Bone Marrow Of Multiple Myeloma Patients Research Articles

CD84: A Potential Novel Therapeutic Target in Multiple Myeloma

Introduction: Multiple Myeloma (MM) cell survival strictly depends on the interaction with multiple cell types in the bone marrow (BM), collectively referred to as the MM microenvironment (MM-ME). CD84 (SLAMF5) belongs to the signaling lymphocyte activation molecule family of immunoreceptors; previous data from our group have shown that CD84 mediates malignant B cells and their ME (Marom et al. 2017); however, its role within the MM-ME has not yet been investigated.Results: Using the MMRF CoMMpass IA9 dataset, we found that CD84 mRNA expression is low or absent in CD138+MM cells isolated from 660 newly diagnosed patients. In agreement with these data, flow analysis showed an absence of CD84 expression in all MM cell lines tested (n=9) and minimal surface expression (5.5-13%) in primary cells (n=3). However, a significantly higher expression of CD84 was detected on the surface of BM and peripheral blood (PB) monocytic fractions (CD14+) (76-97%) compared to that of matched CD14 negative fractions (2-9%) obtained from 16 different MM patients (p<0.001). Since it was recently reported that CD14+ monocytic myeloid derived suppressor cells (Mo-MDSCs) play a pivotal role in supporting MM growth by creating an immunosuppressive ME, we investigated CD84 expression in this population. Using primary PB (n=9) and BM (n=3) samples obtained from MM patients with active disease, we found that the Mo-MDSC population is significantly over-represented (0.34-2.88%) in MM patients compared to the PB from healthy donors (n=7) (0.04-0.45%) (p<0.05) and that CD84 is also significantly up-regulated in this population in MM patients (43-92%) compared to healthy donors (7-21%) (p<0.001).Next, to understand whether the expansion of Mo-MDSC and associated CD84 up-regulation is directly dependent on the MM cells, we investigated Mo-MDSC expansion and CD84 expression in two different MM mouse models. Specifically, 5TGM1 murine MM cells were IV injected into syngeneic immune-competent KaLwRijHsd mice (n=7), and human MM.1S cells were IV injected into immune-deficient NSG mice (n=10). Our data show that MM progression leads to significant Mo-MDSC expansion (p<0.0001) and a subsequent increase in CD84 levels (p<0.05) on this population in both mouse models.Since T cell checkpoint inhibitors PD-1/PD-L1 are tumor immune escape receptors that play a pivotal role in supporting an immunosuppressive MM-ME, we also investigated the expression of PD-L1 on the CD14+ fraction in MM patients. BM-CD14+ cells (n=3) were compared with matched CD14 negative cells. We found that the CD14+ fraction had a higher PD-L1 expression in the BM of MM patients compared to the CD14 negative fraction (60-96% versus 3-8%). Hence, we investigated whether CD84-mediated cell-cell contact may increase the level of PD-L1 expression on Mo-MDSCs and whether MM cells can regulate CD84 and PD-L1 expression on CD14+ cells. Co-culture assays were performed using several human MM cell lines (MM1S, U266 and KMS11) with PB CD14+ cells derived from healthy donors (n=3). Our data show a 1.9-fold increase from the basal level of CD84 (27±16% to 50±9.4%) and a 2.9-fold increase from the basal level of PD-L1 (18±8.5% to 52.3±5.7%).Our recently generated mouse anti-human CD84 blocking antibody (Marom et al. 2017) was used to determine whether CD84 direct targeting can affect MM-induced CD84 and PD-L1 expression on the surface of CD14+cells. Co-culture experiments show that targeting CD84 lowered CD84 (1.6-fold decrease) and PD-L1 expression (1.8-fold decrease) on a CD14+ population co-cultured with MM cells. Moreover, when T cells were included in the co-culture experiments, we observed reduced expression of exhaustion markers PD-1 (1.1-fold decrease) and CTLA4 (1.4-fold decrease) in the presence of the CD84 blocking antibody.Conclusion: We show here that CD84 is expressed on Mo-MDSCs in MM and that the expansion of this myeloid population is strictly associated with MM progression. Disruption of CD84 contacts with a blocking antibody decreases the expression of PD-L1 on CD14+ cells and exhaustion markers on T cells. Thus, our results reveal a novel role for CD84 in regulating PD-L1 in the MM-ME and provides the scientific rationale to investigate whether targeting CD84 expression on Mo-MDSCs can restore T cell functions. To further confirm the role of CD84 regulating T cell activity and Mo-MDSC, in vivo mouse studies are ongoing and results will be presented at the meeting. DisclosuresRosenzweig:Celgene: Speakers Bureau. Krishnan:Sutro: Speakers Bureau; Onyx: Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Equity Ownership, Speakers Bureau; Takeda: Speakers Bureau.

