Different Stages Of Multiple Myeloma Research Articles

Identification of focal lesion characteristics in MRI which indicate presence of corresponding osteolytic lesion in CT in patients with multiple myeloma.

The presence of bone marrow focal lesions and osteolytic lesions in patients with multiple myeloma (MM) is of high prognostic significance for their individual outcome. It is not known yet why some focal lesions seen in MRI, reflecting localized bone marrow infiltration of myeloma cells, remain non-lytic, whereas others are associated with destruction of mineralized bone. In this study, we analyzed MRI characteristics of manually segmented focal lesions in MM patients to identify possible features that might discriminate lytic and non-lytic lesions. The initial cohort included a total of 140 patients with different stages of MM who had undergone both whole-body MRI and whole-body low-dose CT within 30 days, and of which 29 satisfied the inclusion criteria for this study. Focal lesions in MRI and corresponding osteolytic areas in CT were segmented manually. Analysis of the lesions included volume, location and first order texture features analysis. There were significantly more lytic lesions in the axial skeleton than in the appendicular skeleton (p = 0.037). Out of 926 focal lesions in the axial skeleton seen on MRI, 544 (59.3 %) were osteolytic. Analysis of volume and first order texture features showed differences in texture and volume between focal lesions in MRI with and without local bone destruction in CT, but these findings were not statistically significant. Neither morphological imaging characteristics like size and location nor first order texture features could predict whether focal lesions seen in MRI would exhibit corresponding bone destruction in CT. Studies performing biopsies of such lesions are ongoing.

The evolution of immune dysfunction in multiple myeloma.

This review discusses the role of immune dysfunction at the different stages of multiple myeloma (MM). Narrative review. MM is a complex disease and immune dysfunction has been known to play an important role in disease pathogenesis, progression, and drug resistance. MM is known to be preceded by asymptomatic precursor states and progression from the precursor states to MM is likely related to a progressive impairment of the immune system. An understanding of the role of the immune system in the progression of MM is important to guide the development of immunotherapeutic strategies for this disease.

Circulating miR-448 acts as a potential diagnostic biomarker for multiple myeloma

ABSTRACT Objective The current diagnostic methods for multiple myeloma (MM) include bone marrow aspiration and biopsy, which are not only complicated but also invasive, causing great pain to patients. Micro ribonucleotides (miRNA) exist stably in circulating plasma and could be easily detected, making them promising specific diagnostic markers for MM. Methods The expression of plasma miR-448 in MM patients was detected by real-time quantitative PCR analysis. Spearman correlation was used to analyze correlations between miR-448 expression and various clinic pathological characteristics of MM patients. The ROC and the AUC (95% confidence interval) were exploited to evaluate the potential of miR-448 acting as a diagnostic marker for MM patients. Results and discussion In this study, we found that the expression level of miR-448 in patients with MM was significantly higher than that in normal people and showed statistically differential expression in different stages of MM. Besides, we observed that the abundance of miR-448 in the plasma of newly diagnosed MM patients was positively correlated with the proportion of plasma cells (PC) in the bone marrow and the concentration of serum M protein (MP), respectively. In addition, ROC analysis demonstrated the sensitivity and specificity of the diagnostic value of miR-448 for MM are 92.90% and 87.50%, respectively. Conclusion Taken together, these results indicated that miR-448 may serve as a potential molecular diagnostic marker for MM.

Abnormalities in Mitochondrial DNA Copy Number Have Pathogenetic and Prognostic Implications in Multiple Myeloma

Mitochondria are bioenergetic and biosynthetic organelles which control crucial biological pathways such as cell growth, proliferation and apoptosis among others. Over the last two decades, many studies have examined the role of mitochondrial DNA (mtDNA) alterations in carcinogenesis, via inactivating genetic mutations or dysregulation of the mtDNA copy number in the cell. However, the status of the mtDNA copy number and its implication in the pathogenesis of Multiple Myeloma (MM) remains unknown. Here, we have analyzed the mtDNA copy number in asymptomatic and symptomatic stages of MM and evaluated its clinical impact.We have estimated the mtDNA copy number in purified plasma cells from 139 patients across different stages of the disease (4 healthy donor, 19 MGUS, 14 Smoldering MM (SMM), 78 newly diagnosed MM (ndMM) and 24 relapse (rMM). The mtDNA copy number quantification was performed by RT-PCR using 2 Taqman probes which targeted the mitochondrial gene MT-RNR1 and the nuclear gene RPPH1. We have also used whole-genome or whole-exome sequencing data to examine changes in mtDNA copy number in newly-diagnosed or relapsed MM (n=64 samples) including paired sample from SMM patients progressing to ndMM (n=10). FastMitocalc and Samtools were used to extract reads aligning to mitochondrial genome from bam files and calculate de mtDNA copy number by comparing to the number of reads aligning to nuclear genome. Differential gene expression analysis was done by using canEvolve.org ocogenomics web portal (Multiple Myeloma, GSE6477 study).We observed a significant increase in the average mtDNA copy number in myeloma cells compared to healthy plasma cells (39 vs 228 copies; p-value = 0.02). Plasma cells from MGUS cells showed lower mtDNA content than more advanced stages of the disease (129 vs 242 mtDNA copies). Interestingly, amongst 10 paired samples from SMM progressing to MM an increase in the mtDNA copy number was observed in 8 of the 10 patients, being more evident when the time to progression was longer. Additionally, we also observed an increase in the mitochondrial content in 17 of the 21 patient samples examined pre and post relapse, including those patients with three consecutive relapsed samples available. We have evaluated the impact on survival of the mtDNA copy number in 69 newly diagnosed patients and we have found that patients with greater than 400 mtDNA copy number had significantly shorter progression free survival compared to the rest (p-value = 0.024). The differential expression analysis of plasma cells from MGUS, SMM, ndMM and rMM patients compared to normal plasma cells confirmed an overexpression of the mitochondrial biogenesis regulators (TFAM, TUFM, MYC and HNRNPK).We describe for the first time that mtDNA copy number increase across different stages of MM. Our data provides evidence that the malignant transformation of the myeloma cells from asymptomatic stages implies activation of mitochondrial biogenesis resulting in increased mtDNA levels. Functional experiments are ongoing to confirm the metabolic activation of myeloma cells over the time. These results highlight mitochondria as an important component of pathogenesis and progression of MM and as a potential target for therapy. DisclosuresNo relevant conflicts of interest to declare.

