Early Stage Multiple Myeloma Research Articles

Immune microenvironment and immunotherapy resistance in multiple myeloma

Multiple myeloma (MM) is currently treated with combined immunotherapies targeting the immune microenvironment surrounding MM cells. Although novel immunotherapeutic agents for MM have improved complete response rates and survival time, some patients experience repeated relapses and die of MM because its progression is strongly influenced by the immune microenvironment as well as cytogenetic abnormalities of MM cells. Various studies have elucidated the mechanisms underlying the immunosuppressive immune microenvironment of MM. Recently, single-cell RNA sequencing and cytometry by time-of-flight analysis have revealed spatiotemporal crosstalk between MM and immune cells in the bone marrow of patients with MM. These studies showed that immunosuppression appears in the early stages of MM and becomes stronger as the disease progresses, leading to immunotherapy resistance. Understanding the state of the immune microenvironment in patients with MM is important for developing immunotherapeutic strategies for long-term complete response and cure.

Single Cell Analysis of Bone Marrow Stromal Cells Shows That a Pro-Inflammatory and Stress Response Together with Polarized Differentiation of Stromal Elements Characterize the Bone Marrow Microenvironment during Multiple Myeloma Development

Current knowledge of the biological role of the non-hemopoietic components of the bone marrow (BM) niche to the pathogenesis of multiple myeloma (MM) is poorly understood. We utilized a murine model to interrogate niche forming cells early in the natural history of MM, at a precursor stage (MGUS/smoldering-like) in more detail. Studying interactions between the MM clone and the stromal components of the MM niche are challenging as they represent only approximately 0.2% of the total marrow content and are difficult to isolate. However, by isolating the stromal compartment of the BM by depleting hemopoietic cells via flow sorting combined with the use of single cell RNA sequencing (scRNA-seq) it is possible to perform an in-vivo analysis of the individual cellular components of the stromal microenvironment and to characterize their relative abundance, cellular differentiation state, and transcriptomic profile. To comprehensively assess changes in the stromal cells during the early stage of MM, 5TGM1 cells were intravenously injected into aged (6 month old) KaLwRij mice. KaLwRij mice without MM were used as controls. Stromal cells were isolated following tumor engraftment in the marrow at a precursor stage and enriched as previously described (Baryawno et al, Cell 2019). scRNA-seq was performed on 5 libraries of cells using the 10X Genomics Chromium platform. All data analysis was performed in R. After filtering out low quality cells, 45,030 cells remained for analysis. The 5 libraries were integrated with Seurat and run through a standard clustering workflow involving log-normalization, finding variable features, dimensional reduction, and clustering. Cell type annotation for each cluster was performed manually using established markers for both the stromal and immune compartments. After excluding immune cell populations, 14,219 cells remained. Select subpopulations were re-clustered by isolating the cell types of interest and running them through separate standard clustering workflows. For each population of interest, differentially expressed genes were determined using the Wilcox rank sum test in Seurat, trajectory analysis and pseudotime were computed using Monocle3, and enriched gene sets were computed using GSEAPreranked. We identified 7 distinct populations of stromal cells including mesenchymal stromal cells (MSCs) ( Lepr, Adipoq, and Cxcl12), osteo-lineage cells (OLCs) ( Bglap, Spp1, and Sp7), fibroblasts ( S100a4, Fn1, and Dcn), chondrocytes ( Col2a1, Sox9, and Acan), pericytes ( Acta2, Myh11, and Mcam), and two endothelial cell (ECs) populations, arterial (AEC) and sinusoidal (SEC) ( Cdh5, Cd34, and Pecam1). Compared to normal BM, MM engraftment was associated with numerical differences in stromal cell populations between normal and MM. Gene set enrichment analysis showed an inflammatory and oxidative stress signal associated with the MM microenvironment. Sub-clustering analysis showed MSC differentiation was polarized away from osteocyte formation towards adipocytes with the identification of a novel population only seen in MM. Bone marrow endothelial cell populations were also substantially altered at this early disease stage, with differentiation polarized towards sinusoidal endothelial cells generating a pro-angiogenic /pro-inflammatory phenotype. An increase in cells undergoing endothelial to mesenchymal transition (EndMT) was also present. Taken together, we show, for the first time, existence of remodeling of the stromal populations induced by the MM clone characterized by a pro-inflammatory phenotype together with polarized differentiation. These changes result in the expansion of a number of key populations that increase the MM niche contributing to growth and survival signals and shaping of the content of the immune microenvironment. These changes result in a self-perpetuating signaling loop between cells, which needs to be broken therapeutically in order to stop progression, induce remission and long-term disease outcomes. Conclusion - In early stages of MM pathogenesis, MM cells remodel the stromal microenvironment by altering the amount and function of MSCs and endothelial cells. Through favoring an adipocytic fate of MSCs, endothelial mesenchymal transition and altering the balance between arterial and sinusoidal endothelial cells, MM cells promote an inflammatory environment that contributes to MM development and progression.

Whole Exome Sequencing of Paired MGUS/SMM to MM Patients Reveals Novel Subclonal Tumour Evolution Models in Disease Progression of Multiple Myeloma