Detailed Phenotypic, Molecular and Functional Profiling of Myeloid Derived Suppressor Cells (MDSCs) in the Tumor Immune Microenvironment (TIME) of Multiple Myeloma (MM)

Detailed Phenotypic, Molecular and Functional Profiling of Myeloid Derived Suppressor Cells (MDSCs) in the Tumor Immune Microenvironment (TIME) of Multiple Myeloma (MM)

Increased activated regulatory T cell subsets and aging Treg-like cells in multiple myeloma and monoclonal gammopathy of undetermined significance: a case control study

BackgroundAccumulating evidence have indicated that regulatory T cells (Tregs) play an essential role in T cell-mediated immune response and development of multiple myeloma (MM). CD4+FoxP3+ T cells are composed of three phenotypically and functionally distinct subpopulations: CD45RA+FoxP3lo resting Tregs (rTregs), CD45RA−FoxP3hi activated Tregs (aTregs) and CD45RA−FoxP3lo non-suppressive T cells (non-Tregs). We aimed to clarify the frequency and function of these three subpopulations in newly diagnosed multiple myeloma and monoclonal gammopathy of undetermined significance (MGUS) patients. In addition, CD28−CD4+FoxP3+ Treg-like cell is a senescent regulatory T cell subset with partial suppressive function, which could be impaired during myelomagenesis.Methodswe examined 20 patients with MGUS, 26 patients with newly diagnosed MM and 18 healthy volunteers. Flow cytometric analysis in peripheral blood and bone marrow was performed for frequency study. The immunosuppressive function of Treg subsets was assessed by their ability to suppress the proliferation of responder cells in co-culture. Concentration of cytokine from the culture supernatants of proliferation assay was measured using ELISA.ResultsThe proportion of activated Tregs in CD4+ T cells was significantly higher in MGUS and MM patients than healthy controls (P = 0.01, P < 0.001) in both PB and BM; while the proportion of rTregs in MGUS, MM patients was significantly lower than that of controls (P = 0.02, P < 0.01) only in BM. There was no significant difference in frequencies of non-Tregs from MGUS to MM patients with normal controls (P = 0.14, P = 0.88). Significant increase in PB and BM Treg-like cells was observed in MGUS and MM cohort compared with healthy controls (P < 0.01, P < 0.01). Treg-like cells in MM patients were significantly higher than those in MGUS patients (P < 0.01). The inhibition rate of aTreg in bone marrow of MM patients was significantly higher than that of rTreg (P < 0.01), while the inhibition rate of non-Treg was significantly lower than that of rTreg cells (P < 0.01). Functional assays revealed the suppressive and secretory abilities of three Treg subsets were intact in MM patients.ConclusionsIn summary, aTregs and aging Treg-like cells were quantitatively altered in MGUS and MM patients, which might be associated with disease progression and prognosis.

Bone marrow endothelial cells sustain a tumor-specific CD8+ T cell subset with suppressive function in myeloma patients

ABSTRACT Endothelial cells (EC) line the bone marrow microvasculature and are in close contact with CD8+ T cells that come and go across the permeable capillaries. Because of these intimate interactions, we investigated the capacity of EC to act as antigen-presenting cells (APC) and modulate CD8+ T cell activation and proliferation in bone marrow of patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance. We found that EC from MM patients show a phenotype of semi-professional APC given that they express low levels of the co-stimulatory molecules CD40, CD80 and CD86, and of the inducible co-stimulator ligand (ICOSL). In addition, they do not undergo the strong switch from immunoproteasome to standard proteasome subunit expression which is typical of mature professional APC such as dendritic cells. EC can trap and present antigen to CD8+ T cells, stimulating a central memory CD8+ T cell population that expresses Foxp3 and produces high amounts of IL-10 and TGF-β. Another CD8+ T cell population is stimulated by professional APC, produces IFN-γ, and exerts antitumor activity. Thus, two distinct CD8+ T cell populations coexist in the bone marrow of MM patients: the first population is sustained by EC, expresses Foxp3, produces IL-10 and TGF-β, and exerts pro-tumor activity by negatively regulating the second population. This study adds new insight into the role that EC play in MM biology and describes an additional immune regulatory mechanism that inhibits the development of antitumor immunity and may impair the success of cancer immunotherapy.

The expression and role of miR-181a in multiple myeloma.

This study aims to investigate the role of miR-181a in multiple myeloma (MM). Fresh peripheral blood and bone marrows were collected. Expression of miR-181a, BCL-2 mRNA, and NOVA1 mRNA was detected by RT-qPCR. The correlation between miR-181a and clinical features of MM was further analyzed. miR-181a in serum and bone marrow mononuclear cells of MM patients were significantly higher. And, miR-181a level was significantly higher in MM Durie–Salmon stage III than that in stage I+II. miR-181a was positively correlated to Durie–Salmon staging, age, kidney injury, bone injury, β2-MG whereas negatively related to red blood cell, hemoglobin, and albumin. Additionally, BCL-2 and NOVA1 were predicted to be downstream targets of miR-181a. BCL-2 mRNA was significantly higher in the bone marrow mononuclear cells from MM patients. To sum up, the miR-181a expression is increased in peripheral blood and bone marrow of MM patients and is closely related to the clinical pathological indicators of MM.