Pathogenetic and Prognostic Implications of Increased Mitochondrial Content in Multiple Myeloma.

Simple SummaryMonoclonal gammopathies comprise a spectrum of disorders defined by the clonal proliferation of plasma cells and include monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM) and multiple myeloma (MM), which can also evolve from MGUS and SMM. We aimed to analyze the impact of mitochondrial DNA copy number (mtDNACN) and also SNVs and INDELs in frequently mutated mitochondrial-related genes on the disease course of monoclonal gammopathies and MM. We confirmed the increased levels of mtDNA in SMM and MM, their gain as the gammopathy progresses and the similarities in the mitochondrial hallmarks between rapidly-progressing SMM and MM. Our data suggest that mitochondria participate in the malignant transformation of monoclonal gammopathies and contribute to disease progression. Our findings support the clinical importance of mtDNACN evaluation and monitoring to guide clinical decision making in patients with SMM.Many studies over the last 20 years have investigated the role of mitochondrial DNA (mtDNA) alterations in carcinogenesis. However, the status of the mtDNACN in MM and its implication in the pathogenesis of the disease remains unclear. We examined changes in plasma cell mtDNACN across different stages of MM by applying RT-PCR and high-throughput sequencing analysis. We observed a significant increase in the average mtDNACN in myeloma cells compared with healthy plasma cells (157 vs. 40 copies; p = 0.02). We also found an increase in mtDNACN in SMM and newly diagnosed MM (NDMM) paired samples and in consecutive relapses in the same patient. Survival analysis revealed the negative impact of a high mtDNACN in progression-free survival in NDMM (p = 0.005). Additionally, we confirmed the higher expression of mitochondrial biogenesis regulator genes in myeloma cells than in healthy plasma cells and we detected single nucleotide variants in several genes involved in mtDNA replication. Finally, we found that there was molecular similarity between “rapidly-progressing SMM” and MM regarding mtDNACN. Our data provide evidence that malignant transformation of myeloma cells involves the activation of mitochondrial biogenesis, resulting in increased mtDNA levels, and highlights vulnerabilities and potential therapeutic targets in the treatment of MM. Accordingly, mtDNACN tracking might guide clinical decision-making and management of complex entities such as high-risk SMM.

Macrophages in multiple myeloma: key roles and therapeutic strategies.

Macrophages are a vital component of the tumour microenvironment and crucial mediators of tumour progression. In the last decade, significant strides have been made in understanding the crucial functional roles played by macrophages in the development of the plasma cell (PC) malignancy, multiple myeloma (MM). Whilst the interaction between MM PC and stromal cells within the bone marrow (BM) microenvironment has been extensively studied, we are only just starting to appreciate the multifaceted roles played by macrophages in disease progression. Accumulating evidence demonstrates that macrophage infiltration is associated with poor overall survival in MM. Indeed, macrophages influence numerous pathways critical for the initiation and progression of MM, including homing of malignant cells to BM, tumour cell growth and survival, drug resistance, angiogenesis and immune suppression. As such, therapeutic strategies aimed at targeting macrophages within the BM niche have promise in the clinical setting. This review will discuss the functions elicited by macrophages throughout different stages of MM and provide a comprehensive evaluation of potential macrophage-targeted therapies.

