Presence Of Osteolytic Lesions Research Articles

Tuberculous Osteomyelitis of the Fifth Metatarsal Treated Using the Induced Membrane Technique: A Case Report

Tuberculous osteomyelitis of the foot, an uncommon and rare entity, accounts for <0.01% of all tuberculous localizations. Osteomyelitis secondary to tuberculosis is difficult to diagnose and treat, requiring a high suspicion index. We report a case of tuberculous osteomyelitis of the fifth metatarsal bone treated with the induced membrane technique. Here, we report the case of a 40-year-old woman with a history of immunosuppressive treatment presented with a complaint of painful swelling of the left foot. Radiographs showed osteolytic lesions of the fifth metatarsal bone. Initial biopsy suggested tuberculosis. She was surgically treated with the induced membrane technique. Histology confirmed tuberculous osteomyelitis and antituberculous treatment was initiated. Full weight-bearing was allowed after 8 months. At 24 months follow-up, all symptoms were resolved. The radiographs showed an integrated graft. In conclusion, the presence of osteolytic lesions on a plain film should rise high suspicious for a possible infection requiring biopsy, and if confirmed, mandates surgical debridement and reconstruction. The induced membrane technique offers a mean to treat infectious bone defects. Level of Evidence: Level 4.

The Cancer-Related Circrna Cirs-7 (CDR1-AS) Is Differentially Expressed in CD138+ Cells of Patients with Plasma Cell Disorders and Predicts Unfavorable Overall Survival in Multiple Myeloma

Introduction: During the last decades, different non-coding RNAs have been implicated in the biology and prognosis of multiple myeloma (MM). Circular RNAs (circRNAs) constitute a class of non-coding RNAs with a high potential as molecular biomarkers. Recently, ciRS-7 (also known as CDR1-AS), an important circRNA regulating miR-7-5p and other cancer-related miRNAs, was shown to be downregulated in MM patients’ cells exhibiting acquired resistance to immunomodulatory drugs (IMiDs). Our goal was to study the expression of this circRNA in MM, compare the results with those of patients with smoldering myeloma (SMM) and monoclonal gammopathy of undetermined significance (MGUS) and evaluate its prognostic significance. Methods: Bone marrow aspirate (BMA) samples were collected from 177 adult patients with plasma cell disorders. The presence of high-risk cytogenetic abnormalities, namely del(17p), t(4;14), t(14;16), and (+1q), was assessed using fluorescence in situ hybridization (FISH). MM patients were classified according to the ISS and revised ISS (R-ISS) staging systems. Whole-body low-dose computed tomography was used to assess osteolysis. Next, CD138-positive selection of plasma cells among mononuclear cells of BMA samples was performed. Moreover, three human MM cell lines (L-363, H929 and U266) were propagated. Total RNA was extracted from CD138+ enriched BMA samples and the MM cell lines, and reverse-transcribed. A first-round PCR was used to pre-amplify ciRS-7 and GAPDH (i.e. reference gene) transcripts, before their quantification via real-time qPCR. In both reactions for ciRS-7 amplification, divergent PCR primers were used, to achieve high specificity and accuracy. ciRS-7 expression was measured in relative quantification units (RQU). Results: The cohort of patients consisted of 111 newly diagnosed MM, 35 SMM and 31 with MGUS. In total, 107 out of the 177 cases (60.5%) were male and 70 (39.5%) were female. The median age of all patients at diagnosis was 70 years (range: 35-93 years). According to the R-ISS staging system, 24 (21.6%) MM patients had R-ISS stage I, 62 (55.9%) R-ISS stage II and 25 (22.5%) had R-ISS stage III. Seventy-seven (69.4%) MM patients presented with osteolytic disease at diagnosis. Regarding front-line therapy of MM patients, 12/111 (10.8%) received proteasome inhibitor (PI)-based therapy, 7 (6.3%) received IMiD-based treatment, 74 (66.7%) were treated with combinations of PI and IMiD, and 18 (16.2%) received anti-CD38-based treatment. The intracellular levels of the ciRS-7 circRNA were different between MM, SMM and MGUS patients’ CD138+ plasma cells (median values were: for MM patients 0.40 RQU vs. 1.74 RQU for SMM patients vs. 2.79 RQU for MGUS patients; P<0.001). Moreover, ciRS-7 levels in U266 and H929 cells were much lower (2,165- and 26,616-fold) than in L-363 cells. ROC analysis showed that ciRS-7 expression can effectively discriminate between MM and SMM patients (AUC=0.65, 95% CI=0.56-0.75, P=0.007). ciRS-7 levels did not significantly differ among MM patients with different ISS or R-ISS stages and were not associated with any assessed cytogenetic abnormality or the presence of osteolytic lesions. Regarding the progression-free (PFS) and overall survival (OS), out of 106 MM patients with survival data, patients with high levels of ciRS-7 have lower survival probabilities. As shown in Figure 1, the median PFS of MM patients with high ciRS-7 (high: defined as higher of the median value) was 19 months vs. 25 months for patients with low ciRS-7 (lower values than the median) (P=0.046); similarly, the mean OS of ciRS-7 (high) MM patients was 25 months vs. 30.5 months for ciRS-7 (low) MM patients (P=0.008). Bootstrap multivariate Cox regression analysis showed that the unfavorable prognostic value of ciRS-7 expression with regard to OS is independent of R-ISS stage and age of MM patients (HR=2.6, BCa 95% CI=0.91-4.75, bootstrap P=0.041). Conclusions: Expression levels of ciRS-7, a cancer-related circRNA, in CD138+ plasma cells are significantly different among patients with MM, SMM and MGUS. Most likely, high ciRS-7 levels predict an unfavorable prognosis in MM, independently of the R-ISS stage and age. Combining our results with the limited existing literature providing evidence for ciRS-7 implication in response to IMiD therapy, its biological role in MM deserves further elucidation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