Advances in diagnosis and treatment of multiple myeloma with extramedullary disease

Multiple myeloma (MM) with extramedullary disease (EMD) is defined as the infiltration of plasma cells outside the bone marrow in MM patients. The new EMD factor for prognosis of MM patients is added into the DS (Durie-Salmon)-PLUS staging system. It is recommended that whole body MRI and positron emission tomography (PET)/CT should be used to evaluate initial status and treatment efficacy in MM patients with EMD in the clinical. MM patients with EMD, including new diagnosed and relapsed MM patients are associated with adverse prognosis. Prognosis of the MM with EMD is poor, and the median overall survival (OS) of patients with recurrence is less than 6 months. MM with EMD is a high-risk myeloma and should be treated aggressively. In order to enhance the understanding of the diagnosis and treatment in MM patients with EMD, this article summarizes the clinical characteristics, diagnosis and treatment of the MM patients with EMD. Key words: Multiple myeloma; Diagnosis; Therapeutics; Prognosis; Extramedullary disease

MORPHOLOGICAL FEATURES OF TUMORS SUBSTRATE IN MULTIPLE MYELOMA PATIENTS COMPLICATED WITH PLASMACYTOMA

Objective. To study the histological structure of bone marrow (BM) and plasmacytoma tumor substrate in patients with multiple myeloma (MM). Materials and methods. The study included 35 patients (19 men and 16 women) aged 23 to 73 years with newly diagnosed MM. At the first onset of the disease plasmacytoma was diagnosed in 21 patients: bone plasmacytoma, associated with skeletal bones – in 14 patients; extramedullary plasmacytoma, emerging in various organs not connected with bone tissue – in 7 patients. All patients underwent BM trephine biopsy and plasmacytoma biopsy with subsequent histological examination. BM and plasmacytoma histological specimens were studied using LEICA DM4000B microscope. Frequency domain analysis (cross tables, Fisher–Freeman test) was used for data statistical analysis.Results. The analysis showed that the histological features of BM in MM patients with extramedullary plasmacytoma statistically significantly differed from that in MM patients with bone plasmacytoma and without plasmacytoma. As a result of the analysis, the relationship between BM morphological variant and tumor advancement became apparent. When comparing the morphological pattern of the bone and extramedullary plasmacytomas, no significant differences were found, however, the substrate of the extramedullary plasmacytoma was more often represented by tumor cells with immature morphology as compared to the substrate of the bone plasmacytoma.Conclusion. The established differences in the histological structure of the BM in MM patients with extramedullary plasmacytoma suggest that this type of the disease stands apart and requires further detailed pathomorphological study.

Abstract 5324: Intragenic DNA-hypomethylation promotes overexpression of ITGB7 in MF subgroup of multiple myeloma

Abstract Integrin-β7 is part of an extensive family of glycoproteins and is overexpressed in the MF subgroups in Multiple Myeloma (MM), contributing to drug resistance and poor survival. ITGB7 promotes adhesion and proliferation of myeloma cells in bone-marrow stroma by supplementing growth factors like VEGF. Here, we investigate the possible epigenetic mechanism of ITGB7 overexpression in the MF subgroup, comprising of t(14;16) and t(14;20) myeloma. Newly diagnosed MM (NDMM) patient bone marrow aspirates underwent CD138 cell selection to enrich tumor cells to &amp;gt;98%. Samples consisted of those with a t(14;16) (n=17), t(14;20) (n=7), t(4;14) (n=9), t(11;14) (n=10) and hyperdiploidy (n=19, separated into D1 (n=12) or D2 (n=7 subgroups). NDMM patient samples were compared to plasma cells isolated from age-matched healthy donors (n=4), to determine differential changes in epitranscripts. We performed unbiased genome-wide reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs) in CpG islands, which were validated by Infinium MethylationEPIC arrays (Illumina) in conjunction with gene expression array data (U133 Plus 2.0, Affymetrix) to determine the epitranscriptomic profile in all samples. RRBS was performed using 75 bp reads to a minimum of 20 M reads per sample. Data from MM cells at the Blueprint consortium were also used for annotation of epigenetic marks. Using the RRBS data we identified 26 hypomethylated, overexpressed genes in the MF group samples, which were not present in the other subgroups. In comparison, only 1 gene, FUT7, was hypermethylated and down-regulated in the MF cluster. Most interestingly, we identified ITGB7 amongst the hypomethylated, overexpressed genes. With an in-depth analysis we identified 4 significant (p&amp;lt;0.05) DMRs across intragenic regions of ITGB7, within a 2.5 kb region. Mean methylation across the DMRs reduced from 58% to 22% (p&amp;lt;0.01) in t(14;16) and 39% (p=0.02) in t(14;20) with concomitant overexpression of ITGB7 in the t(14;16), 19.5-fold increase, and the t(14;20), 23.4-fold increase, subgroups. These 4 DMRs belong to CpG islands, annotated to be the part of possible intragenic enhancer site with promoter like activity, and enriched for open chromatin structures. The DMRs also align with the hotspot of H3K4me1 marks and a putative binding site of activating transcription factors such as AP1/2 or Sp1. This suggests that the identified DMRs could be within the enhancer that regulates ITGB7 overexpression through DNA-hypomethylation. ITGB7 is a known oncogenic factor in high-risk MM, contributing to cell adhesion, migration and homing. Here we show using combined DNA methylation and expression data that ITGB7 is regulated through hypomethylation of the an enhancer region in the MF subgroup in MM. Citation Format: Samrat Roy Choudhury, Cody Ashby, Ruslana Tytarenko, Yan Wang, Purvi H. Patel, Aneta Mikulasova, Michael Bauer, Shayu Deshpande, Faith E. Davies, Gareth J. Morgan, Brian A. Walker. Intragenic DNA-hypomethylation promotes overexpression of ITGB7 in MF subgroup of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5324.

