scholarly journals Architecture of Sample Preparation and Data Governance of Immuno-Genomic Data Collected from Bone Marrow and Peripheral Blood Samples Obtained from Multiple Myeloma Patients

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Shaadi Mehr ◽  
Daniel Auclair ◽  
Mark Hamilton ◽  
Leon Rozenblit ◽  
Hearn Jay Cho ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Large Scale ◽  
Mass Cytometry ◽  
Data Governance ◽  
Advisory Committees ◽  
Blood Samples ◽  
Data Files

Abstract: Title: Architecture of sample preparation and data governance of Immuno-genomic data collected from bone marrow and peripheral blood samples obtained from multiple myeloma patients In multiple myeloma (MM), the interactions between malignant plasma cells and the bone marrow microenvironment is crucial to fully understand tumor development, disease progression, and response to therapy. The core challenge in understanding those interactions has been the establishment of a standard process and a standard model for handling the data quality workflow and the underlying data models. Here we present the Platform (Figure 1), an integrated data flow architecture designed to create data inventory and process tracking protocols for multi-dimensional and multi-technology immune data files. This system has been designed to inventory and track peripheral blood and bone marrow samples from multiple myeloma subjects submitted for immune analysis under the MMRF Immune Atlas initiative (figure 2), and the processing and storage of Single Cell RNA-seq (scRNA-seq) and Mass Cytometry time-of-flight (CyTOF) data files derived from these immune analyses. While these methods have been previously applied on both tumor and immune populations in MM [2,3], this level of multi-institutional and multi-technology is unique. The Cloud Immune-Precision platform contains standardized protocols and bioinformatics workflows for the identification and categorization of immune cell populations and functional states based upon scRNA-seq gene signatures (ref: Bioinformatics manuscript in submission) and CyTOF protein signatures. Upon further expansion, it will contain high dimensional scRNAseq and CyTOF immune data from both bone marrow and peripheral blood samples from myeloma patients enrolled in the Multiple Myeloma Research Foundation (MMRF) CoMMpass study (NCT01454297) [1] (Figure 3). The architecture covers the automation of data governance protocols, data transformation and ETL model developments that will create an immune proteomic and profiling database and its integration into clinical and genomics databases: e.g. the MMRF CoMMpass clinical trial. This large-scale data integration will establish a cutting-edge Immune-Precision central platform supporting large scale, immune-focused advanced analytics in multiple myeloma patients. This platform will allow researchers to interrogate the relationships between immune transcriptomic and proteomic signatures and tumor genomic features, and their impact on clinical outcomes, to aid in the optimization of therapy and therapeutic sequencing. Furthermore, this platform also promotes the potential to (further) elucidate the mechanisms-of-action of approved and experimental myeloma therapies, drive biomarker discovery, and identify new targets for drug discovery. Figure 1: Cloud Immune-Precision Platform (Integrated data flow architecture designed to create data inventory and process tracking protocols for multi-dimensional and multi-technology immune data files) Figure 2: Sample tracking process architecture Figure 3: Data file creation and repository process tracking References: 1- Settino, Marzia et al. "MMRF-CoMMpass Data Integration and Analysis for Identifying Prognostic Markers." Computational Science - ICCS 2020: 20th International Conference, Amsterdam, The Netherlands, June 3-5, 2020, Proceedings, Part III vol. 12139 564-571. 22 May. 2020, doi:10.1007/978-3-030-50420-5_42 2- Ledergor, Guy et al. "Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma." Nature medicine vol. 24,12 (2018): 1867-1876. doi:10.1038/s41591-018-0269-2 3- Hansmann, Leo et al. "Mass cytometry analysis shows that a novel memory phenotype B cell is expanded in multiple myeloma." Cancer immunology research vol. 3,6 (2015): 650-60. doi:10.1158/2326-6066.CIR-14-0236-T Figure 1 Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder. Dhodapkar:Amgen: Membership on an entity's Board of Directors or advisory committees, Other; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Other; Janssen: Membership on an entity's Board of Directors or advisory committees, Other; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Other; Lava Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other; Kite: Membership on an entity's Board of Directors or advisory committees, Other.

scholarly journals Mass Cytometry Identifies Immunomic Shifts in the Bone Marrow Microenvironment of Multiple Myeloma and Light Chain Amyloidosis after Standard of Care First Line Therapies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1879-1879 ◽  
Author(s):  
Taxiarchis Kourelis ◽  
Jose C Villasboas ◽  
Surendra Dasari ◽  
Angela Dispenzieri ◽  
Shaji K. Kumar
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
Board Of Directors ◽  
Induction Therapy ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Mass Cytometry ◽  
Advisory Committees ◽  
Healthy Donors