Health‐related quality of life of patients with multiple myeloma: A real‐world study in China

PurposeThis study aimed to assess the health‐related quality of life (HRQOL) of Chinese patients with different stages of multiple myeloma (MM) who received various treatments and identify the factors associated with a lower quality of life in China.MethodsA cross‐sectional, anonymous questionnaire was distributed to adults with MM. The measures of quality of life included the European Organization for Research and Treatment of Cancer (EORTC) quality of life questionnaire (QLQ)‐C30, QLQ‐myeloma‐specific module 20 (MY20), and EuroQoL EQ‐5D. The data, including patient factors, difficulties experienced during the diagnosis and treatment processes, psychosocial factors and disease‐ or treatment‐related effects, were collected.ResultsFour hundred and thirty patients with MM were recruited from all 27 provinces of China, and their average age was 55.7 years. Many variables were significantly associated with the HRQOL of the patients with MM. In the multivariate analyses, performance status, psychosocial factors, disease phase, and an early diagnosis were significantly associated with the HRQOL. In the subgroup analysis, the HRQOL of the patients who underwent autologous stem cell transplantation (ASCT) was significantly higher than that of the non‐ASCT patients. Treatment‐related toxicities had a significant impact on the quality of life of the patients with MM, and 91.5% of the patients intended to stop the maintenance treatment.ConclusionsThe quality of life of patients with MM in China is affected by patient factors, difficulties experienced during the diagnosis and treatment processes, psychosocial factors, and disease‐ or treatment‐related effects. Efforts should be exerted to improve the overall quality of life of these patients in China.

Immunoglobulin free light chains and interleukin-6 levels in prediction of kidney injury in patients with multiple myeloma

Background: Multiple myeloma (MM) is a disease of B cell population with excessive secretion of immunoglobulins and presence of free light chains (FLCs) that are by products of immunoglobulin synthesis. Free light chains play crucial role in causing renal damage. Interleukine-6 (IL-6) supports the survival and/or expansion of MM cells by stimulating cells as well as by preventing programmed cell death.
 Aims and Objectives: The aim of this study was to evaluate serum and urine free light chains (FLC)measurement and compare with IL-6 levels in patients with different stages of Multiple Myeloma (MM) and control group of subjects and to determine their relevance in acute kidney injury occurrence.
 Materials and Methods: Recruitment of patients with MM (n=62) made the hematologist that followed clinical Solomon-Durie MM classification. Control group consisted of 20 healthy individuals. Results: Patients with MM and renal function injury had significantly higher concentration of urine κ chains compared to control group and group of MM without renal function injury (p<0.005), whereas this difference was not observed when the patients were divided into disease stages groups. Concentration of IL-6 was significantly higher in patients at MM steady stage compared to control group (p<0.001) and significant difference was also detected in patients with MM at relapse stage and control group (p<0.0005). Concentration of IL-6 in MM patients without renal function and with renal function injury was significantly higher compared to control group (p< 0.001; p<0.0005 respectively). Statistically significant correlation was determined between sera κ and urine κ chains (rho=0.437; p<0.01) as well as between urine λ and sera λ chains (rho=0.505; p<0.01) and between urine κ and urine λ chains (rho=0.364; p<0.01).
 Conclusion: Results showed that urine κ chains, sera κ chains and IL-6 are constructing a fine tuned net and point to conclusion that FLC and IL-6 are important for an early treatment response detection for patients with potentially reversible renal failure.

Diagnostic Value of IGF-I, β2-MG and SF for Patients with Multiple Myeloma and Their Relationship with Clinical Staging

To investigate the diagnostic value of insulin like growth factor I(IGF-I), β2-microglobulin (β2MG) and serum ferritin (SF) in patients with multiple myeloma (MM) and their ralationship with clinical staging. Seventy-seven patients with MM treated in Depertment of Hematology of Shanghai 10th hospital and Oncology of Shanghai Armed Police Hospital from August 2016 to June 2017 were enrolled in MM group, at same period 77 healthy volunteers were enrolled in normal control group. The diagnostic value of IGF-I, β2-MG and SF for MM, and their levels in different stages of MM were compared. The ROC analysis showed that β2-MG and SF alone as well as their combination had the diagnostic significance for MM, moreover the diagnostic value of IGF-I, β2-MG and SF combination was highest, but the single IGF-I did not possess diagnostic significance for MM. The comparison of IGF-I, β2-MG and SF levels in different stages of MM showed that the β2-MG and SF levels in I stage were higher than those in normal control group (P<0.05), but lower than those in II and III stages (P<0.05). The IGF-I level in I stage was not statistically and significantly different from IGF-I level in normal control group (P>0.05), but lower than those in II and III stage (P<0.05). The relationship analysis between IGF-I and β2-MG, SF in different stages showed that the IGF-I related with SF in I stage (r=0.417), but did not relate with β2-MG; the IGF-I in II stage related with β2-MG and SF in II stage (r=0.543, r=0.426); IGF-I related with β2-MG and SF in III stage (r=0.425 and r=0.672). The diagnostic value of IGF-I, β2-MG and SF alone does not high for MM, but their combination can significantly enhance the occurate rate of MM diagnosis. The levels of IGF-I, β2-MG and SF in II and III stages of MM all increase, moreover the level of IGF-I correlates with the levels of β2-MG and SF.

Circulating Adiponectin Levels Differ Between Patients with Multiple Myeloma and its Precursor Disease.

