Human Multiple Myeloma Patients Research Articles

Molecular characterization stratifies VQ myeloma cells into two clusters with distinct risk signatures and drug responses.

Multiple myeloma (MM) is a cancer of malignant plasma cells in the bone marrow and extramedullary sites. We previously characterized a VQ model for human high-risk MM. The various VQ lines display different disease phenotypes and survival rates, suggesting significant intra-model variation. Here, we use whole-exome sequencing and copy number variation (CNV) analysis coupled with RNA-Seq to stratify the VQ lines into corresponding clusters: Group A cells had monosomy chromosome (chr) 5 and overexpressed genes and pathways associated with sensitivity to bortezomib (Btz) treatment in human MM patients. By contrast, Group B VQ cells carried recurrent amplification (Amp) of chr3 and displayed high-risk MM features, including downregulation of Fam46c, upregulation of cancer growth pathways associated with functional high-risk MM, and expression of Amp1q and high-risk UAMS-70 and EMC-92 gene signatures. Consistently, in sharp contrast to Group A VQ cells that showed short-term response to Btz, Group B VQ cells were de novo resistant to Btz in vivo. Our study highlights Group B VQ lines as highly representative of the human MM subset with ultrahigh risk.

Translational Research of PT-112, a Clinical Agent in Advanced Phase I Development: Evident Bone Tropism, Synergy In Vitro with Bortezomib and Lenalidomide , and Potent Efficacy in the Vk*MYC Mouse Model of Multiple Myeloma

Introduction: PT-112 is a first-in-class platinum-pyrophosphate agent under advanced Phase I development in solid tumors. PT-112 was rationally designed to circumvent the toxicity and mechanisms of resistance associated with conventional chemotherapy. In previous preclinical models using PT-112, multiple cell signaling effects have been observed: p16 mediated G1/S cell cycle arrest; MDM2/p53 expression modulation; extrinsic apoptosis initiation; and signals indicative of immunogenic cell death (Ames et al., Eur. J. Cancer 2016). In addition, the ongoing first-in-human PT-112-101 Phase I trial in solid tumors has demonstrated a positive therapeutic index, with a range of biological activity observed at well-tolerated dose levels and a lack of severe hematotoxicity (Karp et al., J. Clin. Oncol. 2017).Due to the pyrophosphate present in PT-112, we hypothesized that PT-112 should localize to bone tissue, and be efficacious against bone marrow malignancies such as multiple myeloma (MM). Biodistribution was measured via laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS), along with translational experimentation in the orthotopic Vk*MYC mouse model of MM. In addition, an assessment of synergy was conducted in vitro in combination with standard of care (SoC) agents such as bortezomib (BOR) and lenalidomide (LEN).Methods: In vitro - Cell viability and IC50 values in MM1R and RPMI-8226 suspension cells were measured after treatment with PT-112, LEN, BOR, or PT-112-containing combinations thereof. Combination indices to assess synergy were calculated using the Chou-Talalay method.Biodistribution - After a single dose with 90 mg/kg PT-112 or vehicle control via tail vein injection, mice were euthanized 45min or 24h post-dose and snap frozen. Slides were prepared and LA-ICP-MS was used to measure and image the presence and concentration of platinum (Pt) in full sagittal plane cross sections, and in individual organs and tissues at higher spatial resolution.In vivo - Genetically engineered de novo Vk*MYC mice with established MM were treated with either 100 mg/kg PT-112 twice weekly (n=2) or 67 mg/kg (n=1) thrice weekly via intraperitoneal injection. In parallel experiments, mice engrafted with the BOR resistant Vk12598 transplantable line received vehicle (n=11) or PT-112 at 62.5 mg/kg (n=12) twice/week. M-spike levels were monitored weekly during treatment and for several weeks after.Results: In vitro, the combination of PT-112 with LEN and BOR resulted in strong synergy in both RPMI-8226 and dexamethasone-resistant MM1R multiple myeloma cells, as indicated by combination indices as low as 0.0029. In vivo, PT-112-derived Pt was observed in kidney, lung, liver and other tissue types, but most prominently in bone. All bone tissue observed had high concentrations of Pt, with the highest amount reaching ~15mM, a concentration >103 fold higher than the IC50 of PT-112 in in vitro cell viability experiments.PT-112 treatment induced a response in all of the de novo Vk*MYC mice with a >50% reductions in M-spike at the 2-week time point, passing the statistically correlated activity threshold indicative of robust activity in human MM patients (Chesi et al., Blood 2012). At this point treatments were discontinued, and M-spikes levels continued to decline for 1-2 weeks, with the lowest level observed at 19% of baseline at 11 days after treatment discontinuation. Importantly, we also noted pronounced single agent PT-112 activity against the BOR refractory Vk12598 tumors, resulting in statistically significant improved overall survival. In vivo combination studies are ongoing.Conclusions: Sophisticated biodistribution imaging techniques reveal the presence of PT-112 derived Pt in several target tissues, with a demonstrably preferential accumulation in bone tissue. These biodistribution results likely explain in part the strong efficacy observed in Vk*MYC mice. Given the predictive power of this model, the likelihood of PT-112 activity in human MM patients is high. Additionally, the potent synergy observed in in vitro systems provides encouraging evidence that PT-112 might combine well with SoC agents in treatment of MM. Taken together, these data provide a strong rationale for study of PT-112 as both a single agent and in combination in MM patients, with a further prospect for investigating other hematological malignancies. DisclosuresAmes:Phosplatin Therapeutics: Employment, Equity Ownership. Sharik:Phosplatin Therapeutics: Research Funding. Rather:Phosplatin Therapeutics: Research Funding. Hochart:Phosplatin Therapeutics: Research Funding. Bonnel:Phosplatin Therapeutics: Research Funding. Linehan:Phosplatin Therapeutics: Research Funding. Stauber:Phosplatin Therapeutics: Research Funding. Wing:Phosplatin Therapeutics: Employment, Equity Ownership. Jimeno:Phosplatin Therapeutics: Employment, Equity Ownership. Medina:Phosplatin Therapeutics: Research Funding. Bertino:Phosplatin Therapeutics: Research Funding. Chesi:Phosplatin Therapeutics: Research Funding. Bergsagel:Phosplatin Therapeutics: Research Funding.