255P Challenges to assess bone metastases in Blinded Independent Central Review (BICR) of breast cancer trials using RECIST 1.1

The most common site of metastases in breast cancer patients is bone with reported incidence around 60–80 %. The skeletal metastases are associated with increased morbidity, but studies have noted a better overall survival compared to patients with visceral organ metastases. Imaging plays a key role in the diagnosis and assessment of bone metastases in clinical practice and trials. The commonly used RECIST 1.1 criteria recognize challenges of assessing bone metasteses. Bone lesions are mainly considered unmeasurable and qualitatively assessed, except in the presence of osteolytic lesions with measurable soft tissue component.

Osteolytic bone lesions in patients with primary myelofibrosis: A systematic review.

Philadelphia negative myeloproliferative neoplasms (MPNs) are classically characterized by excess production of terminal myeloid cells in the peripheral blood. They include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Among this group, primary myelofibrosis is the least common and usually carries the worst prognosis. Bone involvement in primary myelofibrosis has many forms; it affects bone marrow leading to bone marrow fibrosis, it can cause periostitis, in addition to bone and joint pain. A common radiologic finding in primary myelofibrosis is the presence of osteosclerotic lesions. However, the presence of osteolytic lesions in bone imaging was described in few reports. In this review, we searched English literature using the PRISMA guidelines looking for patients with Primary myelofibrosis who had osteolytic bone lesions to assess the impact of such findings on the disease and its effect on prognosis. We found the vast majority of lesions were painful affecting most commonly the vertebral column, pelvis, and ribs, and were detected in patients above 50 years of age with no gender preference, unfortunately they represented advanced disease stages, resulting in inadequate treatment response and poor outcome. (www.actabiomedica.it)

Prognostic value of plasmablastic morphology and p21, p53 and Cyclin D1 expression in myeloma cells: retrospective study of 122 patients with newly diagnosed multiple myeloma

Multiple myeloma (MM) is the most common bone marrow malignancy which is defined with bone marrow infiltration of more than 10% of terminally differentiated plasma cells sive myeloma cells. The aim of this study was to find correlations between the expression of some immunohistochemical markers (Cyclin D1, p53, p21), plasmablastic differentiation and prognosis of MM. Immunohistochemistry was used to detect expression of cell cycle proteins. Bone marrow trephine biopsies of 122 MM patients were analyzed to determine the plasmablastic morphology of myeloma cells and myeloma cells expression of immunohistochemical markers (p21, p53, Cyclin D1). The following clinical data of MM patients were obtained: β2-microglobulin, albumin, haemoglobin, platelet count, glomerular filtration rate (GFR), creatinine, calcium level, M-gradient and presence of osteolytic lesions. These data were compared with the bone marrow histological findings. Plasmablastic differentiation correlated with decreased level of haemoglobin, albumin and GFR. Cyclin D1 expression correlated with significantly higher serum calcium level, more common osteolytic lesions in bones. Patients with p21 expression in myeloma cells had lower albumin levels, higher M-gradient levels and more advanced stage by Salmon–Durie. These results suggest that there are correlations between plasmablastic differentiation, expression of p53, CyclinD1 and p21 and poor prognosis in cases of MM.

The Utility of the Energy Subtraction Method for Thoracic Spine Radiography in Clinical Practice: An Analysis of 25 Patients With Multiple Myeloma.