ObjectiveAn increased risk of multiple myeloma (MM) has been observed among individuals with low pre-diagnostic circulating levels of adiponectin, a metabolic hormone that is typically underexpressed among those who are overweight or obese. To assess whether adiponectin may influence myeloma development or progression to frank MM, we compared circulating adiponectin levels across patients with different stages of MM and its precursor, monoclonal gammopathy of undetermined significance (MGUS).MethodsWe measured adiponectin in 213 patients with MGUS, smoldering MM, or fully developed MM. Differences in adiponectin levels across patient groups were assessed using multivariate linear regression.ResultsRelative to MGUS patients, adiponectin levels were statistically significantly lower among those with smoldering and fully developed MM, both overall (16–20% decrease; P=0.048) and among those with IgG/IgA isotypes (26–28% decrease; P=0.004). Among MGUS patients, adiponectin levels were significantly lower for those with the higher risk IgM isotype compared with those who had IgG/IgA isotypes (42% decrease; P=0.036).ConclusionsThe findings of this study, the largest to investigate adiponectin levels in patients with different stages of MM and the first to evaluate associations with clinical characteristics, suggest that reduced expression of adiponectin may be associated with progression from MGUS to MM.

The Low Expression of IL-37 Involved in Multiple Myeloma - Associated Angiogenesis.

BackgroundAngiogenesis plays a significant role in complex inflammatory and angiogenic processes and is also involved in multiple myeloma (MM) pathogenesis. IL-37 is a proinflammatory cytokine in antitumor activity. Our purpose was to evaluate the IL-37 clinical significance on MM.Material/MethodsWe measured serum levels of IL-37 in 45 patients with different stages of MM and 30 healthy control subjects and correlated IL-37 with numerous cytokines, such as angiogenesis factors including vascular endothelial growth factor (VEGF) and angiotensin-2 (Ang-2). We also measured the tube formation of human umbilical vein endothelial cells (HUVECs) after pretreatment with recombinant human IL-37 (rhIL-37).ResultsSerum IL-37 level was lower in the patients with MM than in the healthy control subjects, whereas VEGF and Ang-2 levels were higher, depending on International Staging System stage. Serum IL-37 level had a negative correlation to VEGF and Ang-2 levels, and VEGF had a positive correlation to Ang-2 level. The tube formation of HUVECs was suppressed by the rhIL-37 pretreatment.ConclusionsOur results indicate that serum level of IL-37 plays a part in the pathophysiology of MM progression. Therefore, IL-37 serum level may be a biomarker for disease stage and angiogenesis processes.

A GRP78-Directed Monoclonal Antibody Recaptures Response in Refractory Multiple Myeloma with Extramedullary Involvement.

Glucose-regulated protein (GRP) 78 is overexpressed in multiple myeloma, and both its surface expression and its biologic significance as key sensor of the unfolded protein response make GRP78 an ideal candidate for immunotherapeutic intervention. The monoclonal antibody PAT-SM6 targets surface GRP78 and leads to disease stabilization when used as single agent in a clinical trial. In this article, we evaluated expression of GRP78 in relapsed-refractory disease and explored PAT-SM6 therapy in combination regimens. GRP78 expression was immunohistochemically analyzed during disease progression and development of drug resistance throughout different stages of multiple myeloma. Activity of PAT-SM6 was evaluated in combination with anti-multiple myeloma agents lenalidomide, bortezomib, and dexamethasone in vitro Finally, we report on a multiple myeloma patient with relapsed-refractory disease treated with PAT-SM6 in combination with bortezomib and lenalidomide. Although sGRP78 expression was present at all stages, it increased with disease progression and was even strongly elevated in patients with drug-resistant and extramedullary disease. Pretreatment with dexamethasone as well as dual combination of PAT-SM6/lenalidomide further increased sGRP78 expression and consecutively showed synergistic anti-multiple myeloma effects with PAT-SM6 in proliferation assays. As proof of concept, a 62-year-old male with triple resistant multiple myeloma treated with PAT-SM6, bortezomib, and lenalidomide experienced partial remission of both intra- and extramedullary lesions. PAT-SM6 therapy in combination regimens showed efficacy in relapsed-refractory multiple myeloma. Clin Cancer Res; 22(17); 4341-9. ©2016 AACR.

The influence of baseline characteristics and disease stage on health-related quality of life in multiple myeloma: findings from six randomized controlled trials.

This descriptive, cross‐sectional analysis evaluated the impact of baseline characteristics on health‐related quality of life (HR‐QoL) at different stages of multiple myeloma (MM). The bortezomib clinical‐trial programme evaluated HR‐QoL early and consistently, producing a large multi‐study dataset. Baseline data, captured using the European Organization for Research and Treatment of Cancer (EORTC) quality‐of‐life questionnaire (QLQ‐C30), were pooled from six bortezomib randomized trials conducted in different disease‐stage categories: ‘New’ (previously untreated; n = 753), ‘Early’ (1–3 prior therapies; n = 1569) and ‘Late’ (≥4 prior therapies; n = 239) disease. Mean EORTC global health scores were similar across the three stages. Unexpectedly, emotional, physical and role functioning were higher in the later stages, indicating better perceived health. Symptom scores, including pain, were largely similar or lower in the later versus earlier stages, signifying a lower symptom burden/better symptom control with more advanced disease. Notable variation in HR‐QoL was observed by age and clinical parameters within and across stages. Multivariate modelling indicated that opioid use and performance status were key factors driving overall HR‐QoL across stages. Using an age‐restricted analysis, transplant eligibility had little impact on HR‐QoL in New disease patients. Thus, changes in HR‐QoL over the treatment course of MM are complex and impacted by baseline factors. A prospective observational international inception cohort study that captures key clinical, HR‐QoL and demographic characteristics, along with safety and supportive care information, is needed.