Introduction: Next Generation Sequencing studies in Multiple Myeloma (MM) have demonstrated that genetic heterogeneity is characteristic of MM at presentation. However, while intraclonal heterogeneity is now an established feature of MM, the subclonal tumour evolution associated with disease progression is not well understood. Here we present the first whole exome sequencing (WES) analysis of 10 paired MGUS-MM or SMM-MM patient samples, providing new insights into the genomic complexity, key molecular mechanisms and subclonal tumour evolution underlying the progression from MGUS/SMM to symptomatic MM.Methods: Fluorescence-activated cell sorting was used to purify CD138+CD38++ plasma cells and matched CD138-38- normal cells from longitudinal MGUS-MM (n = 5) and SMM-MM (n = 5) patient samples, where bone marrow samples were isolated from patients when they were first diagnosed with MGUS/SMM, and then subsequently when they developed MM. Exome libraries were generated using the Nimblegen Hyper Library Prep kit followed by the Agilent Sure SelectXT Clinical Research Exome capture kit before WES using the Illumina HiSeq4000. Bioinformatics analysis was performed using the GATK best practices MuTect2 pipeline to identify the somatic variants, custom in house package to identify copy number changes and PhyloWGS was used to integrate SNVs/CNVs to infer the subclonal evolution associated with MM progression.Results: WES was performed to a minimum depth of 140x and identified a total 4997 somatic non-synonymous single nucleotide variants (NS-SNVs) in the MGUS/SMM samples (range 230-796), with a median 456 per patient. Interestingly, in the MM samples, we identify a total 4127 somatic NS-SNVs (range 221-609), with a median 344 per patient. We observe widespread copy number variations (CNVs), with a total of 82 genes gained or lost with progression across all patients. While we observe some previously identified known “drivers” of MM, we find that the driving events involved in progression are complex and not limited to the known SNVs or CNVs. The RAS/MAPK pathway was found to be the most frequently deregulated pathway, with KRAS and NRAS mutations observed in 40% of patients at MGUS/SMM and 70% of patients at MM. These findings highlight that “driver” mutations can be attained at both the early stages of MM and be maintained with transformation, or attained only at the later symptomatic MM stage.Subclonal reconstruction of the tumour evolution process was carried out for 8 paired patients, to identify both the patterns of evolution and the key genetic changes that occur with MM progression. Our analysis revealed two models of subclonal evolution; firstly, a dominant model (3/8 patients) in which the outgrowth of subclones from MGUS/SMM to MM was observed, and secondly, a maintenance model (5/8 patients) in which subclones that were present at MGUS/SMM are retained at the MM stage. Notably, we observed a decrease in the average number of clones present in MGUS vs. SMM patients, indicative of a reduction in clonal complexity through dominant clonal outgrowth and/or extinction of indolent clones with advancement of disease. The survival of subclonal branches to MM was determined by their clonal fitness, either through their emergence with the acquisition of candidate “driver” gene mutations or outcompeting other subclones that were present. Finally, we describe potential candidate driving events of clonal progression in a range of loci, including ICAM5, DUSP27, HERPUD1, NOD2 and TOP2A.Conclusion: Our genomic analysis of longitudinal MGUS/SMM to MM samples has revealed new insights of the subclonal tumour evolution and identified candidate mutated genes associated with MM transformation. Our analysis has revealed two models of tumour evolution involved in MGUS/SMM to MM transformation; namely the dominant subclonal tumour evolution model, and the maintenance subclonal tumour evolution model. We identified the existence of multiple subclones at the MGUS/SMM stages that are associated with MM progression. Our study suggests that in both circumstances, the subclonal populations involved in MM transformation are already present at the stages of MGUS/SMM diagnosis. Defining potential candidate genes associated with MM disease progression will assist in treatment approaches to arrest MM at the asymptomatic stages. DisclosuresNo relevant conflicts of interest to declare.

Successful Treatment for Solitary Bone Plasmacytoma of the Maxilla: A Rare Case Report

Introduction: Plasma cell tumors are monoclonal neoplastic proliferation of plasma cells which are divided into three groups: solitary bone plasmacytoma (SBP) and extramedullary plasmacytoma (EMP) which are localized forms, and multiple myeloma (MM) which is the disseminated form. Skull, long bones, and vertebrae are common sites for SBP and its presence in maxillofacial area is rare. There is a possibility that SBP converts to MM and some authors believe that it can be the early stage of MM. Case Presentation: Here, we present a 59-year old male patient with a mass in maxillary bone enlarging after tooth extraction. Cone beam computed tomography (CBCT) confirmed the existence of a poorly defined lesion in the left maxilla. The microscopic evaluation and immunohistochemical studies confirmed the diffuse and monoclonal population of cells, indicating the diagnosis of plasma cell tumor. There was also no evidence of bone marrow involvement in the aspiration. Conclusions: Early and precise diagnosis of SBP is a crucial element to rule out the possibility of MM. It must be kept in mind that there is possibility of recurrence or progression to MM after treatment which can completely change the course of disease, and this emphasizes the importance of regular follow up.

First-in-Human Imaging of Multiple Myeloma Using Copper-64-Labeled Daratumumab: Preliminary Results