Abstract INTRODUCTION: Traditional cytometry methods have been unable to capture the immense heterogeneity of the tumor immune microenvironment in various malignancies including multiple myeloma (MM). Cytometry by time of flight (CyTOF) can, to some extent, overcome this limitation. However, the computational challenges that come with analyzing these complex datasets in a reproducible manner remain. In this study, using a large cohort of patients, we compare the bone marrow immunomes from patients with MGUS, multiple myeloma (MM), smoldering MM (SMM) and light chain amyloidosis (AL) at diagnosis, after induction therapy with lenalidomide and dexamethasone and after autologous transplant (ASCT). METHODS: We studied a total of 118 cryopreserved samples as follows: 14 healthy donors, 43 AL (27 newly diagnosed-ND, of which 13 with <10% bone marrow plasma cells, 16 matched samples post ASCT), 12 with ND MGUS, 11 with SMM (of which 6 were ND), 14 with ND MM, 13 paired MM samples post induction therapy and 11 paired MM samples post ASCT. Our CyTOF surface staining panel included the following 33 markers: CD45, HLA-DR, CD19, CD3, CD4, CD8, CD14, CCR6, CD11a, Cd123, CCR5, CD7, ICOS, CD25, CD57, CD45RA, CD163, PD-1, PDL-1, CXCR3, CCR4, CCR7, CD28, CTLA4, CD11c, CD56, CD45RO, CD44, CD27, CD138, CD38, CD-127 and CD16. Data processing and analysis was performed using Cytobank. Live cells were identified based on Pt195 and Ir193 staining. Myeloma cells and CD45- cells were excluded and only CD45+ cells were used for subsequent analyses. Single-cell data were downsampled using VisNE, a permutation of t-Distributed Stochastic Neighbor Embedding (tSNE) and clustered using CITRUS (using 10,000 events per sample with a minimum cluster size of 1%). A Significance analysis of microarrays (SAM) analysis was performed to ascertain differences between groups. Significance was inferred for a false discovery rate <1% . All CITRUS analyses were repeated at least 3 times and only clusters found to differ consistently across runs were considered. RESULTS: The proportions of immune subsets identified to vary by CITRUS before and after induction therapy and ASCT for MM and AL are shown in the table. No differences were identified between MGUS, SMM and NDMM. The proportion of a subset (369850) of CD14+/C16- monocytes, a group of cells shown to correlate positively with survival and response to therapy in solid malignancies, increased after induction therapy in MM. Naïve B cells increased dramatically post ASCT in both AL (429918) and MM (369948), consistent with expected immune reconstitution patterns, although a CCR6+ B Cell subset (429940) shown to mediate effective antibody responses, decreased (in grey). A subset (369980) of functionally exhausted (PD1/CTLA4+) central memory (CM) CD4 T cells decreased after induction therapy in MM but recovered early post ASCT whereas a CM CD4+ subset (369986) lacking major activation markers (CD28, CD25) decreased gradually with therapy and post ASCT. A subset (369953) of naïve CD8 T cells shown to be actively recruited in tumor sites (CXCR3+) decreased after ASCT. CD57+ senescent effector memory (EM) CD8 T cells (369962) decreased with induction therapy but recovered post ASCT. EM CD8 T cells associated with long term immune memory (CCR5+, CD127+, cluster 369959), also decreased after ASCT. LIMITATIONS: Include a) No barcoding b) batch effects were difficult to avoid when processing large number of samples c) use of cryopreserved samples d) need for downsampling to cope with the computational burden. The latter could have been one of the reasons we did not identify any differences between MGUS, SMM and MM. At the time of the meeting we will present confirmatory analyses using conceptually different methods of clustering and of performing across group comparisons as well as analyses that do not include downsampling. CONCLUSIONS: Mass cytometry can provide a more granular view of the bone marrow immunome in plasma cell dyscrasias. Novel agent induction therapy can create an immunologically favorable anti-tumor microenvironment although, in some cases, these favorable immunomic shifts are temporarily reversed by ASCT. Confirmatory analyses, baseline immune profiles, comparisons with healthy donors and correlation with other clinically relevant patient characteristics and outcomes will be reported at the meeting. Disclosures Dispenzieri: Celgene, Takeda, Prothena, Jannsen, Pfizer, Alnylam, GSK: Research Funding. Kumar:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Clonally Expanded Bone Marrow T Cells Show Effector Differentiation and Rarely Recognize Disease-Associated Antigens in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 7-7
Author(s):  
Carlotta Welters ◽  
Meng-Tung Hsu ◽  
Christian Alexander Stein ◽  
Livius Penter ◽  
María Fernanda Lammoglia Cobo ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
T Cell Expansion