Clinical and biological characteristics multiple myeloma with 1q21 amplification

To study the clinical features of multiple myeloma (MM) patients with 1q21 amplification and the detection and biological characteristics of 1q21 copy number variation among different stages of plasma cell dyscrasias. We analyzed the amplification and copy number variation of 1q21 in a cohort of 397 MM patients (290 newly diagnosed patients and 107 relapsed/refractory patients) in Institute of Hematology and Blood Diseases Hospital in the period between January 2009 and December 2012, using fluorescence in situ hybridization (FISH). We compared the incidence and biological characteristics of 1q21 gains among different stages of MM. Among the newly diagnosed MM patients, the cases without 1q21 gains (148 cases) had difference prevalence of q13 deletion (38.3% (56/146) vs 57.7% (82/142), P=0.001), t(4; 14) translocation (17.4% (25/144) vs 30.7% (42/137), P=0.009), and high-risk cytogenetic abnormalities (28.3% (39/138) vs 41.9% (57/136), P=0.018), and International Staging System (ISS) stage (P=0.010) compared to those with 1q21 gains (142 cases); however, there were no significant differences in age(P=0.448), Durie-Salmon clinical stage (P=0.352) and β2-microglobulin level (P=0.414). In the newly diagnosed patients, the incidence of this 1q21 aberration with the percentages of plasma cells involved being ≥10%, ≥20%, and ≥30% was 52.4% (152/290), 49.0% (142/290), and 46.2% (134/290), respectively, lower than that in the relapsed/refractory patients (71.0% (76/107), P=0.001; 68.2% (73/107), P=0.001; 63.6% (68/107), P=0.002). Differences were found between the newly diagnosed MM patients and the relapsed/refractory ones in terms of the incidence of 1q21 copy number gains being 2 (52.2% (145/278) vs 33.3% (34/102), P=0.001), but not in the incidence of 1q21 copy number gains being 3, 4, and >5 (all P>0.05). Amplification of chromosome 1q21 is a common genetic abnormality in MM patients. The copy number varies in patients carrying 1q21 gains, mainly with two or more copies of 1q21. It may therefore be recommended to include testing for 1q21 gains in routine genetic testing of MM patients.

Mimicking Myeloma Niche Ex Vivo

Introduction: Recent studies have elucidated the importance of using 3-dimensional rather than 2-dimensional models in order to create an experimental system recapitulating the specialized properties of the bone marrow microenvironment. Since the neoplastic bone marrow (BM) milieu plays important roles in multiple myeloma (MM) pathogenesis, novel models to study the MM cell in its neoplastic microenvironment are needed.Methods: To mimic the neoplastic BM microenvironment of MM patients, we have established a special hydrogel-based 3-dimensional (3-D) model by ex-vivo culturing MM patient-derived mesenchymal stem cells (MM-MSCs), the predominant cellular component of the marrow niche, which promotes greater mineralization and differentiation than a 2-dimensional (2-D) system.Results: To characterize MM-MSCs in different stages of MM, we utilized an 11 multi-color flow cytometry panel. The percentage of MSCs (CD73+CD90+CD105+lin-CD45-CD34-HLA-DR-) population in BM aspirate samples of 50 MM patients (MGUS, smoldering MM, newly diagnosed MM, and relapsed or relapsed/refractory MM) was evaluated, and correlated with the distribution of (CD38+ CD138+) plasma cells. MSCs were less frequent (10x) than plasma cells, and increased with disease progression to relapsed/refractory MM. We seeded MM-MSCs (N=34) which had been expanded by adhesion methods in 2-D versus 3-D models in order to create an ex-vivo MM niche-like structure. In the hydrogel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections and meshwork-like structures at day 3 to 7. Moreover, calcium mineralization of clusters was observed, associated with the capacity for differentiation towards the osteoblastogenic or adipogenic lineage when cultured with differentiation media. Furthermore, the production of osteopontin (OPN) and angiopoietin-2 (Ang-2) was significantly higher in 3-D vs. 2-D MM-MSCs, assessed by multiplex luminex technology. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of CD73+CD90+CD105+ and lack of expression of CD45, CD34 and HLA-DR, as in to 2-D MM-MSCs. MSC-specific markers including CD166 and HLA-ABC did not reveal any significant changes in 3-D vs. 2-D MM-MSCs; however, 3-D MM-MSCs had significantly decreased expression of CD271 and CD146 compared to 2-D cultures. We also observed significantly higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p<0.001) in 3-D vs. 2-D MM-MSCs. Similarly, activation of integrins including VLA-2, VLA-4 and VLA-5 on the MSCs surface was also increased in 3-D MM-MSCs, as determined by confocal microscopy and flow cytometry analysis. Importantly, MM-MSCs cultured in 3-D vs. 2-D model have higher expression of N-cadherin and CXCL12 and decreased expression of nestin, reflecting the MM BM niche. Gene expression analyses of 3-D MM-MSCs revealed upregulation of BMP-2, MGP, PTGIS, COL14A1 and other genes and down-regulation of DKK1, ADAM9, OPCML genes and others compared to 2-D MM-MSCs. We also measured significantly higher production of IL-6 (p=0.002), IL-8, MCP-1(MCAF), RANTES, VEGF and HGF (p<0.001) in 3-D vs. 2-D MM-MSCs, by multiplex luminex analysis. Next, we co-cultured tumor cells from MM patients (12 MM patients) with either autologous or allogeneic MM-MSCs in 3-D vs. 2-D model. Plasma (CD38/CD138+) cells in 3-D co-culture were increased in 8/12 MM patients and equivalent to 2-D in 4/12 patients. By co-culturing MM cell lines (OPM1, RPMI-S, OCIMY5 and KMS11) labeled with CFSE fluorescent dye with various MSCs, we evaluated expression of side population (SP) cells, identified by Hoechst staining, and gating on CFSE positive MM cells, as low Hoechst stained cells. Our results showed that the SP fraction was significantly lower in 3-D compared to 2-D in co-cultures of various MM-MSCs with all 4 MM cell lines. Finally, we validated drug resistance to melphalan, bortezomib, lenalidomide, and carfilzomib in 3-D co-cultures of CFSE labeled primary tumor cells with various MM-MSCs.Conclusions: This 3-D co-culture system closely mimics the myeloma BM niche, and therefore may be useful to identify and validate novel targeted therapies. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Immune Profile in Patients with Multiple Myeloma Using Cytof Technology