Introduction: 18Fluoro-deoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is one of the most widely used imaging techniques to detect multiple myeloma (MM). Intracellular FDG uptake depicts in vivo metabolic activity, which can be seen in both malignant and non-malignant cells, resulting in limited sensitivity and specificity. Our group showed preclinically that tracing MM dissemination using a CD38-directed human antibody, daratumumab, that is radioconjugated with copper-64 via the chelator DOTA (64Cu-DOTA-Dara), led to improved sensitivity and specificity over that of FDG (Caserta et al, Blood 2018). Herein, we report the results of a Phase 1 trial (NCT#03311828) designed to 1) assess the safety and feasibility of 64Cu-DOTA-Dara PET/CT and 2) to evaluate and characterize the ability of 64Cu-DOTA-Dara to accurately detect or exclude MM lesions compared with FDG PET/CT. Methods: Patients with biopsy-proven MM and/or a plasmacytoma received an FDG PET/CT scan within 60 days of enrollment. On Day 0, patients were infused with unlabeled ("cold") Dara at one of four dose levels (0 mg, 10 mg, 45 mg, 95 mg) to optimize biodistribution of radioconjugated 64Cu-DOTA-Dara, especially in the liver and the spleen. Within 6 hours of unlabeled Dara administration, patients received 64Cu-DOTA-Dara at a dose of 13.63-16.68 mCi (~5 mg). Whole-body PET scans were obtained at 24 hours and at 48 hours (the latter scan encompassing known tumors). 64Cu-DOTA-Dara standardized uptake values (SUV) were evaluated in MM lesions and normal organs, which were then compared with values from standard FDG PET/CT. Biopsies were performed on accessible discordant lesions. Results: A total of 10 Dara-naïve patients were imaged. Patients were treated with 0 (n=3), 10 (n=3), 45 (n=3), or 95 mg (n=1) of unlabeled Dara. Four patients had newly diagnosed disease, one had biochemical relapse, one had a recurrent plasmacytoma by MRI, and four had possible recurrence by standard PET/CT. No significant adverse events were observed from either cold or 64Cu-DOTA-Dara. With the exception of the one patient with a recurrent plasmacytoma, radiolabeled antibody was eliminated from systemic circulation in subjects analyzed at the first three dose levels within 30 min post injection. In the patient with a recurrent plasmacytoma, the radiolabeled antibody was elevated in the blood for over two days. One newly diagnosed patient had extensive disease by FDG PET and had a biopsy-proven target lesion in the sternum, which had an SUVmax of 14.7 on 64Cu-DOTA-Dara PET/CT vs. 3.3 onFDG PET/CT. A second biopsy from the same patient was taken from a discordant iliac crest lesion (positive for 64Cu-DOTA-Dara but negative for FDG PET/CT) that showed 20-30% MM cell infiltration. In another patient, an iliac crest lesion was 64Cu-DOTA-Dara positive and FDG-negative; biopsy revealed 6% plasmacytosis in the bone. A third patient had an FDG PET/CT positive pleural lesion with an SUVmax of 8.98 and negative on 64Cu-DOTA-Dara (Figure 1A). The lesion did not show recurrence upon biopsy. Furthermore, 64Cu-DOTA-Dara PET/CT yielded superior imaging of bone lesions in the calvarium (Figure 1B). Escalating doses of unlabeled Dara decreased liver and spleen uptake by 64Cu-DOTA-Dara. Conclusions: In this ongoing study, 64Cu-DOTA-Dara PET/CT imaging is to date safe and provides whole body imaging of MM. Further dose escalation of cold Dara (145 mg, 195 mg) is planned to optimize background interference. This modality has the potential to improve sensitivity and specificity over FDG PET/CT scanning in early-stage MM as well as in recurrent disease. Disclosures Krishnan: Celgene, Janssen, Sanofi, BMS: Consultancy; Sutro BioPharma, zPredicta: Consultancy; Amgen, Takeda: Speakers Bureau; Celgene, Z Predicta: Other: Stock Ownership; Takeda: Research Funding. Palmer:Gilead Sciences: Consultancy. Rosenzweig:Celgene, Takeda: Speakers Bureau. Wu:ImaginAb, Inc.: Consultancy, Other: Board Member.

Soft computing to determine a Hemoglobin level of an early stage Multiple Myeloma patient by using Rectified Linear Units (ReLu) activation function

International Journal of Computer Sciences and Engineering (A UGC Approved and indexed with DOI, ICI and Approved, DPI Digital Library) is one of the leading and growing open access, peer-reviewed, monthly, and scientific research journal for scientists, engineers, research scholars, and academicians, which gains a foothold in Asia and opens to the world, aims to publish original, theoretical and practical advances in Computer Science,Information Technology, Engineering (Software, Mechanical, Civil, Electronics & Electrical), and all interdisciplinary streams of Computing Sciences. It intends to disseminate original, scientific, theoretical or applied research in the field of Computer Sciences and allied fields. It provides a platform for publishing results and research with a strong empirical component. It aims to bridge the significant gap between research and practice by promoting the publication of original, novel, industry-relevant research.

The use of the Hellwig's method for feature selection in the detection of myeloma bone destruction based on radiographic images

The radiological test is cost-effective, widely available, allows for the visualisation of large areas of the skeleton and can identify long bones potentially at risk for fractures in osteolysis sites. Therefore, radiology is often used in the early stages of multiple myeloma, in the detection and characterisation of complications, and in the assessment of the patient's response to treatment. The accuracy of this method can be improved through the use of appropriate algorithms of computer image processing and analysis. In the study, the feature vector based on humerus CR images was extracted. As a result of the analysis, 279 image descriptors were obtained. Hellwig's method in the selection process was applied. It found the set of feature combinations of the largest integral index of information capacity. To evaluate these combinations, 11 classifiers were built and tested. As a result, 2 feature sets were identified that provided the highest classification accuracy in combination with the K-NN classifier. The 9-NN classifier for the first combination (2 features) was used and 5-NN for the second one (3 features). The classification accuracy (depending on the quality index used) was as follows: overall classification accuracy – 93%, classification sensitivity – 92%, classification specificity – 96%, positive predictive value – 96% and negative predictive value – 93%. Results show that: (1) the use of humerus CR images may be useful in the detection of bone damages caused by multiple myeloma; (2) the Hellwig's method is effective in the feature selection of the analysed kind of images.

The Clinical Impact of Macrofocal Disease in Multiple Myeloma Differs Between Presentation and Relapse