Validation of a Sensitive Flow Cytometry Assay to Assess Minimal Residual Disease of Multiple Myeloma Cells in Apheresis Product

For treatment of patients with multiple myeloma (MM), flow cytometry has become a widely used and valuable method for the evaluation of minimal residual disease (MRD) in bone marrow. Use of an optimized single-tube, 10-color flow cytometry panel for assessment of MM MRD has shown to be beneficial in patient monitoring and has shown correlation to the acknowledged EuroFlow 8-color, 2-tube method. In order to further correlate levels of MM and relapse in patients, validation of a sensitive MM MRD assay that can be applied to testing for residual disease in apheresis product prior to autologous stem cell transplant, a standard treatment for patients with multiple myeloma. This approach can exhibit great value in patient monitoring and treatment. As new combination therapies are developed for treatment of multiple myeloma in concert with autologous stem cell transplantation, evaluation of MRD in apheresis will likely continue to grow in importance. The purpose of this study is to show the validation and sensitivity of a flow cytometry assay designed to detect Multiple Myeloma (MM) cells in G-CSF and other mobilized apheresis samples from human Multiple Myeloma patients. Using GSM-mobilized apheresis product, spiked with different levels of patient de-identified MM plasma cells, validation of a 10-color MM MRD panel can be evaluated, intended to mimic patient apheresis at varying levels of residual disease post treatment. As with any minimal residual disease assessment, a high number of events are required to ensure sensitivity and precision. Typically, acquisition of 3-5 x 107 events is required to ensure precision of MM MRD levels to 0.001%. DisclosuresNo relevant conflicts of interest to declare.

SGN-CD48A: a Novel Humanized Anti-CD48 Antibody-Drug Conjugate for the Treatment of Multiple Myeloma