Interpretation of thoracic spine radiographs is difficult because they cannot clearly depict the vertebrae due to overlap with soft tissues. This study aimed to evaluate whether thoracic spine radiographs obtained using the energy subtraction method could improve the accuracy of a diagnosis of thoracic osteolytic lesions. The authors analyzed 300 thoracic vertebrae from 25 patients with multiple myeloma who underwent thoracic spine radiography. All patients underwent thoracic spine radiography with 2 views. Two sets of images were prepared: computed radiography images (CR images) acquired using conventional processing parameters; and processed images for specifically visualizing bone, using the energy subtraction method (ES images). The CR images (CR group) and paired CR and ES images (CR+ES group) were interpreted in parallel by 5 orthopedic surgeons. The presence of osteolytic lesions was evaluated for each of the 12 thoracic vertebrae, and the sensitivity and specificity of the method were compared with computed tomography (CT), which is considered the gold standard. Subgroup analysis was also performed based on location. Osteolytic lesions were found on CT in 28 (9.3%) vertebrae of 12 patients. The overall sensitivities and specificities of the CR and CR+ES groups were 17.2% and 54.3%, respectively, and 95.6% and 98.0%, respectively, with statistically significant differences. Subgroup analysis showed particular improvement in the sensitivity for the CR+ES group in the middle thoracic spine compared with that at other locations. Thoracic spine radiographs generated using this method may improve the accuracy of diagnosis of thoracic osteolytic lesions. [Orthopedics. 2021;44(1):e31-e35.].

The Clinical Significance of a Novel microRNA Signature in Multiple Myeloma

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.

A Minimum of 15-Year Results of Cementless Total Hip Arthroplasty Using a 28-mm Metal-On-Metal Articulation

BackgroundWe previously reported unexpected early failures of total hip arthroplasty (THA) utilizing a 28-mm Ultima metal-on-metal (MoM) articulation. However, long-term results of small-diameter MoM THAs still remain unclear. The purpose of this study was to evaluate the clinical and radiographic outcomes of the previously reported cohort at a minimum follow-up of 15 years. MethodsThe original cohort consisted of 171 primary THAs (167 patients) using a 28-mm MoM articulation performed between April 2000 and March 2002. Of these, 130 hips (126 patients) were reviewed at an average follow-up of 17.1 (range, 15-18) years. Clinical score, complications, presence of osteolytic lesion, serum metal ion concentrations, and implant survivorships were evaluated. ResultsThe mean Harris Hip Score improved from 44.9 points preoperatively to 90.5 points at the latest follow-up. During the entire period since the original surgery, a total of 5 revisions (3.8%) were associated with adverse reaction to metal debris (ARMD). The last revision surgery for symptomatic ARMD was performed at 6 years postoperatively. The implant survivorships with an end point of revision for aseptic failure and for any reason were 95.4% and 93.8%, respectively. Radiographic osteolytic lesions were detected in 27 hips (20.8%). The average serum metal concentration was 2.50 (range, 0.12-9.86) μg/L for cobalt and 2.81 (range, 0.82-12.3) μg/L for chromium. ConclusionsTHA using a 28-mm MoM articulation showed favorable long-term outcomes with a relatively high revision-free survival rate. There was no significant symptomatic ARMD after the last acetabular revision performed at 6 years postoperatively.

Strong Immunoexpression of Dickkopf-1 Is Associated with Response to Bortezomib in Multiple Myeloma

Background: Although continued improvement in survival in multiple myeloma was observed over the past decade with the introduction of new therapeutic options such as the proteasome inhibitors and immunomodulatory drugs, the prognosis of myeloma patients (pts) with relapsed and refractory and primary refractory to those drugs is significantly worse than that of responders. The predictive significance of various clinicopathologic characteristics and expression status of osteolysis-related proteins in terms of therapeutic response to bortezomib were evaluated in multiple myeloma pts.Methods: In 58 pts treated with bortezomib, the clinicopathologic characteristics was retrospectively collected. Pretreatment biopsy samples were analyzed for receptor activator of NF-kB ligand (RANKL), osteoprotegerin (OPG), macrophage inflammatory protein 1 alpha (MIP-1a), and dickkopf-1 (DKK1) expression by immunohistochemistry (Fig 1.).Results: Bortezomib administrated in the fisrt (11 pts), second (29 pts), third (15 pts), fourth (2 pts) and fifth (1 pt) line of chemotherapy. Overall response rate (RR) of bortezomib treatment was 66%. Eight-year overall survival (OS) rate of all pts were 45.3%. Eight-year OS rate of bortezomib non-responders was significantly inferior compared to that of bortezomib responders (24% vs. 62%, p=0.011). The clinicopathologic characteristics including gender, stage, cytogenetic risk stratification by chromosomal aberration and cellularity of bone marrow plasma cell except age and presence of osteolytic lesion were not correlated with response to bortezomib. Median age of bortezomib responders was higher than that of non-responders (65 vs 60, p=0.034). The presence of osteolytic lesion was associated with higher RR of bortezomib treatment (79% vs 46%, p=0.012). High expression of DKK1 was significantly correlated with presence of osteolytic lesion (p=0.003), while expression status of RANKL, OPG and MIP-1a were not associated with presence of osteolytic lesion. In addition, high expression of DKK1 was associated with female gender (p=0.030, Table 1.), increased cellularity of bone marrow plasma cell (p=0.023, Table 1.) and plasmablatic morphology (p=0.007, Table 1.). In multiple myeloma with high DKK1 expression, higher RR was observed (84% vs 44%, p=0.002, Table 1.). In multivariate analysis, high DKK1 expression were independently associated better response to bortezomib (p=0.005). Pts with high DKK1 expression had significantly longer median PFS (23 months versus 6 months, p=0.022, Fig 2A.) and OS (Not reached versus 26 months, p=0.028, Fig 2B.) after bortezomib treatment compared to those with low DKK1 expression. Multivariate analysis revealed that high DKK1 expression was an independent prognostic factor of favorable PFS (p=0.043) and OS (p=0.045).Conclusion : Despite limitation as retrospective analysis with small sample size, our data indicate that expression status of DKK1 may be a useful predictive and prognostic factor in multiple myeloma pts treated with bortezomib. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Clinical Utility of Bone Resorption and Bone Formation Markers in Multiple Myeloma