IntroductionFunctional imaging of Multiple Myeloma (MM) is redefining our knowledge of disease patterns. A pattern, termed macrofocal MM (macro MM), is defined by the presence of focal lesions and the absence of significant intervening bone marrow (BM) infiltration. At presentation, macro MM constitutes a distinct disease entity likely being associated with a favorable prognosis, although current evidence to support this is limited. Following first-line therapy, macrofocal patterns of disease emerge also in patients that initially presented with classical MM. In these patients the systemic BM involvement disappears in follow up examinations during treatment whereas focal lesions persist. In a third scenario, macrofocal patterns occur at overt relapse representing a patchy type of MM progression (Figure 1). The prognostic impact of a macrofocal pattern at these various disease stages is largely unknown. Therefore, we analyzed the clinical outcome and biological features of macro MM at different treatment stages.Patients and Methods279 patients met the criteria of macro MM. Of those, 56 were at initial presentation, 48 at restaging following first line therapy, and 175 at relapse. Generally, macrofocal lesions were present in both positron emission tomography and magnetic resonance imaging. All first-line patients were treated with multi-agent induction therapy, autologous stem cell transplantation and received maintenance within prospective trials. Outcome results were compared to a set of cases with classical MM matched for age, gene-expression based (GEP) risk group, and treatment protocol.ResultsMacro MM at presentation is rare, constituting 6% of patients in the time period examined. The vast majority showed GEP-based low risk (94%). Age, Ig-type, and sex were not significantly different between macro MM and classical MM. With a median follow up of 8.6 years, only 10 of the macro MM patients relapsed. Compared to a matched-pair MM group, progression-free survival (PFS) and overall survival (OS) were significantly better in the macro MM group (P= 0.01 and 0.04 for PFS and OS, respectively). Thus macro MM at presentation constitutes a low risk form of MM.Focusing on the 10 macro MM cases who relapsed, no specific risk profile could be identified except >26 focal lesions on MRI was associated with a shorter PFS (P=0.04) but not with OS. Of note, although focal lesions frequently responded slowly, the time to response was not associated with outcome.To elucidate whether there are biological differences between MM cells in focal lesions and at differentially involved BM sites, we analyzed a set of 16 patients with paired samples from macrofocal lesions and iliac crest BM aspirates. No difference in a GEP based proliferation index was seen between the two sites. After correction for multiple testing we did not observe gene expression differences between them. A candidate gene study including a set of 27, myeloma relevant, adhesion molecules also did not reveal expression differences.In contrast to the situation at presentation, macrofocal patterns at restaging during initial therapy showed a 70% cumulative 24 months relapse incidence. The outcome of these cases was significantly worse in comparison to matched controls (P=0.02 and 0.02 for PFS and OS, respectively). Of note, all patients with macro MM showed an objective response at the time of imaging with 9 of 46 cases meeting the IMWG criteria for CR.Performing a similar analysis of patients with macro MM at relapse showed that 25% of patients presented with that pattern; a surprisingly high proportion. Extramedullary involvement was common (41%). Of note, 36% of patients repeatedly showed macrofocal patterns at subsequent relapses. PFS and OS at 2 years from macrofocal relapse were 24% and 39%, respectively. A matched group OS comparison was not possible since number of relapses and treatments were too different among the patients.ConclusionsMacro MM at presentation seems to be an early stage of MM with an excellent prognosis. In contrast, a macrofocal pattern at restaging is associated with poor prognosis and early relapse. At this disease stage residual focal lesions may represent drug resistant clones. At overt relapse a macrofocal pattern was frequently seen, highlighting the need to integrate advanced imaging tools into the standard work up and indicating an important confounder of standard minimal residual disease diagnostics in MM. [Display omitted] DisclosuresBarlogie:Signal Genetics: Patents & Royalties. Davies:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding; Univ of AR for Medical Sciences: Employment.

Clinicopathological significance of p15 promoter hypermethylation in multiple myeloma: a meta-analysis.

Published studies reported that loss of function of the p15INK4B gene is caused by hypermethylation; however, whether or not the inactivation is associated with the incidence and clinical significance of multiple myeloma (MM) remains unclear. In this study, we performed a meta-analysis to quantitatively determine the effects of p15 hypermethylation on the incidence of MM. The related research articles in English and Chinese languages were evaluated. The data were extracted and assessed independently. The pooled data were analyzed and odds ratios were calculated and summarized. Sixteen eligible studies were selected for final analysis. We demonstrated that p15 hypermethylation is significantly higher in MM than that in normal bone marrow, as well as monoclonal gammopathy of undetermined significance. However, aberrant p15 hypermethylation was not significantly higher in advanced MM than that in early-stage MM. The results of this study reveal that p15 hypermethylation is correlated with an increased risk in the progression of monoclonal gammopathy of undetermined significance to MM. p15 hypermethylation, which induces the loss of function of the p15 gene, plays a critical role in the early tumorigenesis of MM and serves as a reputable diagnostic marker and potential drug target.

Targeting vasculogenesis to prevent progression in multiple myeloma.

The role of endothelial progenitor cell (EPC)-mediated vasculogenesis in hematological malignancies is not well explored. Here, we showed that EPCs are mobilized from the bone marrow (BM) to the peripheral blood at early stages of multiple myeloma (MM); and recruited to MM cell-colonized BM niches. Using EPC-defective ID1+/- ID3-/- mice, we found that MM tumor progression is dependent on EPC trafficking. By performing RNA-sequencing studies, we confirmed that endothelial cells can enhance proliferation and favor cell-cycle progression only in MM clones that are smoldering-like and have dependency on endothelial cells for tumor growth. We further confirmed that angiogenic dependency occurs early and not late during tumor progression in MM. By using a VEGFR2 antibody with anti-vasculogenic activity, we demonstrated that early targeting of EPCs delays tumor progression, while using the same agent at late stages of tumor progression is ineffective. Thus, although there is significant angiogenesis in myeloma, the dependency of the tumor cells on EPCs and vasculogenesis may actually precede this step. Manipulating vasculogenesis at an early stage of disease may be examined in clinical trials in patients with smoldering MM, and other hematological malignancies with precursor conditions.

BRAF V600E mutation in early-stage multiple myeloma: good response to broad acting drugs and no relation to prognosis

In this study, we analyzed the prevalence and clone size of BRAF V600E mutation in 209 patients with multiple myeloma and related the results to clinical phenotype, response and survival. Biopsies were screened for BRAF V600E by allele-specific real-time PCR (AS-PCR). Positive results were confirmed by immunohistochemistry, Sanger sequencing and, in three patients from whom we had stored purified myeloma cells, whole-exome sequencing. Eleven patients (5.3%) were BRAF V600E mutation positive by AS-PCR and at least one other method. The fraction of mutated cells varied from 4 to 100%. BRAF V600E-positive patients had no characteristic clinical phenotype except for significantly higher levels of serum creatinine (125 versus 86 μmol/l) Seven of eleven patients responded with at least very good partial response to alkylators, immunomodulatory agents or proteasome inhibitors. Progression-free and overall survival were similar in patients with and without the mutation. By this integrated approach, we found that patients with BRAF V600E mutation responded very well to broad acting drugs and there was no relation to prognosis in early-stage myeloma. In particular, a large mutated cell fraction did not correlate with aggressive disease.