Vγ9Vδ2 T Cells in the Bone Marrow of Myeloma Patients: A Paradigm of Microenvironment-Induced Immune Suppression

Vγ9Vδ2 T cells are non-conventional T cells with a natural inclination to recognize and kill cancer cells. Malignant B cells, including myeloma cells, are privileged targets of Vγ9Vδ2 T cells in vitro. However, this inclination is often lost in vivo due to multiple mechanisms mediated by tumor cells and local microenvironment. Multiple myeloma (MM) is a paradigm disease in which antitumor immunity is selectively impaired at the tumor site. By interrogating the immune reactivity of bone marrow (BM) Vγ9Vδ2 T cells to phosphoantigens, we have revealed a very early and long-lasting impairment of Vγ9Vδ2 T-cell immune functions which is already detectable in monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even in clinical remission after autologous stem cell transplantation. Multiple cell subsets [MM cells, myeloid-derived suppressor cells, regulatory T cells, and BM-derived stromal cells (BMSC)] are involved in Vγ9Vδ2 T-cell inhibition via several immune suppressive mechanisms including the redundant expression of multiple immune checkpoints (ICPs). This review will address some aspects related to the dynamics of ICP expression in the BM of MM patients in relationship to the disease status (MGUS, diagnosis, remission, and relapse) and how this multifaceted ICP expression impairs Vγ9Vδ2 T-cell function. We will also provide some suggestions how to rescue Vγ9Vδ2 T cells from the immune suppression operated by ICP and to recover their antimyeloma immune effector functions at the tumor site.

A novel dual-cytokine–antibody fusion protein for the treatment of CD38-positive malignancies

A novel dual-cytokine-antibody fusion protein, consisting of an antibody directed against CD38 [a tumor-associated antigen mainly expressed on the surface of multiple myeloma (MM) cells], simultaneously fused to both tumor necrosis factor ligand superfamily member 10 (TRAIL) and interleukin-2 (IL2), was designed, expressed and purified to homogeneity. The novel fusion protein, termed IL2-αCD38-αCD38-scTRAIL, was able to selectively recognize its cognate antigen expressed on the surface of MM and lymphoma cell lines, as evidenced by flow cytometry analysis. Moreover, the targeted version of TRAIL was able to induce cancer cell death in vitro, both with MM cell lines and with fresh isolates from the bone marrow of MM patients. The experiments provide a rationale for possible future applications of IL2-αCD38-αCD38-scTRAIL for the treatment of patients with MM or other CD38-positive malignancies.

Targeting angiogenesis in multiple myeloma by the VEGF and HGF blocking DARPin® protein MP0250: a preclinical study.

The investigational drug MP0250 is a multi-specific DARPin® molecule that simultaneously binds and neutralizes VEGF and HGF with high specificity and affinity. Here we studied the antiangiogenic effects of the MP0250 in multiple myeloma (MM). In endothelial cells (EC) isolated from bone marrow (BM) of MM patients (MMEC) MP0250 reduces VEGFR2 and cMet phosphorylation and affects their downstream signaling cascades. MP0250 influences the secretory profile of MMEC and inhibits their in vitro angiogenic activities (spontaneous and chemotactic migration, adhesion, spreading and capillarogenesis). Compared to anti-VEGF or anti-HGF neutralizing mAbs, MP0250 strongly reduces capillary network formation and vessel-sprouting in a Matrigel angiogenesis assay. MP0250 potentiates the effect of bortezomib in the same in vitro setting. It significantly reduces the number of newly formed vessels in the choriollantoic membrane assay (CAM) and the Matrigel plug assay. In the syngeneic 5T33MM tumor model, MP0250 decreases the microvessel density (MVD) and the combination MP0250/bortezomib lowers the percentage of idiotype positive cells and the serum levels of M-protein. Overall results define MP0250 as a strong antiangiogenic agent with potential as a novel combination drug for treatment of MM patients.