Introduction: While bone resorption markers such as urinary N-terminal telopeptide (NTx) have long been used in research, serum C-terminal telopeptide (CTx) has become the preferred marker of bone status in multiple myeloma (MM). A full understanding of the clinical utility of CTx, as well as the bone formation markers osteocalcin (OC) and bone-specific alkaline phosphatase (BSAP), in MM is still lacking. In this study, we evaluated levels of CTx, OC, and BSAP in a cohort of MM patients to better understand their roles.Methods: One set of CTx, OC, and BSAP data was obtained by random sampling in patients during a 60-day window at the Weill Cornell Medical College Myeloma Center. Bone marker changes were assessed with respect to type of myeloma therapy, response status, use of anti-resorptive therapy, and presence of osteolytic lesions or fractures. Additionally, we tested the ability of the bone markers to isolate a high fracture risk group, as defined by radiologic evidence of impending fracture. The Wilcoxon-Mann-Whitney test was used to assess clinical variables, and a 2-sided t value of less than 0.05 indicated statistical significance.Results: 50 patients were identified with bone marker data, of which 47 had MM and 3 had MM precursor diseases. The mean age was 64 years, with 25 males and 25 females. All three markers CTx (p=0.001), OC (p=0.004), and BSAP (p<0.001) significantly decreased with myeloma therapy initiation. Only CTx, however, was significantly lower in patients who achieved a partial response or greater, compared to non-responders (p=0.010). Moreover, only CTx decreased significantly in patients using anti-resorptive therapy within one year of bone marker date (p=0.006). Both CTx (p=0.011) and BSAP (p=0.005) were significantly higher in patients with more than 5 osteolytic lesions compared to those without such lesions. Additionally, only CTx predicted which patients were at risk for impending fracture (p<0.001). Of 8 patients in this high-risk group for fractures, 2 developed new pathological fractures within 1 month of the CTx test. CTx values were significantly higher in patients who were taking both proteasome inhibitor (PI) and alkylating agents compared to those only taking PIs (p<0.001). Choice of PI also affected levels of bone markers, with patients receiving bortezomib having significantly higher levels of CTx (p=0.019) than those on carfilzomib. BSAP levels were similarly higher, with a trend toward significance (p=0.064).Discussion: Of the evaluated bone markers, only CTx correlated with response to anti-MM therapy and risk of impending fracture. Additionally, CTx reflects the extent of bone disease. CTx levels decreased with use of anti-resorptive therapy within one year of bone marker date, but not with longer time points. This suggests the clinical benefit of at least annual dosing of anti-resorptive therapy and indicates the dynamic nature of this marker. The high CTx values in the PI plus alkylating group may also suggest that PI alone is more effective in addressing bone disease in such patients. Differences in CTx between individual proteasome inhibitors points to agent-specific effects of PI on bone remodeling, which warrant further investigation. The potential increase in BSAP with bortezomib versus carfilzomib treatment is consistent with recent findings, which show that bortezomib promotes osteoblastic differentiation and bone formation. As opposed to our CTx data, OC and BSAP did not prognosticate disease response or future fractures or correlate with use of anti-resorptive therapy, suggesting limited utility of these bone formation markers in MM.Table 1Bone MarkerCategoryMM Therapy InitiationMM Therapy RespondersART Use Within 1 YearART Use Before 1 YearLytic Lesions Present5+ lytic Lesions PresentRisk of Future FracturePI Plus AlkylatorCTxBone Resorption↓↓↓↓↓NS↑↑↑↑↑↑↑↑OCBone Formation↓↓NSNSNSNSNSNSNSBSAPBone Formation↓↓NSNSNS↑↑↑NS↑Abbreviations: CTx, C-terminal telopeptide; OC, osteocalcin; BSAP, bone-specific alkaline phosphatase; MM, multiple myeloma; ART, anti-resorptive therapy; NS, not significant; PI, proteasome inhibitor. Single arrow represents a change by a factor of less than 2. Double arrows represent a change by a factor of greater than 2. Triple arrows represent a change by a factor of greater than 3. DisclosuresPearse:Celegen: Consultancy. Rossi:Calgene: Speakers Bureau. Mark:Calgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Niesvizky:Celgene: Consultancy, Speakers Bureau. Lane:Agnovos Healthcare, LLC: Consultancy; Bone Therapeutics: Membership on an entity's Board of Directors or advisory committees; CollPlant Holdings, Ltd.: Consultancy; D'Fine, Inc.: Consultancy; Gradtys: Membership on an entity's Board of Directors or advisory committees; ISTO Technologies, Inc: Membership on an entity's Board of Directors or advisory committees; Kuros: Membership on an entity's Board of Directors or advisory committees; Royal Consulting & Marketing: Consultancy.