Understanding multiple myeloma

Understanding multiple myeloma

Therapeutic Potential Of Targeting Sphingosine-1-Phosuphate and Sphingosine Kinases In Multiple Myeloma

Multiple myeloma (MM) is one of the common hematological malignancies and is a uniformly fatal disorder of B cells characterized by accumulation of abnormal plasma cells in the bone marrow.Clinical progression of patients with MM is improved with the proteasome inhibitor (PI) (e.g. bortezomib) and the immunomodulatory drugs (IMiDs) such as thalidomide and lenalidomide. Although PI and IMiDs have considerably changed the treatment paradigm of MM, many patients show disease relapse due to developing into drug resistance of MM cells. Since the prognosis remains poor for patients with refractory disease, the new therapeutic strategies are required to treat against these patients. Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid. Two isoforms of sphingosine kinases (SphKs), SK1 and SK2, catalyze the formation of the S1P in mammalian cells. SphKs have also been shown to be up-regulated in the variety of cancer types. SphKs/S1P/S1P receptor (S1PR) axis is involved in multiple biological processes. It has been reported that S1P is involved in cell proliferation, angiogenesis and inflammation. S1P is also involved in cancer progression including cell transformation, oncogenesis and cell survival in hematological malignancies such as multiple myeloma. Therefore, S1P and SphKs may present attractive targets for MM treatment. One of the S1P analog, fingolimod (FTY720), which is an orally active immunomodulatory drug, is developed for the treatment of multiple sclerosis. SKI-I, which is a non-lipid pan-SphK inhibitor and ABC294640, selective inhibitor of SK2, are currently investigated in a pivotal phase 1 clinical trial against solid tumors. In this study, we investigated the efficacy of fingolimod, SKI-I, and ABC294640 by using the MM cell lines, RPMI8226, MM1.S and MM1.R. 72 hours treatment of fingolimod exhibited cell growth inhibition of MM cell lines in a dose dependent manner. Treatment of SKI-I and ABC294640 also exhibited cell growth inhibition in a dose dependent manner. Since S1P is the ligand for a family of five G-protein-coupled receptors with distinct signaling pathways that regulate angiogenesis and chemotaxis, we next evaluated the chemotactic response of human umbilical vein endothelial cells (HUVEC). We found that 4 hours treatment of S1P significantly induced the migration of HUVECs compared to control medium. Treatment of HUVECs with fingolimod inhibited S1P-stimulated chemotaxis in a dose dependent manner. We also found that S1P-induced chemotaxis was abolished by the SKI-I and ABC294640. These results suggest that intracellular SK1 and SK2 may play the important role in S1P induced chemotaxis of HUVEC. We next investigated the S1P concentrations in MM patient by enzyme-linked immune sorbent assay (ELISA), because S1P is a potent tumorigenic growth factor that is likely released from tumor cells. We found that serum concentrations of S1P were significantly higher in patient with MM compared with normal samples. The average S1P levels of MM and normal control are 1503.431 and 1103.38 (p <0.05). We also found that conditioned medium from MM cell line had chemotactic activity for HUVECs. These results implicate that S1P may be a novel biomarker for early stage of MM and that S1P is an important bioactive sphingolipid involved in angiogenesis. In this study, we also demonstrate that fingolimod, SKI-I and ABC294640 have potent preclinical anti-tumor activity in MM. These agents possibly inhibit angiogenesis with relation to MM cell growth and offer unique opportunities for novel therapeutic strategies for the treatment of multiple myeloma. Disclosures:No relevant conflicts of interest to declare.

Myeloid-Derived Suppressor Cells Regulate Growth of Multiple Myeloma by Inhibiting T Cells in Bone Marrow

Myeloid-derived suppressor cells (MDSC) are one of the major factors limiting the immune response in cancer. However, their role in bone marrow (BM), the site of primary localization of multiple myeloma (MM), is poorly understood. In this study, we found a significant accumulation of CD11b(+)CD14(-)CD33(+) immunosuppressive MDSC in BM of patients with newly diagnosed MM. To assess the possible role of MDSC in MM, we used immunocompetent mouse models. Immunosuppressive MDSC accumulated in BM of mice as early as 1 wk after tumor inoculation. S100A9 knockout (KO) mice, which are deficient in their ability to accumulate MDSC in tumor-bearing hosts, demonstrated reduced MDSC accumulation in BM after injection of MM cells compared with wild-type mice. Growth of the immunogenic MM cells was significantly reduced in S100A9KO mice. This effect was associated with the accumulation of Ag-specific CD8(+) T cells in BM and spleens of S100A9KO mice, but not wild-type mice, and was abrogated by the administration of anti-CD8 Ab or adoptive transfer of MDSC. Thus, the accumulation of MDSC at early stages of MM plays a critical role in MM progression and suggests that MDSC can be considered a possible therapeutic target in this disease.

Functional P2X7 Receptors in Myeloma Cell Lines and Myeloma Patient Bone Marrow Samples Can Be Blocked by Specific P2X7 Receptor Antagonists: Implications for Myeloma Therapy and Chemotherapy Induced Inflammation.