Abstract 53: Expression of EBV in the non-neoplastic cell population in trephine biopsies of patients with multiple myeloma is associated with a significant survival advantage

Abstract Introduction: Multiple myeloma (MM) is a plasma cell (PC) disorder of the elderly, affecting predominantly males. Therapeutic advancement during the last decade has improved overall survival. Crosstalk between malignant PCs and the microenvironment is considered to play an important role in the development of resistance to treatment and relapse. Epstein Barr virus (EBV) is the first B-lymphotropic herpes virus identified for its association with human tumors. Reports relating an association of EBV to MM are few and inconsistent. It has been proposed that initiation of viral lytic cycle in EBV infected B cells is preceded by X-Box binding Protein-1 (XBP-1) mediated differentiation, resulting in a subset of EBV expressing infected PCs. Increased incidence of EBV expressing MM cases have been observed in immune-compromised patients. However, the significance of EBV in the bone marrow (BM) of MM patients has not been fully elucidated. The aim of our study was to evaluate prevalence of EBV in association with MUC1, XBP-1, ALDH1, and B-cell lineage markers (CD20, CD45, CD56, and CD138) in bone marrow trephine biopsies of patients with MM and to assess if there was any association with prognosis in a cohort of 151 MM patients at a single tertiary teaching hospital institution in Pakistan. Methodology: Formalin-fixed, paraffin-embedded trephine blocks of 151 MM cases diagnosed from 2007-2015 were evaluated for immunohistochemical (IHC) expression of MUC1, XBP-1, and EBV along with stem cell marker (ALDH1) and B-cell lineage markers (CD20, CD45, CD56, and CD138). Clinicopathologic details and follow-up data were collected from patients' medical records. Data was analyzed on SPSS 19. Five-year survival was estimated by Kaplan Meier curve for patients with follow-up available for 5 years or more (n=75). Results: Mean age at diagnosis was 57 years (range 30-89) with male preponderance (68.2%). Women (60.4%) with MM presented at an earlier age (median age 55 years, range 42-78) and difference in age of presentation was found to be significant (p value 0.04). Bone lesions were identified in 68.9% of patients at presentation. Stage I, II, and III disease was identified in 32.4%, 23.4%, and 44.1 % of patients, respectively (ISS staging system). Relapse was observed in 79.6% of patients and was significantly correlated with the expression of XBP-1 (p value 0.035). CD56 was expressed in 60.3% of cases and was significantly associated with cases diagnosed after the age of 57 years (p value 0.003). Expression of MUC1 was observed in 62.3% of tumors and 70% of MUC1-positive tumors also expressed XBP-1 (p value 0.06). Although the number of cases expressing EBV in the MM tumor cell population was only 7.2%, we detected EBV in 80.2% of the cases in the adjacent non-neoplastic cells with conspicuous expression in megakaryocytes. Five-year survival analysis (n=75) showed that expression of EBV+ in non-neoplastic cells was associated with significantly improved outcome with an advantage of 1.8 years (95% CI 3.32-4.4) as compared to EBV- cases (p value &amp;lt; 0.005). Among the XBP-1+ tumors, lack of EBV expression in non-neoplastic cells significantly correlated with adverse outcome (95%CI, 0.70-2.6) (p value &amp;lt; 0.005). Conclusions: To the best of our knowledge, this is the first study reporting expression of EBV in non-neoplastic cell population in BM trephines of MM patients. Salient conclusions of our study are as follows: 1. Expression of EBV in the surrounding tumor microenvironment of trephine biopsies of patients with MM is associated with a significant survival advantage; 2. Patients with MM in Pakistan present at a comparatively earlier age; 3. XBP1 expression was associated with relapse. Citation Format: Sheerien Rajput, Khurram Minhas, Iqbal Azam, Sadia Habib, Azhar Hussain, El-Nasir Lalani. Expression of EBV in the non-neoplastic cell population in trephine biopsies of patients with multiple myeloma is associated with a significant survival advantage [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 53.