Multiple myeloma is a malignancy of monoclonal plasma cells accumulating in the bone marrow. The critical influence of tumor-infiltrating T cells on disease control and therapeutic responses has been shown in a variety of malignancies, however, the role of multiple myeloma bone marrow-infiltrating T cells is incompletely understood. Although it has been shown that multiple myeloma neo-antigen-specific T cells can be expanded in vitro, little is known about functions and specificities of clonally expanded multiple myeloma-infiltrating bone marrow T cells. Here we asked at the single cell level whether clonally expanded T cells i) were detectable in multiple myeloma bone marrow and peripheral blood, ii) showed characteristic immune phenotypes, and iii) recognized antigens selectively presented on multiple myeloma cells. A total of 6,744 single bone marrow T cells from 13 treatment-naïve patients were index-sorted and sequenced using our methodologies for determination of paired T cell receptor (TCR) αβ sequences along with immune phenotype, transcription factor and cytokine expression. Clonal T cell expansion occurred predominantly within the CD8+ compartment. Phenotypes of clonally expanded T cells were distinctive of cytolytic effector differentiation and significantly different from non-expanded CD8+ T cells. Less than 25% of expanded CD8+ T cell clones expressed the immune checkpoint molecules programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), or T cell immunoglobulin and mucin-domain containing-3 (TIM-3), while B and T lymphocyte attenuator (BTLA) was expressed on more than half of the expanded clones. Clonal T cell expansion did not correlate with neo-antigen load as determined by whole exome and RNA sequencing of purified multiple myeloma cells. Furthermore, peripheral blood TCRβ repertoire sequencing from five selected patients with substantial bone marrow T cell expansion identified 90% of expanded bone marrow T cell clones overlapping with peripheral blood. To determine whether clonally expanded bone marrow T cells recognized antigens selectively presented on multiple myeloma cells, 71 dominant TCRs from five selected patients with substantial clonal T cell expansion were re-expressed in 58α-β- T-hybridoma reporter T cells and co-incubated with CD38-enriched multiple myeloma cells from the same patients. Only one of these TCRs recognized antigens selectively presented on multiple myeloma cells and this TCR was not neo-antigen-specific. Hypothesizing that the target antigen was a non-mutated self-antigen, we could show that this TCR also recognized the plasma cell leukemia cell line U-266 in an HLA-A*02:01-restricted manner. In summary, clonally expanded T cells in multiple myeloma bone marrow of newly diagnosed patients show cytolytic effector differentiation. In the majority of patients, clonally expanded bone marrow T cells do not recognize antigens presented on multiple myeloma cells and are not neo-antigen-specific. Our findings are relevant for the design of future therapeutics and clinical trials. The identified TCR, which recognizes a multiple myeloma antigen shared with U-266 in an HLA-A*02:01-restricted manner, could be a promising candidate for T cell therapy. Disclosures Bullinger: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Carfilzomib Thalidomide and Dexamethasone Is Safe and Effective in the Treatment of Relapsed/Refractory Multiple Myeloma: Preliminary Outcome from the Open Label Phase II ALLGMM018/AMN003 Study

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1955-1955
Author(s):  
Hang Quach ◽  
Simon J. Harrison ◽  
Slavisa Ninkovic ◽  
Jane Estell ◽  
Noemi Horvath ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Open Label ◽  
Advisory Committees ◽  
Open Label Phase ◽  
Grade 3

Abstract Background: Carfilzomib lenalidomide and dexamethasone (KRd) is FDA-approved for the treatment relapsed/refractory multiple myeloma (RRMM) based on data from the ASPIRE study (Stewart K et al. NEJM 2015). Thalidomide, a first generation immunomodulatory drug (IMiD) is less costly than lenalidomide and is synergistic in combination with proteasome inhibitors in the treatment of MM. ALLG MM018/ AMN003 is an open label phase II study of carfilzomib thalidomide and dexamethasone (KTd) for patients with RRMM. The primary end point is progression free survival (PFS). Secondary endpoints include overall response rate (ORR), duration of response (DOR), safety and health related quality of life. Method: Eligible patients were those with RRMM who have had 1-3 prior lines of treatment. The KTd regimen consisted of carfilzomib [20mg/m2 IV C1D1 and 2, 56mg/m2 (36mg/m2 for patients age ≥75 years) from C1D8 onwards], thalidomide (100mg po nocte) and dexamethasone [40mg (20mg for patients age ≥75 years) po weekly], in a 28-day cycle. After 12 cycles, thalidomide was omitted and Kd [carfilzomib 56mg/m2 (36/m2 for patients age ≥75 years) on days 1,2,15,16 and dexamethasone 40mg (20mg for patients age ≥75 years) on days 1,15 every 28 days]was continued for a further 6 cycles. Peripheral blood and bone marrow aspirate and trephine for correlative studies were collected from the first 30 patients, at baseline, after cycle 6 and at confirmed disease progression. The aim of the correlative study was to assess for immunological correlates to clinical outcome. Immunological parameters that will be assessed include NK and T cells subsets on peripheral blood via mass cytometry (CyTOF). On the bone marrow trephine, NK cells, T cells, GRP78 expression within CD38 positive plasma cells, PD1 and PDL1 expression will be assessed at the myeloma site and the surrounding microenvironment using OPAL multiplex immunohistochemistry technology. Results: Between March 2017 to June 2018, 56 patients (median age 66 years, range 56-79; 77% Caucasian and 23% Asian) out of the planned 100 were enrolled, with a median follow up of 4.9 (range, 1.0-13.7) months. Response rates in 39 evaluable patients were ≥MR (97%), ≥PR (89%) and ≥VGPR (66%). Median PFS is not reached, and no patients with ≥MR have relapsed. Grade ≥3/4 AEs occurred in 56% of patients, the most common of which were peripheral sensory neuropathy (13%), dyspnoea (13%) and infections (7%). All grade cardiovascular AEs included dyspnoea (27%), cardiac complications (5%), systemic-hypertension (9%) and pulmonary-hypertension (1.9%), however very few were grade ≥3. Three patients have died on study from disease complications, haemorrhage, and primary cardiac ischaemic event. Thus far, we have not found a significant difference in rates or profile of adverse events between the Caucasian versus Asian subgroups of patients. Conclusion: This preliminary analysis demonstrates that the KTd combination is a tolerable regimen for patients with RRMM with a safety profile in line with previous reports for each of carfilzomib and thalidomide. Initial response rates appear very promising and durable with responses up to 13.7 months thus far in some patients. Patient accrual is ongoing. Disclosures Quach: Janssen Cilag: Consultancy; Sanofi Genzyme: Research Funding; Celgene: Consultancy, Research Funding; Amgen: Consultancy, Research Funding. Harrison:Janssen-Cilag: Other: Scientific advisory board. Mollee:Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Durie:Takeda: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy. Chng:ASLAN Pharmaceuticals: Research Funding.