Relationship Between Presence of Osteolytic Lesions, Cytogenetic Features and Bone Marrow Levels of Cytokines and Chemokines in Multiple Myeloma Patients: Role of Chemokine (C-C motif) Ligand 20 as new marker of bone disease

Relationship Between Presence of Osteolytic Lesions, Cytogenetic Features and Bone Marrow Levels of Cytokines and Chemokines in Multiple Myeloma Patients: Role of Chemokine (C-C motif) Ligand 20 as new marker of bone disease

LSD1 Impairs the Epithelial-Mesenchymal Transition (EMT) and Osteoclastogenesis Potency in Multiple Myeloma and Synergistically Induces Cytotoxicity with HDAC Inhibitors

Lysine-specific demethylase 1 (LSD1) is a FAD-dependent histone demethylase, which selectively removes mono- and di-methyl groups from histone 3 lysine 4 or 9 residues (H3K4, H3K9) leading to either repression or activation of transcriptome. Previous studies have shown that lenalidomide and pomalidomide cause cell cycle arrest in Multiple Myeloma (MM) by modifying the chromatin structure of the p21WAF-1 promoter through LSD1 demethylation. LSD1 forms a co-repression complex with HDAC1 and HDAC2, mSin3a, and MMSET. However, the functional role of LSD1 in MM and its contribution in aggressive traits of the disease is largely unknown.First, we evaluated the expression of LSD1 in different datasets of MM patients (GSE2113, GSE16122) and observed significant overexpression in patients with symptomatic MM and Plasma Cell Leukemia (PCL) (p<.001). The expression of LSD1 in a panel 45 HMCLs was also pronounced. We confirmed the expression and both its nuclear and cytoplasmic localization by immunoblotting analysis in 4 different HMCLs and primary bone marrow plasma cells from newly diagnosed, relapsed MM and PCL patients (N=8).We further evaluated the LSD1-mediated effect on proliferation and survival by performing loss- and gain of function studies. LSD1 knockdown in LP1 and MM1S cells resulted in modest cytotoxicity. After a combination silencing of JARID1 members and LSD1 we were able to observe a further significant decrease in survival of MM cells lacking JARID1C and LSD1, indicating that the overlapping demethylation of H3K4 is of high importance for the cell survival.We examined the post-translational histone modifications by immunobloting after LSD1 knockdown and as expected, we observed significant increase of K4me2/3 and K9me2 marks, but more interestingly, alteration of acetylation status of K9. Therefore, we performed cytotoxicity and proliferation experiments in MM after knockdown of LSD1 in combination with HDAC inhibitors (SAHA, LBH589) and we observed that LSD1 depletion enhances the cytotoxicity effect of HDACs inhibitors. LSD1 depletion resulted in significant reduction of mRNA levels by using real-time PCR and protein expression by immunoblotting of HDAC1 and HDAC2.Furthermore, based on findings of higher expression of LSD1 in more aggressive types of MM, we sought to investigate the impact of LSD1 in epithelial-mesenchymal transition (EMT). LSD1 depletion in MM1S and LP1 cells inhibited significantly the migratory ability estimated by transwell migration assay, invasion and wound healing assays. More importantly, MM cells lacking LSD1 expressed significant lower levels of E-cadherin, N-cadherin and Vimentin evaluated by immunoblotting and immunocytochemistry. We confirmed the suppression of EMT-involved gene expression by performing a PCR-microarray assay.Finally, given the presence of osteolytic lesions as a hallmark of disease, and consequent impact on outcome, we evaluated the impact of LSD1 on osteoblast differentiation and osteoclastogenesis. LSD1 depletion/ and pharmacological inhibition (S2101) resulted in significant inhibition of osteoclastogenesis and RANKL-induced resorption evaluated by double TRAP/ALP staining, survival of OCs, and mRNA expression level of osteoblast markers (APL, BSP, OC). In contrast, LSD1 overexpression confirmed the upregulation of Wnt/b-catenin pathway suggesting a possible underlying mechanism for the osteoclastogenesis potency in MM patients with high expression of LSD1.Taken together, our findings demonstrate a promising epigenetic approach in myeloma therapeutics by targeting the deregulated LSD1-methylome in MM patients earlier than aggressive disease phase. DisclosuresNo relevant conflicts of interest to declare.

Role of osteocytes in multiple myeloma bone disease.