JAM-A as a prognostic factor and new therapeutic target in multiple myeloma

Cell adhesion in the multiple myeloma (MM) microenvironment has been recognized as a major mechanism of MM cell survival and the development of drug resistance. Here we addressed the hypothesis that the protein junctional adhesion molecule-A (JAM-A) may represent a novel target and a clinical biomarker in MM. We evaluated JAM-A expression in MM cell lines and in 147 MM patient bone marrow aspirates and biopsies at different disease stages. Elevated JAM-A levels in patient-derived plasma cells were correlated with poor prognosis. Moreover, circulating soluble JAM-A (sJAM-A) levels were significantly increased in MM patients as compared with controls. Notably, in vitro JAM-A inhibition impaired MM migration, colony formation, chemotaxis, proliferation and viability. In vivo treatment with an anti-JAM-A monoclonal antibody (αJAM-A moAb) impaired tumor progression in a murine xenograft MM model. These results demonstrate that therapeutic targeting of JAM-A has the potential to prevent MM progression, and lead us to propose JAM-A as a biomarker in MM, and sJAM-A as a serum-based marker for clinical stratification.

ID: 1075 Concentration and κ/λ ratio of serum free light chain in multiple myeloma patients at Cho Ray Hospital

Introduction: multiple myeloma (MM) is a B cell malignancy which characterized by accumulation of plasmocyte in bone marrow, immunoglobulins and free light chain in serum (sFLC). According to IMMWG (International multiple myeloma working group), sFLC is a criterion in diagnosis of MM. In this study, we investigate the concentration and κ/λ ratio of sFLC in newly diagnosed MM patients at Cho Ray hospital. Methods: cross-sectional study. Serum samples of 36 newly diagnosed MM patients were analyzed by Binding Site Freelite method to measure the amount of κ and λ sFLC.&#x0D; &#x0D; Results: the median age of 36 MM patients was 61 yrs (41 – 88 yrs), with the male/female ratio was 1.2/1. MM stage III, stage II and stage I accounting for 63.9% (23/36), 27.8% (10/36), and 8.3% (3/36), respectively. Clonal IgG, IgA and IgE MM accounting for 61.1% (22/36), 30.6% (11/36), and 8.3% (3/36), respectively. We reported the median of plasmocyte in 36 cases was 35.4% (95%CI: 27.6 – 43.3). The median of concentration of κ sFLC, λ sFLC, and involved/uninvolved sFLC ratio were 1601.8 mg/L (95%CI: 104.9 – 3098.7), 900.1 mg/L (95%CI: 10.7 – 1789.5), and 191.0 (95%CI: 87.6 – 294.4), respectively. The abnormally high in sFLC concentration was reported in 97.2% MM cases (35/36), in which 55.6% cases (20/36) had increased κ sFLC, 19.4% cases (7/36) had increased λ sFLC, and 22.2% cases (8/36) had increased both κ+λ sFLC concentration. Increased involved/uninvolved sFLC ratio was found in 94.4% MM cases (34/36), in which 36.1% cases (13/36) had sFLC ratio &gt; 100, with all involved sFLC concentration &gt; 100 mg/L. In 3 cases of MM with kidney failure, 2 cases had increased both κ+λ sFLC, 1 case had increased κ sFLC concentration; and all of 3 cases had involved sFLC level &gt; 5300 mg/L with sFLC ratio &gt; 100. No significant difference of sFLC concentration and sFLC ratio among gender, stage of disease, Ig clone, or plasmocyte percent in bone marrow of MM patients. Conclusions: due to the abnormally high of sFLC concentration and sFLC ratio in MM, it is necessary to monitor frequently these parameters during treatment to prevent the risk of kidney failure for patients.

High levels of FLT3-ligand in bone marrow and peripheral blood of patients with advanced multiple myeloma.

IntroductionMultiple myeloma (MM) is still incurable due to resistance against various therapies. Thus, the identification of biomarkers predicting progression is urgently needed. Here, we evaluated four biomarkers in bone marrow and peripheral blood of MM patients for their prognostic significance.Materials & methodsBone marrow- and peripheral blood plasma levels of FLT3-L, soluble TIE2, endostatin, and osteoactivin were determined in patients with monoclonal gammopathy of undetermined significance (MGUS, n = 14/n = 4), patients with newly diagnosed MM (NDMM, n = 42/n = 31) and patients with relapsed/refractory MM (RRMM, n = 27/n = 16) by sandwich ELISA.ResultsMedian FLT3-L expression increased from MGUS (58.77 pg/ml in bone marrow; 80.40 pg/ml in peripheral blood) to NDMM (63.15 pg/ml in bone marrow; 85.05 pg/ml in peripheral blood) and was maximal in RRMM (122 pg/ml in bone marrow; 160.47 pg/ml in peripheral blood; NDMM vs. RRMM p<0.001). A cut-off value of FLT3-L >92 pg/ml in bone marrow and >121 pg/ml in peripheral blood was associated with relapse or refractoriness in MM patients. FLT3-L was found to be a high predictive marker for discrimination between NDMM and RRMM as well in bone marrow as in peripheral blood (AUC 0.75 in bone marrow; vs 0.84 in peripheral blood).ConclusionHigh levels of FLT3-L in bone marrow and peripheral blood of MM patients identify patients with progressive disease and are associated with relapse or refractoriness in MM patients. FLT3-L could be useful as a marker to identify RRMM patients and should be evaluated as target for future therapies.