scholarly journals Multiple Myeloma Cells Induce Lipolysis in Adipocytes and Uptake Fatty Acids through Fatty Acid Transporter Proteins

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 41-42
Author(s):  
Cristina Panaroni ◽  
Keertik Fulzele ◽  
Tomoaki Mori ◽  
Chukwuamaka Onyewadume ◽  
Noopur S. Raje
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Fatty Acid ◽  
Board Of Directors ◽  
Metabolic Reprogramming ◽  
Glycerol Production ◽  
Advisory Committees ◽  
Myeloma Cells ◽  
Long Chain

Multiple myeloma (MM) originates in the bone marrow where adipocytes occupy 65% of the cellular volume in a typical myeloma patient. Cancer associated adipocytes support the initiation, progression, and survival of solid tumors via mechanisms including adipokine secretion, modulation of the tumor microenvironment, and metabolic reprogramming of cancer cells. Although MM cells are surrounded by abundant bone marrow adipocytes (BMAd), the nature of their interaction remains unclear. Recent studies have elucidated the role of BMAds in supporting the survival of MM cells, in part, through secreted adiponectin. Increased fatty acid (FA) metabolism may result in metabolic reprogramming of cancer cells impacting their growth and survival. Here, we hypothesize that MM cells extract FA from adipocytes for their growth. We first characterized mesenchymal stem cells (MSCs) from MGUS, smoldering MM (SMM), and newly diagnosed MM (NDMM) patients by flow cytometry analysis. MSCs showed significant increase in Pref1, leptin receptor and perilipin A, suggesting increased adipogenic commitment. MSCs from healthy donors (HD), MGUS, SMM, and NDMM patients were induced to differentiate into adipocytes and then co-cultured with human MM MM.1S cells. After 72 hr of co-culture, CyQUANT assay demonstrated significant increase in proliferation of MM.1S cells in the presence of BMAd from HD; this was further increased in the presence of BMAd from MGUS/SMM and NDMM. These data suggest that the BMAd support the growth of MM cells and this effect is more pronounced in patient derived BMAd. A PCR-array targeting lipid metabolism on BM fat aspirates showed significant deregulation of genes involved in FA synthesis and lipolysis. Taken together, our data suggest that BMAd in MM patients are altered to further support the aggressive expansion of MM cells. The proliferative-supportive role of adipocytes was further validated in co-culture of OP9 murine BM stromal preadipocytes with 5TGM1 murine MM cells. To study the bidirectional interaction of MM/ BMAd, mature OP9 adipocytes were co-cultured with 5TGM1 or human OPM2 MM cells for 24 hr. Intracellular lipid droplets were labelled with Deep Red LipidTox stain. The lipid droplet sizes were significantly decreased in the presence of both 5TGM1 and OPM2 cells compared to OP9 alone. The decrease in lipid size suggested that MM cells may induce lipolysis in adipocytes. Indeed, 24hr co-culture of 5TGM1 cells with OP9 mature adipocytes significantly increased lipolysis 3-fold as measured by glycerol secretion in conditioned media. Co-culture of OP9 adipocytes with other MM cell lines of human origin, MM.1S, INA6, KMS-12 PE, and OPM2 also significantly increased the glycerol production as much as 4-fold. Taken together these data indicate that MM cells induce lipolysis in adipocytes. In contrast, treatment of 5TGM1 cells with synthetic catecholamine isoproterenol did not induce lipolysis, or glycerol production, indicating lack of triglyceride storage. Next, we hypothesized that the free FAs released from adipocytes are taken up by MM cells for various biological processes. To test this, 5TGM1, MM.1S and OPM2 cells were incubated with BODIPY-C12 and BODIPY-C16, the BODIPY-fluorophore labelled 12-carbon and 16-carbon long chain FA. All MM cells showed saturated uptake of the FA within 10 minutes suggesting that MM cells have efficient FA transporters. To confirm this uptake, unstained 5TGM1, OPM2 and KMS12 PE cells were co-cultured with the LipidTox-labelled OP9 mature adipocytes. After 24 hours, flow cytometric analysis showed LipidTox signal in MM cells. These data demonstrate that FAs released by MM induced adipocyte lipolysis are taken up by MM cells. Long-chain FAs such as BODIPY-C12 and BODIPY-C16 are transported into cells through FA transporter protein (FATP) family of lipid transporters. We therefore analyzed patient samples which showed that CD138+ plasmacells and myeloma cells expressed high levels of FATP1 and FATP4 whereas, their expression was absent in lineage-sibling T-cells. Moreover, pretreatment with Lipofermata, a FATP inhibitor, was able to decrease the uptake of BODIPY-C12 and -C16 in 5TGM1 cells. Taken together, our data show that myeloma cells induce lipolysis in adipocytes and the released free FAs are then uptaken by myeloma cells through FATPs. Inhibiting myeloma cell induced lipolysis or uptake of FA through FATPs may be a potential anti-tumor strategy. Disclosures Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Raje:Amgen: Consultancy; bluebird bio: Consultancy, Research Funding; Caribou: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immuneel: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Immuneel: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