Introduction: CyTOF (time-of-flight mass cytometry) is a novel high-dimensional technology which permits immunophenotyping and analysis of signaling in single cells. This approach enables simultaneous evaluation of up to 40 parameters using antibodies tagged with distinct elemental isotopes, by combining flow cytometry with atomic mass spectrometry. Since multiple myeloma (MM) is characterized by immune dysfunction, we used CyTOF technology to define the complex immune profile in MM patient bone marrow (BM) samples.Methods: We used 40 different markers to define various B, T, natural killer (NK) subsets, as well as cells of monocytic, granulocytic, erythroid and platelet lineages. Our preliminary data are results from 10 patients with MGUS/ smoldering MM (SMM); 10 newly diagnosed MM; 20 relapsed/refractory MM; and 15 WM patients (5 newly diagnosed and 10 receiving treatment) in comparison with age-matched healthy donors’ BM (HD). A significantly larger cohort of MM (N=150) and WM (N=50) patients is being similarly analyzed and will be presented. To evaluate phenotypic abnormalities in various B cell subsets, we used B lineage markers CD10, CD19, CD20, CD22, CD27, CD34, CD38, CD45, IgA, IgD, IgG and IgM to define B cells maturation stages from hematopoietic stem cells (HSC) to naïve to mature B lymphocytes (pro-B; pre-B-I; pre-B-II; immature B; and mature (naïve) B cells), as well as memory non-switched and memory switched B cells, plasmablasts, normal (CD138+CD38+CD19+CD45+) and clonal plasma cells (CD138+CD38+CD19-CD45-/low), which reside in the specific BM niche. Furthermore, natural killer (NK) subsets (such as NK and NKT cells) and T cells (such as memory CD4T, naive CD4T, memory CD8T, naive CD8T, T regulatory cells and Tg/d cells) were examined. High-dimensional data was obtained using CyTOF technology and analyzed by SPADE and viSNE software.Results: Our data showed significantly decreased HSC in patients with newly diagnosed and relapsed/refractory MM compared to HD (P<0.025). A significant increase in pre-B-I cells was detected in relapsed/refractory MM vs. MGUS/SMM (P<0.028), but the opposite trend was observed in the pre-B-II subpopulation (P<0.005). No differences in immature B cell populations were observed in different stages of MM. However, a significantly higher percentage of immature B cells was present in relapsed/refractory MM compared to HD (P=0.008), and transitional B cells were significantly decreased in newly diagnosed MM compared to HD (P<0.001). Moreover, memory B cells were significant decreased in all MM stages compared to HD (P<0.003). Non-switched memory B cells were significantly increased in MGUS and SMM compared to newly diagnosed MM, while a significant increase of switched memory B cells was present in newly diagnosed MM compared to relapsed/refractory MM. A significant increase in plasmablasts was seen in relapsed/refractory MM in comparison with other MM stages (P<0.011) by CyTOF analyses. Malignant plasma cells (PC) were defined as CD19-, CD38++, CD45-/dim, CD138+ and either cyk or cyl positive. Importantly, a significant increase in clonal PC was found in all MM stages vs. HD, as well as in newly diagnosed MM compared to relapsed/refractory MM (P<0.01). The percentage of PC from CyTOF analyses correlated with % of PC obtained using flow cytometry by Bland-Altman method comparison. We also observed significant differences in T cell subsets including naïve, central memory, effector, and effector memory CD4+ and CD8+T populations between MGUS and newly diagnosed MM, but no significant changes in T regulatory and Tg/d cells. Furthermore, plasmacytoid dendritic (pDC) cells were significantly increased in newly diagnosed MM, and PD-1 expressed on pDC was significantly decreased in newly diagnosed MM compared to MGUS (P=0.007). Interestingly, PD-1 and its ligand PD-L1 were variably expressed on B cells (2-9% and 3-27%) and PC (0.5-46% and 3-41%) from MM BM samples. Other surface molecules including CD269 (4-32%), CD289 (1-8%), CD362 (0.5-1%) and CD329 (1-4%), were variably expressed in PC.Conclusion: A better understanding of the neoplastic BM milieu will provide the framework for identifying and validating novel targeted therapies directed against MM. CyTOF technology represents a novel diagnostic tool to assess the status not only of MM, but also of host immunity, and may allow for the development of rational personalized therapies. DisclosuresNo relevant conflicts of interest to declare.