Abstract 1845Inflammation is a driving factor in the pathogenesis of many cancers including multiple myeloma (MM). The active role that cytokines play, IL-1β in particular, during the early stages of MM has been extensively characterized. Myeloma is a plasma cell tumor whose early stages are characterized by cytokine dependent growth, predominantly driven by IL-6. We have demonstrated that IL-1β is a key inducer of IL-6 expressed by bone marrow stromal cells in a paracrine fashion during the progression of multiple myeloma from smoldering myeloma (SMM) to active disease (Mayo Clinic Proc 84:114 (Feb. 2009)). Elevated ATP levels have been documented in vivo in tumor micro-environments and is a potential contributing factor to IL-1β induced inflammation. The P2X7 receptor (P2X7R) is an ATP-gated ion channel expressed by cells of the hematopoetic lineage that has been shown to play a critical role in IL-1b processing and secretion. We investigated the role this receptor may have in the release of IL-1β in the bone marrow microenvironment using bone marrow samples from both chemotherapy treated and untreated patients.Myeloma cell lines were evaluated by RT-PCR for the expression of P2X7R mRNA. Expression was detected and confirmed by sequencing in ANBL-6, MM1.S, U266, RPMI-8226 and KAS-6/1 and was comparable to U937, a previously characterized P2X7R positive cell line. A newly derived plasmacytoma cell line, PCYT3, showed very low levels of message expression. The ability of ATP, the P2X7R agonist, to trigger the processing and release of IL-1β by activation of the P2X7 channel was studied using KAS-Pro, a myeloma cell line stably transfected with a pro- IL-1β gene. IL-1β, measured by ELISA, was released in a dose dependent fashion in response to increasing amounts of ATP ranging from.6mM to 5mM. Four P2X7R specific antagonists were tested for their effect on ATP stimulated IL- 1β release from KAS-Pro; all 4 compounds demonstrated inhibition in a dose dependent manner with varying degrees of potency correlating with P2X7R inhibition.In addition fresh patient samples (8 MGUS/7SMM/9 newly diagnosed MM as well as 12 Treated MM) were evaluated for receptor expression and function. All stages of disease expressed the receptor by RT-PCR. Analysis of samples from all stages of MM, as measured directly by IL-1β ELISA as well as indirectly by measurement of IL-1β induced IL-6 production from normal bone marrow stromal cells, showed functional receptor activation in response to ATP stimulation. IL-6 production detected from MGUS patient samples had a mean result of 2,253 pg/ml, the mean for SMM was 5,495 pg/ml, MM were 6,875 pg/ml while treated MM samples showed a mean result of 15,219 pg/m. Greater than 90% inhibition of IL-1β release by the P2X7R antagonists was seen for all patients who demonstrated a positive ATP induced IL-1β release (see figure). We observed a greater than 2 fold increase in IL-1β release between the treated MM samples and the newly diagnosed MM samples, suggesting a chemotherapy induced inflammation process may be ongoing in these patients. [Display omitted] These results confirm the activity of functional P2X7 receptors on myeloma cell lines and in fresh patient samples and suggest a role for the use of P2X7 receptor antagonists in the therapy of myeloma, particularly in patients undergoing chemotherapy. Disclosures:Hromockyj:Pfizer Corporation: Employment. Meyer:Pfizer Corporation: Employment.

Molecular and cellular effects of multi‐targeted cyclin‐dependent kinase inhibition in myeloma: biological and clinical implications

Cell cycle regulators, such as cyclin-dependent kinases (CDKs), are appealing targets for multiple myeloma (MM) therapy given the increased proliferative rates of tumour cells in advanced versus early stages of MM. We hypothesized that a multi-targeted CDK inhibitor with a different spectrum of activity compared to existing CDK inhibitors could trigger distinct molecular sequelae with therapeutic implications for MM. We therefore studied the small molecule heterocyclic compound NVP-LCQ195/AT9311 (LCQ195), which inhibits CDK1, CDK2 and CDK5, as well as CDK3 and CDK9. LCQ195 induced cell cycle arrest and eventual apoptotic cell death of MM cells, even at sub-μmol/l concentrations, spared non-malignant cells, and overcame the protection conferred to MM cells by stroma or cytokines of the bone marrow milieu. In MM cells, LCQ195 triggered decreased amplitude of transcriptional signatures associated with oncogenesis, drug resistance and stem cell renewal, including signatures of activation of key transcription factors for MM cells e.g. myc, HIF-1α, IRF4. Bortezomib-treated MM patients whose tumours had high baseline expression of genes suppressed by LCQ195 had significantly shorter progression-free and overall survival than those with low levels of these transcripts in their MM cells. These observations provide insight into the biological relevance of multi-targeted CDK inhibition in MM.

A Phase III Randomized Trial of Thalidomide (THAL) Plus Zoledronic Acid (ZLD) Versus Zoledronic Acid Alone In Patients with Early Stage Multiple Myeloma (MC0289)

AbstractAbstract 3053 Background:Patients with smoldering multiple myeloma (SMM) have a higher chance of progression to active MM than patients with monoclonal gammopathy of undetermined significance (MGUS). Since active MM remains incurable and since patients with SMM can have a long time to requiring treatment, observation remains an option for these patients. Bisphosphonates can prevent the bone complications of myeloma. The immunomodulatory (IMiD) drugs are well-tolerated and have documented anti-tumor activity in active MM. We hypothesized that treatment with the IMiD THAL and a bisphosphonate (ZLD) would prolong the time to progression (TTP) over the control arm of ZLD alone. This is the first report of this phase III trial of THAL/ZLD vs ZLD alone for patients with untreated SMM. Goals:The primary goal of this trial was to compare the TTP between patients treated with THAL/ZLD versus ZLD alone in asymptomatic MM. Secondary goals were progression rate at one year, response rate, duration of response, time to next therapy, and toxicity. Methods:Patients were required to have measurable disease as defined by either a serum monoclonal protein >1.0 g by protein electrophoresis or nephelometry or >200 mg of monoclonal protein in the urine on 24 hour electrophoresis or a measurable soft tissue plasmacytoma; >10% plasma cells as measured on the bone marrow aspirate, bone marrow biopsy, or labeling index; absolute neutrophil count >1500/μL; platelet count >100,000/μL; creatinine <2.0 mg/dL, and a performance status of 0, 1, or 2. Patients could not have symptomatic MM that required chemotherapy. The statistical plan was to accrue 120 eligible patients (60 per arm) over 4 years and after a minimum follow-up of 12 months the study would provide >90% power at a type I error rate of 0.05 to detect an increase in the median TTP from 12 months (ZLD) to 24 months (THAL/ZLD), and >80% power to detect an increase in median TTP from 12 to 21 months). Results:The study was activated in July 2003 and closed March 2009 due to slow accrual. Sixty-eight patients (35 Thal/ZLD; 33 ZLD) with a median age 63 years (range, 47–84) were randomized. The median TTP for Thal/ZLD versus ZLD was 2.4 years versus 1.2 years, respectively, P=0.02 one-sided log-rank. After adjusting for pre-specified stratification factors, the hazard ratio for TTP was 2.2 (one-sided p value = 0.01, univariate stratified Cox PH model) for ZLD compared to Thal/ZLD. 89% of patients on the Thal/ZLD arm were progression-free survival (PFS) at one year compared to 55% on ZLD alone (one-sided p<0.001, chi-square). Similar results were obtained (Table and Figures) when the CRAB (calcium, renal, anemia, bone) criteria were applied. Confirmed response was evaluated using the first 12 months of treatment. In the Thal/ZLD arm the response rate was 31% with a median duration of response of 5.1 years (95% CI: 1.9 - NA). There were no confirmed responses in the ZLD alone arm. The median overall survival has not been reached for either arm. In regards to toxicity, no grade 5 adverse events (AEs) have been reported. Thirty patients have reported grade 3+ AEs (17 Thal/ZLD; 13 ZLD, Fisher's exact p-value 0.47). Eight patients have reported grade 4 adverse events (5 Thal/ZLD; 3 ZLD, Fisher's exact p-value 0.71). Overall, only one grade 4 event was felt to be at least possibly related to study treatment – grade 4 neutropenia in Thal/ZLD. Conclusions:While the trial did not meet its planned accrual goals, there was a significant improvement in improved in outcomes in the Thal/ZLD arm compared to control (ZLD). Thal/ZLD produces anti-tumor responses that are quite durable with the median response duration of over 5 years. ZLD alone did not produce any anti-tumor responses. This study indicates that a non-chemotherapy approach can be effective in SMM and may be a useful strategy to test in future studies.Table:TTP & PFS Estimates for Protocol and CRAB Progression CriteriaCriteriaThal/ZLD (n=35)ZLD (n=33)p-value (unadjusted, log-rank, one-sided)p-value (adjusted for stratification factors using a univariate Cox model, one-sided)TTP Protocol2.4 years1.2 years0.020.01# of Events2124TTP CRAB4.1 years3.1 years0.030.02# of Events1520PFS Protocol2.4 years1.2 years0.020.005# of Events2325PFS CRAB3.8 years2.3 years0.030.008# of Events1822 [Display omitted] [Display omitted] Disclosures:Off Label Use: Thalidomide for smoldering myeloma. Lacy:Celgene: Research Funding. Dispenzieri:Celgene: Honoraria, Research Funding; Binding Site: Honoraria. Kumar:Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding. Gertz:Celgene: Honoraria; Millennium: Honoraria.