scholarly journals Phase II Trial of the Combination of Ixazomib, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 804-804 ◽  
Author(s):  
Mark Bustoros ◽  
Chia-jen Liu ◽  
Kaitlen Reyes ◽  
Kalvis Hornburg ◽  
Kathleen Guimond ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
High Risk ◽  
Disease Progression ◽  
Board Of Directors ◽  
Rna Sequencing ◽  
Research Funding ◽  
Response Rate ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Background. This study aimed to determine the progression-free survival and response rate using early therapeutic intervention in patients with high-risk smoldering multiple myeloma (SMM) using the combination of ixazomib, lenalidomide, and dexamethasone. Methods. Patients enrolled on study met eligibility for high-risk SMM based on the newly defined criteria proposed by Rajkumar et al., Blood 2014. The treatment plan was designed to be administered on an outpatient basis where patients receive 9 cycles of induction therapy of ixazomib (4mg) at days 1, 8, and 15, in combination with lenalidomide (25mg) at days 1-21 and Dexamethasone at days 1, 8, 15, and 22. This induction phase is followed by ixazomib (4mg) and lenalidomide (15mg) maintenance for another 15 cycles. A treatment cycle is defined as 28 consecutive days, and therapy is administered for a total of 24 cycles total. Bone marrow samples from all patients were obtained before starting therapy for baseline assessment, whole exome sequencing (WES), and RNA sequencing of plasma and bone marrow microenvironment cells. Moreover, blood samples were obtained at screening and before each cycle to isolate cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Stem cell collection is planned for all eligible patients. Results. In total, 26 of the planned 56 patients were enrolled in this study from February 2017 to April 2018. The median age of the patients enrolled was 63 years (range, 41 to 73) with 12 males (46.2%). Interphase fluorescence in situ hybridization (iFISH) was successful in 18 patients. High-risk cytogenetics (defined as the presence of t(4;14), 17p deletion, and 1q gain) were found in 11 patients (61.1%). The median number of cycles completed was 8 cycles (3-15). The most common toxicities were fatigue (69.6%), followed by rash (56.5%), and neutropenia (56.5%). The most common grade 3 adverse events were hypophosphatemia (13%), leukopenia (13%), and neutropenia (8.7%). One patient had grade 4 neutropenia during treatment. Additionally, grade 4 hyperglycemia occurred in another patient. As of this abstract date, the overall response rate (partial response or better) in participants who had at least 3 cycles of treatment was 89% (23/26), with 5 Complete Responses (CR, 19.2%), 9 very good partial responses (VGPR, 34.6%), 9 partial responses (34.6%), and 3 Minimal Responses (MR, 11.5%). None of the patients have shown progression to overt MM to date. Correlative studies including WES of plasma cells and single-cell RNA sequencing of the bone microenvironment cells are ongoing to identify the genomic and transcriptomic predictors for the differential response to therapy as well as for disease evolution. Furthermore, we are analyzing the cfDNA and CTCs of the patients at different time points to investigate their use in monitoring minimal residual disease and disease progression. Conclusion. The combination of ixazomib, lenalidomide, and dexamethasone is an effective and well-tolerated intervention in high-risk smoldering myeloma. The high response rate, convenient schedule with minimal toxicity observed to date are promising in this patient population at high risk of progression to symptomatic disease. Further studies and longer follow up for disease progression are warranted. Disclosures Bustoros: Dava Oncology: Honoraria. Munshi:OncoPep: Other: Board of director. Anderson:C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; BMS: Consultancy.

Inhibition of the Proteasome in Bone Marrow-Derived CD138+ Tumor Cells Following Carfilzomib Administration in Relapsed or Refractory Myeloma Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1845-1845
Author(s):  
Suzanne Trudel ◽  
Susan Lee ◽  
Christopher J. Kirk ◽  
Nashat Gabrail ◽  
Sagar Lonial ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Whole Blood ◽  
Research Funding ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Advisory Committees ◽  
Post Dose ◽  
Blood Samples