Analysis of the serum levels of selected biological parameters in monoclonal gammopathy of undetermined significance and different stages of multiple myeloma

Protein kinase C was found to be significantly over-expressed in cancer samples compared to adjacent normal cervical tissues by proteomics in our previous study. The aim of this study was to examine protein kinase C expression and to analyze the expression patterns of protein kinase C isoforms in squamous cervical cancer at the protein levels and their associations with clinical and pathologic factors of squamous cervical cancer. First, Western blotting was used to examine protein kinase C expression in the specimens of tumors and matched adjacent normal tissues which were collected from 12 patients with squamous cervical cancer. Protein kinase C isoforms (α, δ, θ and ζ) expression were then detected by immunohistochemistry in other 43 cases of squamous cervical cancer tissues, 32 cases of corresponding adjacent normal cervical squamous epithelial tissue and 31 cases of cervical intraepithelial neoplasia. Western blot analysis revealed that protein kinase C expression was positive in squamous cervical cancer while it was not expressed in normal cervical tissues. On the other hand, immunohistochemical analysis suggested that, protein kinase C isoforms (α, δ, θ and ζ) expression was significantly higher in squamous cervical cancer compared to cervical intraepithelial neoplasia, as well as in cervical intraepithelial neoplasia compared with normal tissues, respectively.High levels of protein kinase C α expression were associated with cellular differentiation(P<0.05). Protein kinase C δ was significantly associated with tumor stage (P<0.05) and protein kinase C ζ was associated with lymphatic metastasis (P < 0.05). These findings indicate that protein kinase C isoforms expression in cervical lesions was associated with carcinogenesis and might play important roles throughout the process of cervical cancer development.

The Utility of the New Instrument for Comprehensive Symptom Profile Assessment In Patients with Multiple Myeloma

AbstractAbstract 4757Increased importance should be placed on the comprehensive symptom assessment in patients with multiple myeloma to accurately document the broad range of physical and psychological disease manifestations given from the patient perspective. The new developed symptom assessment tool - Comprehensive Symptom Profile in Multiple Myeloma Patients (CSP-MM) aims to provide an in-depth view of patient's problems. It is currently undergoing content validation through a structured, iterative process that conforms to the FDA industry guidance on the use of patient-reported outcome measures. We aimed to evaluate the utility of the new instrument providing evidence of its content validity.Pilot sample of twenty two patients with different stages of multiple myeloma were included in this qualitative research study. Patients underwent either conventional chemotherapy or autologous stem cell transplantation. Mean age was 58 years old; male/female distribution –11/11. The CSP-MM is a self-reported tool which consists of 51numeric rating scales (where “0” - no symptom, “10” - most expressed symptom). Evidence of adequate tool content was collected from multiple sources: literature review, pilot CSP-MM completion, cognitive interviews with patients and clinicians, expert evaluation, and data quality control. Patients filled out the CSP-MM before and at different time-points of treatment.The utility of the CSP-MM was demonstrated. After the completion of the pilot, patients were asked about their overall assessment of the questionnaire. Open-ended patient interviews provided a full understanding of the patient's perspective and showed that saturation of the items has been reached. The patients acknowledged the comprehensiveness of the tool. All of the items were easy for the patients to read and understand. Total number of completed surveys was 79 with only 1.6% missing items throughout all forms. Completion of the CSP-MM in paper and pencil format took 7–9 min. The data produced by the tool were clear for interpretation by physicians. According to the clinicians' interviews, changes in symptom severity captured by the tool at different time-points of treatment gave the physicians information about patient experience and were used by them in day-to-day decision making.Thus, the CSP-MM is an appropriate and practical tool to assess the symptom severity in myeloma patients. The utility of the questionnaire was shown. Pending further validation, the CSP-MM can be used to assess symptoms in MM patients and the patient benefit from the treatment. Disclosures:No relevant conflicts of interest to declare.