Despite the increased knowledge of osteocyte biology, the contribution of this most abundant bone cell to the development and progression of multiple myeloma in bone is practically unexplored. Multiple myeloma bone disease is characterized by exacerbated bone resorption and the presence of osteolytic lesions that do not heal because of a concomitant reduction in bone formation. Osteocytes produce molecules that regulate both bone formation and resorption. Recent findings suggest that the life span of osteocytes is compromised in multiple myeloma patients with bone lesions. In addition, multiple myeloma cells affect the transcriptional profile of osteocytes by upregulating the production of pro-osteoclastogenic cytokines, stimulating osteoclast formation and activity. Further, patients with active multiple myeloma have elevated circulating levels of sclerostin, a potent inhibitor of bone formation which is specifically expressed by osteocytes in bone. Understanding the contribution of osteocytes to the mechanisms underlying the skeletal consequences of multiple myeloma bone disease has the potential to provide important new therapeutic strategies that specifically target multiple myeloma-osteocyte interactions.

Radiographic Analysis of Bone Remodeling around Hydroxyapatite Coated Femoral Stems

The study presented in the current paper was designed to investigate periprosthetic bone remodeling associated with two cementless HA coated stem models, ABG-II (Stryker) and Corail (DePuy), randomly implanted in 40 active patients at Colentina Clinical Hospital,Romania. Anteroposterior with neutral rotation and Lequesne profile radiographs of both hips were taken and several data were recorded: acetabular angle, rotation center of the hip, rotation center height with respect to the greater trochanter tip, femoral cortical thickness, cortico-medullary index at three areas (lesser trochanter (LT), 8 and 12 mm distal to LT), and osteoporosis severity according to the Singh index. The presence of osteolytic lesions in the proximal femur was assessed according to the scale proposed by Goetz in the radiographs obtained at the end of the follow-up (1 to 5 years). The radiological results showed that all stems were stable, and no evolutive radiolucent lines have been observed. We also have noticed minimal remodelling changes in 7 hips (36.61%), with minor cancellous bone densification mainly in zones 2 and 6, without cortical hypertrophy or osteoporosis. A decreased bone density in zones 1 and 7, secondary to stress-shielding, has been evident in 5 hips. Stem subsidence has been similar to that reported in literature with other hydroxyapatite coated designs.

Osseous sarcoid with lytic lesions in skull

Sarcoidosis bone is uncommon, and involvement of the skull is exceptionally rare. We present a 65-year-old obese female who presented with a 2-month history of dryness of mouth, polyuria, fatigue, and anorexia. She had generalized lymphadenopathy, organomegaly, and hypercalcemia, and a skeletal survey revealed extensive osteolytic lesions in the skull and phalanges. Both lymph node biopsy from the cervical lymph node and bone marrow examination revealed non-caseating granulomas, suggesting sarcoidosis. She was started on 1 mg/kg oral corticosteroids; during a follow-up of 6 months, she achieved normocalcemia; however, the punched-out lesions in the skull remained unchanged. This case reiterates several important issues that all lymphadenopathy in emerging nations may not be tubercular, and presence of osteolytic lesions in skull are unusual for sarcoid, at an elderly age, necessitates evaluation for more common etiologies like metastases and myeloma. Finally, patients with osseous sarcoid should be on a close follow-up since due to the rarity of this presentation, no definite consensus on the management of such cases exists in the literature.

Abstract 2664: Alpharadin inhibits osteoclast differentiation in vitro and progression of established breast cancer bone metastases in vivo

Abstract Alpharadin is an alpha-emitting radium isotope currently in phase III clinical study for the treatment of castration resistant prostate cancer bone metastases. As a calcium mimetic Alphapharmaceutical, radium localizes to bone, where the emitted alpha-particles provide an efficient and localized radiation treatment to the metastatic skeletal tumor lesion. While promising clinical data in predominately osteoblastic bone metastases such as prostate cancer have already been obtained (Nilsson, Lancet Oncol 2007), robust clinical evidence for the activity of Alpharadin in osteolytic bone metastases associated with other cancer entities is lacking. As previously reported (Henriksen, Cancer Res 2002), Alpharadin increases symptom-free survival in an osteolytic breast cancer bone metastasis model in nude rats. Besides this study on the therapeutic efficacy in a preclinical skeletal metastasis tumor model it is still unclear how Alpharadin affects the vicious cycle of tumor growth and osteolysis. We report here the effects of Alpharadin on differentiation and activity of human osteoclasts in vitro and on treatment of established breast cancer bone metastases in vivo. The in vitro effects of Alpharadin (50-1600 Bq/ml, n=8/group) on osteoclasts were studied using human osteoclast precursor cells cultured on bovine bone slices. Alpharadin was added in the osteoclast differentiation assay at day 0, and in the activity assay at day 7. Secreted tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) was measured at day 7 as a marker of osteoclast differentiation and secreted C-terminal cross-linked telopeptides of type I collagen (CTX) at day 10 as a marker of resorption during days 7-10. The in vivo effects were studied using a model where human MDA-MB-231SA/GFP cells were inoculated intracardially in nude mice. The animals were randomized to two groups (n=15) based on body weight and presence of osteolytic lesions at day 14. Either vehicle or Alpharadin (300 kBq/kg) were administered iv at day 15. Radiography and fluorescence imaging were performed at day 14 and at sacrifice (day 25). Alpharadin inhibited dose dependently differentiation of osteoclasts in vitro at all tested concentrations, but had no effect on resorption activity of mature osteoclasts. In vivo, Alpharadin decreased whole body tumor burden by 43% as quantified by fluorescence imaging and osteolysis by 56% compared to the vehicle control group. Furthermore, Alpharadin treatment completely prevented tumor associated cachexia observed in the control group. In summary, Alpharadin inhibits differentiation of osteoclasts and progression of established, osteolytic breast cancer bone metastases. These preclinical findings strongly support the clinical development of Alpharadin in additional cancer indications which form predominantely osteolytic bone metastasis, e.g. breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2664. doi:10.1158/1538-7445.AM2011-2664