Abstract Abstract 1845 Poster Board I-871 Background: Proteasome inhibition is an effective strategy for the treatment of multiple myeloma. In patients, proteasome inhibition has primarily been measured in peripheral blood samples (whole blood or mononuclear cells). However, it is unknown whether myeloma cells in the bone marrow (BM) are equally sensitive to proteasome inhibitors such as bortezomib (BTZ) and carfilzomib (CFZ). Aim: To measure proteasome inhibition in purified tumor cells from BM samples taken from patients enrolled in two ongoing Phase 2 trials of single agent CFZ in relapsed or refractory myeloma: PX-171-003 (003) and PX-171-004 (004). Methods: CFZ was administered as an IV bolus of 20 mg/m2 on Days 1, 2, 8, 9, 15 and 16 of a 28-day cycle on both trials. Bone marrow samples, from an optional sub-study of both trials, were taken during screening and Day 2 (post-treatment) and sorted into CD138+ and CD138− cells. Proteasome activity was measured by an enzymatic assay using a fluorogenic substrate (LLVY-AMC) for the chymotrypsin-like (CT-L) activity and an active site ELISA (ProCISE) to quantitate levels of the CT-L subunits of the constitutive proteasome (Beta5) and immunoproteasome (LMP7) and the immunoproteasome subunit MECL1. Results: Whole blood samples from patients treated with CFZ showed inhibition of CT-L activity of ∼80+, similar to values obtained in Phase 1 studies. A total of 10 CD138+ screening samples, 6 from 004 and 4 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed for proteasome levels and activity. In addition, 15 CD138−screening samples, 7 from 004 and 8 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed. When compared to the average base-line activity, CFZ treatment resulted in 88% CT-L inhibition in CD-138+tumor cells from 004 patients (P = 0.0212 by unpaired t-test) and 59% CT-L inhibition in CD-138+ tumor cells from 003 patients (P = 0.25). Baseline CT-L activity in CD138+ tumor cells was 3-fold higher in 004 than 003, which includes a more heavily pre-treated patient population with greater prior exposure to BTZ. Higher specific enzymatic activity was due to increased levels of both constitutive and immunoproteasomes in tumor cells, where immunoproteasomes account for >75% of total cellular proteasomes. No differences between trials were seen in baseline CT-L activity from non-tumor (CD138−) cells. Inhibition in CD138− cells was 84% (P = 0.0380 and 42% (P = 0.38) in 004 and 003, respectively. Using ProCISE, we measured inhibition of LMP7 (66%), beta5 (48%) and MECL1 (64%) in CD138+ tumor cells from 004 patients. Three patients from 004 and one from 003 had both a screening and post-dose tumor cell samples available for analysis. Inhibition of CT-L activity was >80% in two of the 3 patients on 004; the third patient showed no proteasome inhibition by ProCISE and was unavailable for analysis by CT-L. CT-L activity in the CD138+ tumor cells in the 003 patient was not inhibited, however, inhibition was seen in non-tumor cells. Conclusions: CFZ inhibits the proteasome activity of myeloma cells in the bone marrow of relapsed and refractory myeloma patients. The levels of inhibition were similar to those measured in whole blood samples, supporting the use of the blood-based assay as a surrogate marker for proteasome inhibition in tumor cells. CFZ treatment resulted in inhibition of both CT-L subunits as well as additional subunits of the immunoproteasome in tumor cells. Reduced baseline activity in the more heavily pretreated 003 patients may reflect reduced tumor-dependency on the proteasome and may be related to prior treatment with BTZ in these patients. More samples are needed in order to make correlations between levels of proteasome inhibition in bone marrow tumor cells and prior therapies or response. These observations support further evaluation of proteasome activity and the effects of this promising new agent in primary tumors cells from myeloma patients. Disclosures: Trudel: Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Lee:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc.: Employment. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Wang:Proteolix, Inc.: Research Funding. Kukreti:Celgene: Honoraria. Stewart:Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. McDonagh:Proteolix: Research Funding. Zonder:Celgene: Speakers Bureau; Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen: Consultancy; Millennium: Research Funding. Bennett:Proteolix: Employment.

Phase 1/2 Study of Elotuzumab in Combination with Bortezomib in Patients with Multiple Myeloma with One to Three Prior Therapies: Interim Results.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3876-3876 ◽  
Author(s):  
Andrzej J Jakubowiak ◽  
William Bensinger ◽  
David Siegel ◽  
Todd M. Zimmerman ◽  
Jan M. Van Tornout ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Clinical Response ◽  
Board Of Directors ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Phase 1 ◽  
Surface Glycoprotein ◽  
Advisory Committees ◽  
Cell Surface Glycoprotein