Regulation of Multiple Myeloma Survival and Progression by CD1d

Downregulation of conventional HLA molecules from the surface of tumour cells is an important mechanism for tumour immune evasion, survival and progression. Whether CD1d, a non-conventional, glycolipid-presenting HLA class I-like molecule can affect tumour cell survival is not known. To test this we studied expression of surface CD1d on plasma cells from different stages of multiple myeloma (MM) using flow-cytometry. Expressing results as the ratio of the Geo MFI CD1d/isotype IgG1 we found that while CD1d expression was comparable between MGUS (n=8) and newly diagnosed MM patients (n=14; Geo MFI MGUS: 8.61±4.3 vs new MM: 7.1±4.72, p>0.05), in relapsed/advanced disease CD1d was significantly lower (Geo MFI:1.92±0.9, p<0.003 vs MGUS and new MM) and completely lost in 4 out of 5 myeloma cell lines at protein and RNA level. Further, 4 out of 8 paired, same-patient trephine biopsies stained with anti- CD1d showed drastic loss of CD1d expression in advanced compared to early disease. These results confirmed loss of CD1d expression during disease progression and suggested that CD1d impacts negatively on myeloma cell survival. Consistent with this, we found that engagement of CD1d by 2 different anti-CD1d mAbs and as compared to isotypic IgG or media control, induces cell death (i.e., Annexin+) of the CD1d-expressing B lymphoblastoid cell line C1R-CD1d, of myeloma cell lines with retrovirally restored expression of CD1d and purified, CD1d-expressing primary myeloma cells in a dose- and time-dependent manner, coincident with loss of mitochondrial membrane potential (MMP) as assessed by DioC3 staining. Biochemical analysis of relevant cell death pathways showed that MMP loss is associated with overexpression of the pro-apoptotic protein Bax but as demonstrated by immunoblotting and pharmacological inhibition it is caspase- independent. By introducing appropriate CD1d retroviral constructs into CD1d- myeloma cell lines we showed that anti-CD1d-induced cell death requires the cytoplasmic tail but not a Tyr residue critical for lysosomal sorting of CD1d. Finally, we found that anti-CD1d co-operates with anti-myeloma agents in the killing of myeloma cells. Thus, these findings provide evidence linking a novel function of CD1d in the regulation of cell death with tumour survival and progression and might have pathogenetic and therapeutic implications for other CD1d-expressing hematopoietic malignancies as well as myeloma.

Cancer: Stress Link Redefined

As the battle against cancer continues, doctors, researchers, and patients continue to investigate new avenues for prevention and treatment. But the struggle isn’t getting any simpler. Researchers in a handful of laboratories have recently documented that, among many other triggers and promoters, stress hormones are a direct contributing factor in the progression of ovarian and nasopharyngeal cancer. Now a team from The Ohio State University has discovered a similar link with a third cancer, multiple myeloma. Multiple myeloma, which involves lymphoid tumors, kills more than 10,000 Americans annually, usually within 3 to 4 years of diagnosis. Ovarian and nasopharyngeal cancers are characterized by solid epithelial tumors and result in about 15,000 and 650 U.S. deaths per year, respectively. Until recently, stress hormones were thought to simply weaken the immune system and impair the body’s ability to fight cancer. Instead, the new evidence is showing that stress hormones such as norepinephrine, epinephrine, and cortisol, whether induced by psychological stimuli or environmental agents such as cold temperatures or infectious diseases, may play a much different role in cancer progression than generally presumed. The Ohio State researchers began their study, published online 5 November 2007 ahead of print in Brain, Behavior, and Immunity, with a few givens: multiple myeloma has been linked with increased growth of supporting blood vessels, and vascular endothelial growth factor (VEGF) has been shown to play a role in that vessel growth. The team evaluated 3 human cell lines from 3 patients, representing different stages of multiple myeloma, to see if either of 2 doses of norepinephrine played a role in the expression of VEGF in the cell lines. Based on their assessment of selected adrenergic receptors in the cell lines, they found that norepinephrine stimulated all 3 cell lines. The FLAM-76 cell line taken from bone marrow, which represented the earliest stage of multiple myeloma, showed the most significant stimulation. Responses in all 3 cell lines were dose- and time-dependent, but there were no distinct patterns. Depending on the circumstances, either the higher or lower dose induced the most stimulation or suppression, and there were large differences over 1-, 3-, 6-, and 24-hour periods. Eric Yang, lead author of the study and a research scientist at The Ohio State University Institute for Behavioral Medicine Research, points out that the effect on multiple myeloma of chronic stress from single or multiple sources is unknown. In addition, he says that the lowest concentration of norepinephrine tested was 10 times higher than levels typically detected in stressed humans. However, he says the team’s attempts to test at more realistic concentrations were stymied by erratic results that were difficult to interpret. But analogous research on ovarian cancer has shown similar effects at realistic doses. Anil Sood, a professor of gynecologic oncology and cancer biology at the University of Texas M.D. Anderson Cancer Center, says that for stress and cancer the effects of other variables such as age, sex, race, ethnicity, and genetic makeup also remain a puzzle, and that there is little information on how best to manage or treat chronic stress in order to reduce its potential effects on tumor growth. Another concern is that the cell lines were grown in a tissue culture that can’t represent what actually occurs in the body, says Bruce Rabin, a professor of pathology, psychiatry, and psychology at the University of Pittsburgh School of Medicine. However, Rabin says the evidence from the experiments is a good first indication of possible effects of stress hormones on multiple myeloma.