Abstract 5394: Sequential treatment with zoledronic acid and doxorubicin has no additive effects in an established mouse model of bone metastasis

Abstract Bone metastases still present an unmet need for effective treatment. One preclinical study has suggested a beneficial effect with sequential treatment of doxorubicin (DOX) and zoledronic acid (ZOL). Furthermore, bisphosphonates are used as adjuvant therapy, but controversy of their anti-tumor effects remains. We tested clinically relevant doses of DOX, ZOL and their sequential combination in a treatment setting in a widely used nude mouse model of breast cancer bone metastasis. GFP-transfected MDA-MB-231(SA) human breast cancer cells were inoculated into five-week-old female nude mice via left cardiac ventricle. Subsequent tumor growth and development of osteolytic bone lesions were quantitated by fluorescence imaging and radiography, respectively, on days 14 and 25. The mice were randomized to four groups (n=8/group) on day 14 according to body weight and the presence of osteolytic lesions. Starting on day 15, groups received either weekly dose of vehicle (control group), DOX 2.5 mg/kg i.p. weekly, ZOL 0.1 mg/kg s.c. on day 15 and vehicle weekly, or weekly dose of DOX and a single injection of ZOL 24 hrs after the first dose of DOX (combination group). Bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b) was determined in serum samples collected on days 1, 9, 17 and 24. Bone samples were collected on day 25, and tumor area, trabecular bone area and apoptotic index (TUNEL and apoptotic morphology) were quantitated by histomorphometry. Statistical analysis was performed using One-way ANOVA followed by Dunnett's test or Kruskal-Wallis test followed by Mann-Whitney U-test. All animals in the control and DOX groups had lesions at sacrifice, whereas 12.5% of the mice in the ZOL group and 42.9% in the combination group had lesions. Osteolytic lesion area at sacrifice was significantly lower in both groups treated with ZOL compared to the control group, with no increase from day 14. Lesion area in the DOX-treated group was not different from control. Whole body tumor burden or the tumor area in hind limbs did not differ between the groups, indicating no inhibition on tumor growth. Apoptotic index was increased in groups treated with DOX. ZOL inhibited the increase in serum TRACP 5b, although less effectively in the combination group. Taken together, clinically relevant doses of DOX, ZOL or a sequential combination treatment of these two were not able to inhibit tumor growth in the established disease in this aggressive model of breast cancer bone metastasis. However, DOX increased the apoptosis of cancer cells and ZOL potently inhibited cancer-induced osteolysis, but the sequential treatment did not have additive affects on either tumor growth or osteolytic lesion area. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5394.

Hypercalcemia Following Treatment with Lenalidomide in Chronic Lymphocytic Leukemia (CLL).