Abstract Abstract 3876 Poster Board III-812 Background Elotuzumab is a humanized monoclonal IgG1 antibody directed against CS1, a cell surface glycoprotein, which is highly and uniformly expressed in multiple myeloma (MM). In mouse xenograft models of MM, elotuzumab demonstrated significantly enhanced anti-tumor activity when combined with bortezomib compared to bortezomib alone (Van Rhee et al., Mol. Cancer Ther., in press, 2009). This phase 1/2 trial will determine the maximum tolerated dose (MTD), overall safety, pharmacokinetics (PK) and clinical response of elotuzumab in combination with bortezomib in patients with relapsed MM following 1-3 prior therapies. Methods The study consists of 4 escalating cohorts of elotuzumab (2.5 mg/kg to 20 mg/kg) administered on Days 1 and 11 and bortezomib (1.3 mg/m2) administered on Days 1, 4, 8 and 11 of a 21-day cycle. Patients with progressive disease at the end of Cycle 2 or 3 also receive oral dexamethasone (20 mg) on Days 1, 2, 4, 5, 8, 9, 11 and 12 of each subsequent cycle. Patients with stable disease or better at the end of 4 cycles will continue treatment for 6 or more cycles unless withdrawn earlier due to unexpected toxicity or disease progression. Key entry criteria: age ≥ 18 years; confirmed diagnosis of MM and documentation of 1 to 3 prior therapies; measurable disease M-protein component in serum and/or in urine; and no prior bortezomib treatment within 2 weeks of first dose. Results To date, a total of 16 MM patients with a median age of 64 years have been enrolled in the study. The median time from initial diagnosis of MM was 3.5 years and patients had received a median of 2 prior MM treatments. Patients have been treated in four cohorts; 3 each in 2.5, 5 and 10 mg/kg elotuzumab cohorts, and 7 in the 20 mg/kg elotuzumab cohort. No dose limiting toxicity (DLT) was observed during the first cycle of the study and the MTD was not established. Five SAEs have been reported in four patients in later treatment cycles; two events, chest pain and gastroenteritis, occurring in one patient, were considered elotuzumab-related. Other SAEs include grade 3 sepsis, vomiting, pneumonia and grade 2 dehydration. The most common AEs reported include Grade 1-3 diarrhea, constipation, nausea, fatigue, thrombocytopenia, neutropenia, anemia and peripheral neuropathy. The best clinical response (EBMT criteria) for the 16 patients who have received at least two cycles of treatment is shown in the table below. Preliminary PK analysis suggests a serum half-life of 10-11 days at higher doses (10 and 20 mg/kg). Preliminary analysis of peripheral blood mononuclear cells and bone marrow of patients on study indicates that objective responses in the study correlate well with complete saturation of CS1 sites by elotuzumab on bone marrow plasma and NK cells. Conclusions The combination of elotuzumab with bortezomib has a manageable adverse event profile and shows promising preliminary efficacy with ≥PR in 44% and ≥MR in 75% of all enrolled patients. Accrual is ongoing in the expanded 20 mg/kg cohort. Updated safety, efficacy, and PK data will be presented at the meeting. Disclosures: Jakubowiak: Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Centocor Ortho Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Exelixis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Bortezomib in combination with elotuzumab for the treatment of relapsed/refractory multiple myeloma. Bensinger:Millennium: Membership on an entity's Board of Directors or advisory committees. Siegel:Millennium: Speakers Bureau; Celgene: Speakers Bureau. Zimmerman:Millennium: Speakers Bureau; Centecor: Speakers Bureau. Van Tornout:BMS: Employment. Zhao:Facet Biotech: Employment. Singhal:Facet Biotech: Employment. Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 133-133 ◽  
Author(s):  
Patricia Maiso ◽  
AbdelKareem Azab ◽  
Yang Liu ◽  
Yong Zhang ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Bone Marrow Microenvironment ◽  
Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Multiple Myeloma In Complete Remission After Autologous Stem Cell Transplantation: Impact of Bone Marrow Plasma Cell Assessment by Conventional Morphology on Disease Progression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2946-2946
Author(s):  
Carlos Fernández de Larrea ◽  
Natalia Tovar ◽  
María Rozman ◽  
Laura Rosiñol ◽  
Juan I. Aróstegui ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stem Cell ◽  
Complete Remission ◽  
Board Of Directors ◽  
Research Funding ◽  
Bone Marrow Aspirate ◽  
Advisory Committees ◽  
Bone Marrow Plasma ◽  
Serum And Urine

Abstract Abstract 2946 Background: The achievement of complete remission (CR) is the crucial step for a long-lasting response and prolonged survival after autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM). The European Group for Blood and Marrow Transplantation (EBMT) criteria for CR include the negativity of serum and urine immunofixation (IFE) and less than 5% of bone marrow plasma cells (BMPCs). Additionally, the International Myeloma Working Group (IMWG) has even proposed a stringent CR category, which requires to rule out the clonal nature of the BMPCs. However, few studies have addressed this issue in patients with MM and negative IFE. The aim of the present study was to determine the impact of plasma cell count in the bone marrow aspirate on the long-term outcome of patients with MM with negative IFE after ASCT. Methods: Thirty-five patients (16M/19F; median age at ASCT 55 years, range 26–68) with MM who underwent ASCT from March 1994 to December 2008, were studied. All patients had achieved a negative serum and urine IFE after high dose therapy with melphalan-based regimens. Bone marrow aspirate was performed when negative serum and urine IFE was achieved and at least three months from ASCT (median 3.24 months). The analysis was based on microscopic revision for May-Grünwald-Giemsa stained bone marrow smears performed according to standard procedures. BMPC percentage was calculated independently by two observers counting 500 bone marrow total nucleated cells in random areas from two different slides (1000 cells on each patient). Results: Median BMPCs percentage was 0.8 (range 0.1–5.8). Only two patients had more than 3% BPMCs. These results are in contrast with a recent report from the Mayo Clinic group, where 14% of the patients with MM and negative IFE had 5% or more BMPCs. In univariate Cox-model regression analysis, the number of BMPCs significantly correlated with progression-free survival (PFS)(p=0.021) with no impact on overall survival (OS)(p=0.92). This statistical significance on PFS was retained in the multivariate analysis, when baseline prognostic factors such as age, hemoglobin level, serum creatinine, β2-microglobulin and Durie-Salmon stage were added to the model (p=0.003). To establish the best predictive cut-off for progression and survival, a receptor-operator curve (ROC) analysis was developed. It showed the value of 1.5% BMPCs, with a sensitivity of 53%, specificity of 90% and area under the curve of 0.66 for predicting progression. Ten patients had more than 1.5% BMPC, and 25 equal or less than 1.5% BMPC. Median PFS was 8.5 years (CI 95% 2.6 to 14.3) and was not reached in patients with ≤1.5% BMPCs versus 3.1 years in patients with >1.5% BMPCs, with a hazard ratio probability to progression of 3.02 (CI 95% 1.18 to 9.71)(p=0.016) in the group with more than 1.5% of BMPCs (Figure 1). Median OS was not reached in patients with ≤1.5% compared with a median of 9.7 years in those with more than 1.5% BMPCs (p=0.195) (Figure 2). It is likely that serological CR with very low percentage of BMPCs (i.e. ≤1.5%) is equivalent to negative MRD assessed by MFC or molecular studies. In fact, all 8 patients in continued CR between 9 and 16 years beyond ASCT (“operational cures”) are in the group with ≤1.5% BMPCs, while all patients in the group with >1.5% BPMC have relapsed within the first 9 years from ASCT (Figure 1). Conclusion: The percentage of BMPCs in patients with MM in CR after ASCT is a strong predictor of progression. Bone marrow morphology examination is an easy, inexpensive, and non-time consuming test and it should be the first step in the estimation of the residual tumor mass in patients with MM in CR after ASCT. Disclosures: Rosiñol: Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Cibeira:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Blade:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Dynamic Regulation of the Level of Hypoxia In the Bone Marrow Regulates Cell Dissemination In Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4035-4035
Author(s):  
Abdel Kareem A. Azab ◽  
Phong Quang ◽  
Feda Azab ◽  
Brian Thompson ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Progression ◽  
Board Of Directors ◽  
Tumor Burden ◽  
Secondary Antibody ◽  
Advisory Committees ◽  
Hypoxic Conditions ◽  
Mild Hypoxia

