immunology ◽

10.1155/2013/289458 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 38

Author(s):

Angela Oranger ◽

Claudia Carbone ◽

Maddalena Izzo ◽

Maria Grano

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Disease ◽

Plasma Cells ◽

Negative Impact ◽

Current Knowledge ◽

Hematologic Malignancy ◽

Cell Activity ◽

Bone Lesions ◽

Pathologic Fractures

Multiple myeloma (MM) is a hematologic malignancy of differentiated plasma cells that accumulates and proliferates in the bone marrow. MM patients often develop bone disease that results in severe bone pain, osteolytic lesions, and pathologic fractures. These skeletal complications have not only a negative impact on quality of life but also a possible effect in overall survival. MM osteolytic bone lesions arise from the altered bone remodeling due to both increased osteoclast activation and decreased osteoblast differentiation. A dysregulated production of numerous cytokines that can contribute to the uncoupling of bone cell activity is well documented in the bone marrow microenvironment of MM patients. These molecules are produced not only by malignant plasma cells, that directly contribute to MM bone disease, but also by bone, immune, and stromal cells interacting with each other in the bone microenvironment. This review focuses on the current knowledge of MM bone disease biology, with particular regard on the role of bone and immune cells in producing cytokines critical for malignant plasma cell proliferation as well as in osteolysis development. Therefore, the understanding of MM pathogenesis could be useful to the discovery of novel agents that will be able to both restore bone remodelling and reduce tumor burden.

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The Clinical Significance of a Novel microRNA Signature in Multiple Myeloma

Blood ◽

10.1182/blood-2019-129218 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5529-5529

Author(s):

Pinelopi I Artemaki ◽

Aristea-Maria Papanota ◽

Paraskevi Karousi ◽

Christine Liacos ◽

Christos K. Kontos ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Bone Disease ◽

Untranslated Region ◽

Research Funding ◽

Plasma Cells ◽

Predictive Biomarker ◽

Lymphocytic Leukemia ◽

Osteolytic Lesions ◽

Expression Levels

Multiple myeloma (MM) is a hematologic malignancy characterized by infiltration of the bone marrow (BM) by malignant plasma cells. Multiple myeloma diagnosis is made by the presence of one or more of the CRAB criteria or one of the recently added biomarkers of malignancy. Smoldering MM (SMM) is a plasma cell dyscrasia preceding multiple myeloma, characterized by bone marrow infiltration of 10-60% and/or serum monoclonal protein ≥3g/dL or urinary monoclonal protein ≥500 mg per 24h, along with the absence of myeloma-defining events. MicroRNAs (miRNA) are single-stranded, small non-coding RNA molecules (~21 nt) that regulate protein-coding gene expression at the post-transcriptional level, mainly through interactions with the 3′-untranslated region of target mRNAs. The results of such interactions can be mRNA degradation and/or translational repression, depending on the complementarity of the miRNA seed sequence with the mRNAs 3′-untranslated region. They can function as oncogenes or tumor suppressors, possessing a vital role in several aspects of all stages of tumorigenesis and cancer progression. In the present study, we have investigated the clinical value of a molecular signature consisting of 10 cancer-related miRNAs in MM: miR-15a, miR-16, miR-21, miR-221, miR-222, miR-25, miR-125, miR-155, miR-223, and miR-181a. These molecules were selected due to their well-documented role and clinical significance in numerous human malignancies. More specifically, miR-15a and miR-16 expression levels have been associated with chronic lymphocytic leukemia. The deletion 13q14, the most prevalent alteration in CLL, leads to the deletion of these miRNAs, which act on cell proliferation and in the process of apoptosis. miR-221 and miR-222 form a cluster that has been correlated with tumorigenesis and unfavorable prognosis in human malignancies, while miR-155 is a pro-inflammatory, oncogenic molecule, with a potential role in chronic lymphocytic leukemia. Bone marrow aspiration samples were collected from 94 patients with MM and SMM, and CD138+ plasma cells were positively selected using magnetic beads coated with an anti-CD138 antibody. Total RNA was isolated using TRIZol. Thereafter, 200ng RNA of each sample were polyadenylated at the 3´ end and reversely transcribed. An in-house developed real-time quantitative PCR assay was conducted and the results were biostatistically analyzed. For the normalization of the expression levels of each miRNA, the mean expression of two small nucleolar RNAs (RNU43 and RNU48) was used as reference. Seventy six out of the 94 BM aspiration samples were derived from MM patients and 18 of them from SMM patients, at the time of diagnosis. The MM patients were classified, according to the R-ISS staging system, as follows: 15 patients had stage I disease, 42 patients had stage II, and 19 patients stage III MM. Forty nine myeloma patients presented with osteolytic lesions at diagnosis. The statistical analysis revealed significantly lower expression levels of miR-16 (p=0.036) and miR-155 (p=0.045) in CD138+ cells of MM patients, compared to those from SMM patients, highlighting their potential value to discriminate MM from SMM. Furthermore, miR-221 and miR-222 expression levels were negatively correlated with R-ISS; thus, miR-221 and miR-222 expression was significantly downregulated in MM patients with R-ISS stage III (p=0.004 and 0.034, respectively). This tendency reveals a potential favorable prognostic value of miR-221/222 cluster in MM. Next, miR-15a and miR-16 expression was shown to be associated with the presence of osteolytic lesions. In this regard, the expression levels of miR-15a (p=0.048) and miR-16 (p=0.047) were decreased in MM patients with bone disease, compared to those without bone disease. The observed decreased expression of these two miRNAs in symptomatic MM patients could constitute a predictive biomarker for the occurrence of bone disease and, hence, a putative predictive biomarker of SMM patients at high risk of evolution to symptomatic disease with bone lesions. We conclude that miR-221/222 correlate with more favorable R-ISS stage, while miR-15a and miR-16 correlate with the presence of osteolytic disease in MM. This ongoing study will further reveal the possible prognostic significance of this 10 miRNAs signature studied, when response to therapy, progression-free and overall survival is available. Disclosures Kastritis: Genesis: Honoraria; Prothena: Honoraria; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria. Gavriatopoulou:Takeda: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Amgen: Honoraria; Genesis: Honoraria, Other: Travel expenses. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Takeda: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Other: Travel expenses, Research Funding; Genesis: Honoraria, Other: Travel expenses, Research Funding.

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