Multiple myeloma (MM) is an incurable hematologic malignancy of transformed plasma cells. New targeted biological therapeutics are needed to increase the stringency and durability of remissions. In this study we describe SGN-CD48A, a potent CD48-targeting antibody-drug conjugate (ADC) utilizing a novel glucuronide-monomethylauristatin E (MMAE) linker, under development for the treatment of MM. CD48, or SLAMF2 (Signaling Lymphocyte Activation Molecule family member 2), is a GPI-anchored membrane protein in the SLAM family of immunoreceptors. CD48 is expressed on B and T lymphocytes, natural killer (NK) cells, and other immune cell types where it functions to modulate immune cell activation, proliferation, and differentiation. CD48 is also a tumor antigen broadly expressed in MM. We observed CD48 expression on the surface of malignant plasma cells in 90% (90/100) of human multiple myeloma patient samples examined by flow cytometry. Monoclonal antibodies (mAbs) specific for human CD48 were evaluated and a lead antibody was selected based on binding characteristics and cytotoxic activity against myeloma cells as an auristatin ADC. SGN-CD48A is a humanized anti-CD48 mAb to which eight molecules of MMAE, a potent microtubule disrupting cytotoxic drug, have been conjugated via a β-glucuronidase-cleavable linker. This novel glucuronide-MMAE drug-linker incorporates a PEG side chain and self-stabilizing maleimide to achieve homogenous drug-to-antibody ratio (DAR) 8 conjugates with decreased plasma clearance and increased preclinical antitumor activity. Following binding of CD48 at the myeloma cell surface, SGN-CD48A internalizes and traffics to lysosomal vesicles. Intracellular MMAE drug released from SGN-CD48A in myeloma cells induced cell cycle arrest at G2/M phase, phospho-histone H3 (Ser-10) phosphorylation, and caspase 3/7 dependent apoptotic cell death. SGN-CD48A demonstrated potent cytotoxic activity (EC50 values 1.0 - 11 ng/mL) against a panel of human MM cell lines, with CD48 expression levels of 135,000 - 480,000 receptors per cell. In contrast, SGN-CD48A had negligible cytotoxic activity against normal resting human B, NK, and T lymphocytes. We evaluated the in vivo antitumor activity of SGN-CD48A in disseminated MM cell line mouse xenograft models. In the NCI-H929 and EJM xenograft models, a single intraperitoneal dose of 0.3 mg/kg SGN-CD48A produced durable complete remissions in 8/8 and 6/8 mice, respectively. Similarly, in the U-266 xenograft model, a single dose of 1.0 mg/kg SGN-CD48A produced durable complete remissions in 7/8 mice. Neither unconjugated mAb nor a non-binding control MMAE ADC were active in these MM xenograft models, demonstrating that targeted delivery of MMAE drug through CD48 binding is required for activity. In summary, CD48 is a highly expressed new multiple myeloma target and the novel glucuronide-MMAE ADC SGN-CD48A shows potent antitumor activity against cell line models of MM. DisclosuresLewis:Seattle Genetics, Inc.: Employment, Equity Ownership. Olson:Seattle Genetics, Inc.: Employment, Equity Ownership. Gordon:Seattle Genetics, Inc.: Employment, Equity Ownership. Sandall:Seattle Genetics, Inc.: Employment, Equity Ownership. Quick:Seattle Genetics, Inc.: Employment, Equity Ownership. Finn:Seattle Genetics, Inc.: Employment, Equity Ownership. Westendorf:Seattle Genetics, Inc.: Employment, Equity Ownership. Linares:Seattle Genetics, Inc.: Employment, Equity Ownership. Leiske:Seattle Genetics, Inc.: Employment, Equity Ownership. Nesterova:Seattle Genetics, Inc.: Employment, Equity Ownership. Law:Seattle Genetics, Inc.: Employment, Equity Ownership.

Abstract 1195: SGN-CD352A: A novel humanized anti-CD352 antibody-drug conjugate for the treatment of multiple myeloma

Abstract Multiple myeloma (MM) is a hematologic malignancy of transformed plasma cells. In spite of recent advances, MM remains an incurable disease, underscoring the need to develop new targeted biological therapeutics to augment existing treatments. In this study we describe SGN-CD352A, a potent new CD352-targeting antibody-drug conjugate (ADC) under development for the treatment of MM. CD352, or SLAMF6 (Signaling Lymphocyte Activation Molecule family member 6), is a type 1 membrane protein in the SLAM family of immunoreceptors. Like other SLAM family members, CD352 is a positive regulator of natural killer (NK) cell functions. CD352 is also a tumor antigen expressed on B cell malignancies such as MM, chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). We observed CD352 expression on the surface of malignant plasma cells in 87% (13/15) of human multiple myeloma patient samples examined by flow cytometry. Monoclonal antibodies (mAbs) specific for human CD352 were produced and a lead antibody was selected based on affinity, endocytic internalization rate, and tumor cell cytotoxic activity as an ADC. SGN-CD352A is a humanized anti-CD352 engineered cysteine (ec) mAb (h20F3ec) to which two molecules of pyrrolobenzodiazepine (PBD) dimer, a potent DNA damaging cytotoxic drug, have been conjugated. Upon binding CD352 at the MM cell surface, SGN-CD352A undergoes rapid clathrin-dependent endocytosis (< 2 hours) and traffics to lysosomal vesicles. PBD dimers released from SGN-CD352A in lysosomes induce a dose dependent DNA damage signaling response in MM cells, activating ATM and ATR kinases, and caspase 3/7 dependent apoptotic cell death results within 48 hours. SGN-CD352A demonstrated potent cytotoxic activity (EC50 values 6.6 - 52.6 pM) against a panel of human myeloma and lymphoma cell lines, with efficient cancer cell killing observed at CD352 expression levels as low as 3,500 receptors per cell. In contrast, SGN-CD352A had no effect on the viability of resting human T lymphocytes and minimal cytotoxic activity on B lymphocytes. We evaluated the in vivo antitumor activity of SGN-CD352A in disseminated MM cell line mouse xenograft models. In the MM.1R xenograft model, a single intraperitoneal dose of 30 μg/kg SGN-CD352A produced durable complete remissions in 10/10 mice. Similarly, in the U-266 xenograft model, a single dose of 100 μg/kg SGN-CD352A produced durable complete remissions in 9/10 mice and significant (P <0.0001) tumor delay at a single dose of 30 μg/kg. Neither unconjugated h20F3ec mAb nor a non-binding control PBD dimer ADC produced remissions in these MM xenograft models, demonstrating that targeted delivery of PBD dimer drug through CD352 binding is required for activity. In summary, CD352 is a newly validated multiple myeloma tumor antigen and the novel PBD dimer ADC SGN-CD352A shows potent antitumor activity against cell line models of MM at clinically relevant doses. Citation Format: Tim Lewis, Devra J. Olson, Kristine A. Gordon, Sharsti L. Sandall, Jamie Miyamoto, Lori Westendorf, Germein Linares, Chris Leiske, Heather Kostner, Ivan Stone, Martha Anderson, Albina Nesterova, Mechthild Jonas, Che-Leung Law. SGN-CD352A: A novel humanized anti-CD352 antibody-drug conjugate for the treatment of multiple myeloma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1195.