Abstract 4413 BACKGROUNDIn concert with emerging effectiveness data, we are learning that the side effect profile of lenalidomide (L) in CLL may be different from that observed with other malignancies, such as multiple myeloma (MM) or myelodysplastic syndrome (MDS). In particular, at lower doses than typically used for MM or MDS, a unique tumor flare response (TFR) has been observed for which the mechanism is still unclear, but may be related to a “cytokine release” phenomenon. CASE REPORTHere we would like to describe 3 cases of hypercalcemia that occurred in 3 of 17 patients with fludarabine refractory CLL treated at the Moffitt Cancer Center: 2 patients who were on a clinical trial of lenalidomide plus rituximab (LR), and 1 patient who was treated with L outside of a clinical trial setting. Case 1Pt #1 was initiated on (LR) on 12/2007 for CLL with bulky LAN, elevated wbc (110K), and 90% bone marrow (BM) involvement. By day 6, his calcium peaked at 11.78 and was accompanied by a drop in wbc to 15K. He had no other evidence of TFR, and was specifically without complaints of back pain or tender/progressive LAN. He was treated with IV fluids and steroids which promptly corrected his calcium, after which L was resumed. His course has been complicated by neutropenia requiring G-CSF. Although LAN remains stable, he has had an excellent BM response, with only 40% CLL seen on reevaluation in 06/2009. Case 2Pt #2 was initiated on LR on 8/2008 for CLL with bulky LAN, mildly elevated wbc (20.5K) and 60% BM involvement. By day 4 his calcium peaked at 16.94, at which time he presented to our urgent care center with weakness, sweats, back pain, nausea and vomiting. No tender or progressive LAN was appreciated. He was treated with IV fluids, pamidronate, calcitonin and steroids which normalized his calcium. An attempt was made to restart L, which again produced hypercalcemia after 4 days. No further attempts were made to resume this medication. A workup performed during the first episode of hypercalcemia was notable for a slight elevation in the PTHrp (8.5). Case 3Pt #3 was initiated on L alone on 6/2008 for CLL with bulky LAN, elevated wbc (283.6K), and 95% BM involvement. By day 4, his calcium peaked to 14.64, and was associated with a TFR, including a rise in wbc (306K), LDH (1285), and enlarging/painful LAN. The hypercalcemia was treated with IV fluids, zoledronate, and steroids. Bendamustine was immediately initiated for the treatment of CLL with dramatic improvement on lymph nodes and WBC counts. Following 1 cycle of bendamustine, L was resumed, with stabilization of his disease.Workup in these patients was unremarkable, with normal VitD/PTH levels, and only a mild elevation of PTHrp in Pt#2 (see table), in whom hypercalcemia had reoccurred upon rechallenge with L. Pt#1 and #3 were successfully retreated without recurrent hypercalcemia, though, Pt#3 received prior cytoreductive therapy with bendamustine. CONCLUSIONHypercalcemia is a rare complication of advanced B-cell malignancies seen with lymphokine/cytokine mediated bone disruption. Increased production of l,25-(OH) 2D by lymphocytes may also lead to hypercalcemia, and in CLL it is mainly associated with Ritcher's transformation or presence of osteolytic lesions. Here we report on 3 cases of hypercalcemia, which were identified during L treatment in CLL, and which had not been observed in other studies of L. We hypothesize that the hypercalcemia could be a direct reflection of increased cytokine levels, including IL-6, TNFα, and, MIP-1α, which are known to influence osteoclastogenesis. Interestingly, IL-6 is known to act synergistically with PTHrp in experimental models to drive hypercalcemia, which may be relevant for the patient who developed recurrent hypercalcemia on rechallenge with L. Levels of cytokines at different time points are now under investigation and are anticipated for presentation at the meeting. These events indicate that serum chemistries of patients initiating treatment with lenalidomide should be closely monitored during the first weeks of treatment.Table 1:Pt_DoseCalcium level before treatmentMaximum CalciumPhosPTH/PTH-rpVit DFISHWBC ChangeLDH Change12.5mg9.411.7857 / 018del 11q□ 94.83□ 12122.5mg9.916.9446 / 8.551del 13q□ 7.39□ 17310mg9.314.644.66 / --del 11q; del 13q14□ 23.14□ 753 Disclosures:Off Label Use: Lenalidomide is an immune modulator drug currently being studied for efficacy in conjunction with Rituxan in chronic lymphocytic leukemia. This abstract summarizes a unique side effect, hypercalcemia, seen among a small cohort of patients on this study.. Lancet:Celgene: Research Funding. Pinilla:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

Dickkopf-1: a suitable target for the management of myeloma bone disease

Bone disease remains a major problem in the management of patients with multiple myeloma (MM) and is characterized by the presence of lytic lesions due to increased osteoclastic activity and reduced osteoblast function. Wingless-type and integrase 1 (Wnt)/β-catenin signaling is a central pathway for bone development and homeostasis. Dickkopf-1 (Dkk-1) is a soluble inhibitor of Wnt, which disrupts osteoblast differentiation and action. Dkk-1 is produced by myeloma cells and overexpressed in myeloma microenvironment of patients with extensive bone disease. In addition to its direct inhibitory effect of Dkk-1 on osteoblasts, Dkk-1 disrupts the Wnt3a-regulated osteoprotegerin and receptor activator of NF-κB ligand (RANKL) expression in osteoblasts and thus it indirectly enhances osteoclast function in MM. Dkk-1 serum and bone marrow plasma levels are increased in MM patients and correlated with advanced International Staging System stage and presence of osteolytic lesions. Preclinical studies in mouse myeloma models showed that targeting Dkk-1 with neutralizing anti-Dkk-1 antibodies resulted in increased numbers of osteoblasts, reduced numbers of multinucleated osteoclasts and increased bone volume. The bone anabolic effect of anti-Dkk-1 may also be associated with reduced myeloma burden. These data show that Dkk-1 has a pivotal role in bone health and disease and is a novel target for the management of myeloma patients with lytic bone disease.

Platelet is a major contributor to circulating levels of Dickkopf‐1: clinical implications in patients with multiple myeloma

Platelet is a major contributor to circulating levels of Dickkopf‐1: clinical implications in patients with multiple myeloma