Abstract Abstract 4035 INTRODUCTION: The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis, implying that progression of MM occurs through continuous interaction between the BM and MM cells, which controls the ability of MM cells to egress out of the BM and home into new BM niches. We have previously shown that the CXCR4/SDF1 axis as well as Rho GTPases downstream of the receptor was important for chemotaxis, adhesion, homing and egress of MM cells. However, the driving force for MM cells to leave the BM and metastasize to other BM sites is not well understood. Regions of severe oxygen deprivation (hypoxia) arise in tumors due to rapid cell division and are associated with poor patient prognosis, cell motility, associated angiogenesis and metastasis. In this study, we tested the role of hypoxia in the dissemination of MM cells in vivo, as well as regulation of the retention/egress of MM cells in and out of the BM. METHODS: To test the effect of hypoxia on induction of MM egress, MM1s-GFP+/Luc+ cells were injected into 12 SCID mice, and then mice with different stages of tumor development (based on the tumor size detected by bioluminescence) were treated with the hypoxia marker pimonidazole. Blood was drawn and BM was obtained from the femur. Mononuclear cells were then fixed, permeabilized, and stained with antibodies against pimonidazole, followed with an APC- secondary antibody, PE-mouse-anti-human CXCR4, and anti-cadherin antibody followed by an Alexa-Fluor-594 secondary antibody. MM cells in BM and peripheral blood were identified by gating on cells with high GFP signal. To confirm the effects of severe hypoxia found in vivo compared to physiologic mild hypoxia found in the BM, we tested the effect of mild hypoxic conditions (6% O2) and severe hypoxic conditions (0.5% O2) on MM expression of cadherins and CXCR4, as well on functional adhesion of MM cells to stromal cells and chemotaxis. RESULTS: Twelve mice with different stages of MM tumor progression were used. A bi-phasic correlation between tumor progression and the percent of hypoxic cells in BM was found, showing that severe hypoxic conditions in the BM correlated with tumor burden. The correlation between the tumor burden and the number of circulating cells was not linear; however, a direct linear correlation was observed between the number of circulating MM cells and hypoxia in the BM. Moreover, hypoxia in BM correlated directly with the expression of CXCR4 and negatively correlated with the expression of cadherins in MM cells isolated from the BM. To test the effect of the severe hypoxic conditions induced by tumor progression compared to mild hypoxic conditions found physiologically in the BM, we tested the effect of 0.5% O2 (severe hypoxia) and 6% O2 (mild hypoxia) compared to normoxia (21%) on MM cell adhesion to BMSCs, as well as on chemotaxis in response to SDF1, as well as expression of CXCR4 and cadherins. We found that severe hypoxic conditions decreased MM expression of cadherins and adhesion to BMSCs, as well as increased expression of CXCR4 and chemotaxis to SDF1 compared to cells in normoxia. In contrast, mild hypoxic conditions did not alter the expression of CXCR4 and cadherins, adhesion of MM cells to BMSCs, or chemotaxis of MM to SDF1 compared to normoxic cells. CONCLUSION: Hypoxia in the BM directly correlates with the number of circulating MM cells, and with changes in expression of cadherins and CXCR4 in vivo. Severe hypoxic conditions, but not mild hypoxic conditions, induce hypoxic responses in MM cells. Based on these findings, further studies to manipulate hypoxia in order to regulate tumor dissemination as a therapeutic strategy in MM are warranted. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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