Interest of Diffusion Weighted Magnetic Resonance Imaging for the Evaluation of Bone Marrow Infiltration in Multiple Myeloma, Smoldering Myeloma and Monoclonal Gammopathy of Undetermined Significance.

Abstract 4874 IntroductionMonoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) are two asymptomatic early stages of multiple myeloma, and are characterized by the absence of bone involvement. In contrast, bone lytic lesions are common complications of multiple myeloma. Magnetic resonance imaging (MRI) has improved the evaluation of bone involvement in multiple myeloma. Moreover MRI allows visualization of the medullary cavity and a direct assessment of the degree of myeloma cell infiltration before bone destruction becomes visible on plain radiographs. Diffusion weighted magnetic resonance imaging (DWI) probes the diffusion of water in the body. This sequence yields qualitative and quantitative information that reflects the tissue cellularity and cell membrane intregrity and thus complements morphological information obtained by conventional MRI. It shows considerable promise for detecting and characterizing tumors and evaluating treatment response. ObjectiveOur objective was to study the potential interest of diffusion weighted MRI for the evaluation of bone marrow infiltration in multiple myeloma, SMM and MGUS. Diagnostic criteria used were those defined by the International Myeloma Working Group. Patients and methodsThis retrospective study was conducted over a period of 8 months (November 2008 - June 2009) in Internal Medicine and Hematology departments of our institution. All patients who had a whole body MRI for evaluation of multiple myeloma, SMM or MGUS were included. We choose to study simultaneously 3 complementary sequences: T1 weighted, short time inversion recovery (STIR) and diffusion weighted. ResultsSeventy patients were included in this study: 9 MGUS, 19 SMM and 42 symptomatic multiple myeloma (16 at diagnosis and 27 at relapse). They were 30 men and 40 women. The median age was 64 years (36 - 89 years). Thirty one of the 42 multiple myeloma patients have focal bone lesions. Focal lesions were observed in the 3 MRI sequences and diffusion did not detect new lesions. Regarding bone marrow infiltration assessment by MRI, four groups of patients could be identified: - Group 1 (20 patients – 28.6%): normal signal in T1, STIR, and diffusion. - Group 2 (19 patients – 27.1%): abnormal signal involving the whole spine on the STIR or diffusion weighted sequences and with a normal signal of the spine on T1.- Group 3 (25 patients – 35.7%): abnormal signal involving the whole spine on the STIR and diffusion weighted sequences and with a normal signal of the spine on T1.- Group 4 (6 patients – 8.6%): abnormal signal involving the whole spine on the 3 sequences. For the 28 patients with either MGUS or SMM, the distribution in the 4 groups was respectively 13 patients (46.4%), 7 patients (25.0%), 7 patients (25.0%) and 1 patient (3.6%) for group 1,2 3 and 4. Regarding the 42 cases of symptomatic multiple myeloma, 7 patients (16.7%) were classified in group 1, 12 (28.6%) in group 2, 18 (42.9%) in group 3 and 5 (11.9%) in group 4. There was no correlation between number of focal lesions and degree of diffuse infiltration. The degree of bone marrow infiltration evaluated by MRI was not correlated with Durie and Salmon stage, ISS, monoclonal component concentration or bone marrow plasmocytosis. DiscussionDiffusion weighted magnetic resonance imaging combined with conventional sequences (T1 and STIR) can distinguish 4 groups of patients with different aspects of bone marrow infiltration. Fifteen patients (53.6%) with either MGUS or SMM had various degree of bone marrow infiltration without any focal lesions. A prospective study on a large population of MGUS and SMM is needed to assess the impact of these asymptomatic infiltrations on the risk of malignant transformation. The degree of bone marrow infiltration evaluated by MRI is very heterogeneous between patients with multiple myeloma and not correlated with classical prognostic factors. Further prospective studies are necessary to investigate the possible prognostic impact of these different groups on the evolution of myeloma. DisclosuresNo relevant conflicts of interest to declare.

The Functional Role of Microrna 15a/16-1 as Tumor Suppressor Genes in Multiple Myeloma.