Blockade of deubiquitylating enzyme Rpn11 triggers apoptosis in multiple myeloma cells and overcomes bortezomib resistance.

Proteasome inhibition is an effective therapy for multiple myeloma (MM) patients; however, the emergence of drug resistance is common. Novel therapeutic strategies to overcome proteasome inhibitor resistance are needed. In this study, we examined whether targeting deubiquitylating (DUB) enzymes upstream of 20S proteasome overcomes proteasome inhibitor resistance. Gene expression analysis, immunohistochemical studies of MM patient bone marrow, reverse transcription-PCR and protein analysis show that Rpn11/POH1, a DUB enzyme upstream of 20S proteasome, is more highly expressed in patient MM cells than in normal plasma cells. Importantly, Rpn11 expression directly correlates with poor patient survival. Loss-of-function studies show that Rpn11-siRNA knockdown decreases MM cell viability. Pharmacological inhibition of Rpn11 with O-phenanthroline (OPA) blocks cellular proteasome function, induces apoptosis in MM cells and overcomes resistance to proteasome inhibitor bortezomib. Mechanistically, Rpn11 inhibition in MM cells activates caspase cascade and endoplasmic stress response signaling. Human MM xenograft model studies demonstrate that OPA treatment reduces progression of tumor growth and prolongs survival in mice. Finally, blockade of Rpn11 increases the cytotoxic activity of anti-MM agents lenalidomide, pomalidomide or dexamethasone. Overall, our preclinical data provide the rationale for targeting DUB enzyme Rpn11 upstream of 20S proteasome to enhance cytotoxicity and overcome proteasome inhibitor resistance in MM.

Telomere length is positively associated with the expression of IL‑6 and MIP‑1α in bone marrow mesenchymal stem cells of multiple myeloma.

Potential roles of mesenchymal stem cells (MSCs) in the pathogenesis of multiple myeloma (MM) are largely unknown. In the current study, the authors analyzed telomere length and the expressions of interleukin (IL)-6 and macrophage inflammatory protein (MIP)-1α in MSCs derived from the bone marrow (BM) of MM patients and controls. The current results demonstrated that there was no significant difference in cell surface expression of CD73 and CD90, and the capacity to differentiate into bone tissue were identified between the BM MSCs derived from MM patients and controls. Interestingly, telomere length (TL) and mRNA expressions of IL-6 and MIP-1α were significantly longer or higher in BM MSCs of MM than those of controls. Moreover, TL is positively associated with the expressions of IL-6 and MIP-1α at the mRNA level in BM MSCs of MM. Additionally, IL-6 and MIP-1α expression were significantly upregulated when MSCs from MM patients were cultured in the myeloma associated condition medium. The present study indicated that myeloma-associated elongation of TL of BM MSCs may be a key element contributing to the increased IL-6 and MIP-1α expression, by which MSCs in the tumor microenvironment may facilitate MM and/or MM bone disease development.

Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: Role of HSP70/TLR2/NF-kB axis

ABSTRACTExosomes are a class of nanovesicles formed and released through the late endosomal compartment and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Here, we investigated whether genotoxic stress could promote the release of exosomes from multiple myeloma (MM) cells and studied the immunomodulatory properties they exert on NK cells, a major component of the antitumor immune response playing a key role in the immunosurveillance of MM. Our findings show that melphalan, a genotoxic agent used in MM therapy, significantly induces an increased exosome release from MM cells. MM cell-derived exosomes are capable of stimulating IFNγ production, but not the cytotoxic activity of NK cells through a mechanism based on the activation of NF-κB pathway in a TLR2/HSP70-dependent manner. Interestingly, HSP70+ exosomes are primarily found in the bone marrow (BM) of MM patients suggesting that they might have a crucial immunomodulatory action in the tumor microenvironment. We also provide evidence that the CD56high NK cell subset is more responsive to exosome-induced IFNγ production mediated by TLR2 engagement. All together, these findings suggest a novel mechanism of synergism between chemotherapy and antitumor innate immune responses based on the drug-promotion of nanovesicles exposing DAMPs for innate receptors.

The Utilization of Karyotyping, iFISH, and MLPA for the Detection of Recurrence Genetic Aberrations in Multiple Myeloma

Multiple myeloma (MM) is a hematological malignancy characterized by abnormal accumulation of clonal plasma cells in the bone marrow. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for detection of common genetic alterations in MM patients. Here, we aimed to confirm MLPA utility for this purpose and furthermore to test the feasibility of a combination of karyotyping, interphase fluorescence in situ hybridization (iFISH) and MLPA methods for diagnosis, prognostic assessment and risk stratification of MM. Thirty-five genomic DNA samples isolated from CD138-enriched plasma cells from bone marrow of MM patients were analyzed using the MLPA method. We found that amp (1q) was the most frequent genetic alteration (48.6%) in the tested samples, followed by del (1p) and del (13q) (34.3%). Moreover, concordant results between sensitivity and specificity of iFISH and MLPA for the detection of del (13q) (p-value >0.05) and del (17p) (p-value >0.05) were obtained. In summary, we could provide evidence of MLPA assay utility for the detection of common genetic alterations in MM. The combination of karyotyping, iFISH, and MLPA proved very helpful for clinical risk stratification.