Abstract 1963Poster Board I-986 Background:Multiple Myeloma (MM), a cancer of plasma cells is characterized by frequent chromosomal alterations. Deletion of chromosome 13, especially band 13q14, is commonly observed in early stages of MM, suggesting the presence of tumor suppressor genes within this region. When studied in the context of CLL, the miR 15a and 16-1 cluster was associated with a distinct miR signature and clinical outcome. Over-expression of miR16 caused induction of apoptosis and downregulation of the anti apoptotic gene BCL2 in a megakaryocytic leukemia cell line and induced growth arrest in MM cells. Nonetheless, being lost in CLL, MM, MCL and LPL, their functional role has not been studied using a loss-of-function approach in any of these lymphoid malignancies. Here, we describe the generation of an in vivo system for the long term, stable knockdown of miR 15a/ 16-1 expression in myeloma cells to recapitulate the conditions seen in chromosome 13q14 deleted MM. Methods:Using lentiviral vectors to stably express a competitive sponge miR16 inhibitor we set up a system to functionally validate the role of microRNA 15a/16-1 cluster. Pure populations of lentivirally transduced MM cell lines were sorted by flow cytometry using GFP marker. Decreased miRs 15a and 16 expression levels were assessed by Northern blot and R-luciferase reporter system. Cell growth rate was measured using trypan blue counting, and thymidine incorporation. Cell cycle analysis was measured by flow cytometry after staining with PI. Intracellular signal modulation was demonstrated by Western blotting. RNA from MM cell lines expressing the control sponge or sponge16 were hybridized on an Affymetrix U133A 2.0 array chip, and validated using quantitative real time PCR. Xenograft murine models were performed using the stable MM cell lines injected into 6-week old NOD.CB17-PrkdcSCID/J irradiated mice. Results:Selected stable miR knockdown MM cell lines exhibited significantly reduced expression of miRs15a/16-1 as assessd by both by mRNA levels and miR luciferase reporter assays. The knockdown cells showed a significant increase in growth rates (1.5-2 fold) compared to control cells, as measured by viable cell counts and proliferation by thymidine incorporation in vitro. Importantly, miR16 inhibition decreased animal survival in a xenograft model of MM by increasing tumor load, invasiveness and host angiogenesis. To further delineate the role of miR15a/16 in MM and to gain additional insight into the possible target genes regulated by this cluster, we performed gene expression-profiling analysis in controls and miR16 deficient MMS1 and RPMI cell lines. Since our sponge system produces downregulation of the miRs, we focused on the upregulated probes. Expression profiling analysis of miR16 deficient cells identified a surprisingly large number of downstream target-genes such as FGFR1, PI3KCa, MDM4, VEGFa, as well as secondary affected genes such as JUN and Jag1. These results were verified both at the mRNA level and the protein level, as well as in other MM cell lines. Moreover, we were able to show that these knockdown cells were partially addicted to some of these pathways using specific drug inhibitors. We further validated designated genes as direct miR16 targets by showing binding sites within the conserved 3' UTR and also within the mRNA coding region, thus indicating that the miRs may have many more possible targets than anticipated by conventional prediction methods. Conclusions:Using this loss-of-function system, which mimics the pleiotropic chronic effects of microRNA loss at the 13q chromosomal deletion, provides a valuable tool to investigate their function as tumor suppressor genes in MM pathogenesis, affecting multiple targets, and may represent a novel potential for therapeutic targeting in MM and other lymphoid malignancies. Disclosures:Munshi:Seattle Genetics, Inc.: Research Funding.

Expression of MAGE-A1 and MAGE-A3 in Bone Marrow from Monoclonal Gammapathy to Myeloma Patients.

Multiple myeloma (MM) is an incurable tumor characterized by multifocal clonal expansion of malignant plasma cells within bone marrow (BM), with sometimes extramedullary location, i.e. mainly in peripheral blood (PB), pleural effusion or ascites. Cancer/testis antigens (CTA) are a category of tumor antigens with normal expression restricted to a male germ cells in the testis but not in adult somatic tissues. CTA are promising candidates for cancer immunotherapy. Presently, 44 distinct CT-antigen families have been identified. Presence of RNA transcripts encoding members of the MAGE gene family, as well as NY-ESO-1, CT7/MAGE-C1 in myeloma tumor cells and cell lines has been documented. In MM, cancer germ-line specific genes are preferentially expressed in advanced disease (up to 50%) and in almost all cell lines. Most of the genes of the MAGE-A family were found to be expressed in myeloma cells, although with various incidences. The aim of this study was to evaluate the posibility of using these genes as molecular markers of the progression MGUS to MM and the early relapse of the MM. Due to rare extramedullary location of malignant plasma cells we tried to undercover if expression is detecable in PB simultaneously with expression in the BM. Total of 151 samples from BM were evaluated: 83 samples from advanced myeloma, 12 samples of patients with early stage of MM who did not required treatment (smoldering MM 4x, stage IA 8x), 41 samples of MGUS patiens and 15 samples of normal healthy donors served as control group. In 15 advanced myeloma patients we simultaneously assessed BM sample and PB sample. Total RNA was evaluated by RT-PCR and then by real-time PCR using FRET probes. For relative quantification we used G6PDH housekeeping gene as external standard. As positive control we used myeloma cell line U266. Only 4,8% (2 /41) samples from MGUS patient showed expression of MAGE-A1 or MAGE-A3. 50% (6/12) patients with early stage of MM (IA and smoldering) showed expression of MAGE-A1 or MAGE-A3. 39,7% (33/83) of the samples from the advanced myeloma patients expressed at least 1 of these genes, or both (20 cases). We tested 15 BM from normal donors. All were negative. In 15 advanced myeloma BM samples we have observed expression in 8/15 cases but no expression in PB sample. We have confirmed that expression of MAGE is not present in samples of healthy donors. There is an obvious correlation between expression of the MAGE genes and early-late stage of the disease. Our evaluation confirmed the detection of low expression levels of MAGE-type mRNA in BM from patients with MGUS and early stage of MM. It is possible that MAGE antigen monitoring may predict the evolution towards more advanced disease. But due to need of separated myeloma cells or BM samples this metod can not be used for monitoring minimal residual disease in patients with MM.