The Effect of Lenalidomide on Multiple Myeloma Associated Macrophages

Introduction: In multiple myeloma (MM), accessory cells, such as monocytes and macrophages, located in the bone marrow (BM) tumor microenvironment play a crucial role in the fate of malignant cells. Under the influence of the surrounding milieu, monocytes can change their migratory capacity and differentiate into tumor-associated macrophages (TAMs). In the tumor site, TAMs can alter their profile from M1 macrophages with antitumor activities to M2-like macrophages that support tumor growth.Lenalidomide (Len), an immunomodulatory drug, used for MM treatment, is known to target different immune components inducing inflammatory responses; however, its direct influence on monocyte migration, macrophage differentiation and function in the tumor microenvironment is still unclear. The current study has aimed to explore the effect of Len on monocyte recruitment, macrophage polarization and pro-tumor functions.Methods: Monocytes were isolated from peripheral blood mononuclear cells of healthy donors, using anti-CD14 microbeads. To assess their migration capacity, monocytes were allowed to migrate through transwell insert towards the conditioned medium (CM) obtained from the BM of newly diagnosed MM patients or from MM cell lines (RPMI or U266) in the presence or absence of Len (10µM); the percentage of migrating monocytes was determined by FACS. For macrophage generation, monocytes were cultured with M-CSF followed by incubation with IL-4 to obtain M2 macrophages. To generate TAMs, CM obtained from the BM of MM patients was used. Len was added to the culture every 24 hours. The phenotype and functional properties of the generated macrophages were assessed. Endocytosis was evaluated by an antigen uptake assay. Macrophages were incubated with FITC-dextran at 37°C or 4°C, as a control, for 60 minutes, and analyzed by FACS. To test T cell proliferation, autologous lymphocytes labeled with CFSE, were stimulated with PHA and co-cultured with macrophages. T cell (CD3+) division was assessed by FACS. For IFN-γ secretion evaluation, lymphocytes co-cultured with macrophages were stimulated with PMA and ionomycin. The percentage of T cells expressing IFN-γ was quantified by FACS.Results: Monocyte migration towards CM obtained from MM cell lines (RPMI or U266) or from BM of MM patients (80.89%; n=4; p<0.01, 57.17%; n=4; p<0.01 and 42.9%; n=9; p<0.05, respectively) was significantly higher compared to migration towards normal BM CM (25.39%; n=2). Monocytes treated with Len demonstrated significantly decreased migration toward CM of MM cell lines (RPMI or U266) compared to untreated monocytes (45% vs. 80.8%; n=4 and 30.2% vs. 57.1%; n=4, respectively; p<0.01). The effect of Len on monocyte migration toward patient- derived CM was diverse. While 4 samples demonstrated decreased migration compared to untreated cells [51.1% vs. 59.6%; n=4; p<0.01], in 5 samples it increased [39.8% vs. 29.7%; n=5; n=5; p<0.01].To evaluate the effect of Len on macrophage polarization we examined their phenotype and functions. Both M2 macrophages and TAMs treated with Len expressed higher levels of M2 markers CD206, CD163, and cytokine IL-10 compared to untreated macrophages. Functional assays showed that Len increased endocytosis of both M2 macrophages [50% vs. 20%; n=5; P<0.01] and TAMs [47.4% vs. 41.4%; n=6; NS]. Exposure to Len led to suppression of T cell proliferation, when T cells were co-cultured with either autologous M2 macrophages [31% vs. 16%; n=4; p<0.05] or TAMs [39.7% vs. 31.5%; n=3; p<0.01]. In addition, M2 macrophages treated with Len demonstrated a reduction in IFN-γ secretion from T cells compared to untreated M2 macrophages (10.6% vs. 7.1%; n=4; NS).Conclusions: This study has demonstrated that Len has a direct effect on monocyte/macrophage behavior in the microenvironment generated by MM cells. Len is found to reduce monocyte migration, support polarization of macrophages towards the M2 phenotype and promote macrophage immunosuppressive functions, such as endocytosis, reduction of T-cell proliferation and inhibition of IFN-γ production. These findings need to be further investigated in in vivo experiments and could support the benefit of using agents targeting specific pathways associated with TAM development in the treatment of MM patients. DisclosuresNo relevant conflicts of interest to declare.