Characterization of the Role of Regulatory T Cells (Tregs) in Inducing Progression of Multiple Myeloma

Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis.Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry.Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs.Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P<0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P<0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P<0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P<0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P<0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs.Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. DisclosuresNo relevant conflicts of interest to declare.

Relationship between tumour necrosis factor-related apoptosis inducing ligand (TRAIL) and vascular endothelial growth factor in human multiple myeloma patients.

Tumour necrosis factor-alfa (TNF-α) is an inflammatory cytokine with a wide spectrum of biological activity, including angiogenesis. Tumour necrosis factor-related apoptosis inducing ligand (TRAIL), which belongs to the TNF family of proteins, plays a role in the regulation of vascular responses, but its effect on the formation of new blood vessels (angiogenesis) is unclear. We analysed TRAIL concentrations in parallel with pro-angiogenic cytokines in serum and their expression in trephine biopsy (TB) in 56 patients with newly diagnosed IgG MM and 24 healthy volunteers. The study showed statistically higher concentrations of TRAIL and TNF-α, as well as of VEGF and its receptor, in MM patients compared to healthy volunteers and patients in advanced stages of the disease. Furthermore, we observed a significant decrease in all studied pro-angiogenic cytokines and significant increase of TRAIL concentration after anti-angiogenic therapy, with meaningful differences between responders (at least partial remission) and patients with progression during the induction treatment. It was also established that TRAIL correlated statistically and negatively with pro-angiogenic cytokines such as VEGF with its receptor and expression of VEGF and syndecan-1 in TB. In summary, our data indicate that in MM patients, both clinical course and treatment responsiveness are associated with dynamic yet corresponding changes of levels of TRAIL parallel pro-angiogenic mediators such as VEGF with its receptor and expression of VEGF and syndecan-1 in TB.

Vitamin A: An unexpected role in B cell cancers

T deregulation of B cell differentiation has been shown to contribute to autoimmune disorders, hematological cancers, and aging. We provide evidence that the retinoic acid-producing enzyme aldehyde dehydrogenase 1a1 (Aldh1a1) is an oncogene suppressor in specific splenic B cells populations. Aldh1a1 regulated transcription factorsand oncogene suppressorsretinoic acid receptor alpha (Rara), zinc finger protein Zfp423, and peroxisome proliferator-activated receptor gamma (Pparg) insplenocytes. In Aldh1a1-/mice, splenic IgG1+/CD19and IgG1+/CD19+ B cells acquired expression of proto-oncogenic genes c-Fos, c-Jun, and Hoxa10 that resulted in splenomegaly. Human multiple myeloma patients and multiple myeloma B cell lines also lack Aldh1a1 expression. Ectopic Aldh1a1 expression rescued these cells. Our data highlight a mechanism by which vitamin A metabolism protects B cellsfromoncogenesis.

Low Expression of CXCR4 in Bortezomib-Resistant Multiple Myeloma Correlates with Extramedullary Disease in a Murine Mouse Model

AbstractAbstract 442The tumor microenvironment is a critical component of cancer growth involved in initiation, progression, metastasis and chemoresistance. This interplay has been described in the plasma cell neoplasm, multiple myeloma (MM), in which the relationship between transformed cells and the bone marrow milieu aids in maintaining cell viability and, in the case of dexamethasone, confers resistance (Abe, M. Int. J. Hematol. (2011) 94: 334). With the advent of new drugs for the treatment of MM, it is critical that we further define what role the MM niche plays in chemosensitivity.The proteasome inhibitor bortezomib/VELCADE® (Bz) is approved for the treatment of MM and relapsed mantle cell lymphoma and has been widely used, with much success, in patients. However, despite these advances in the clinic, the majority of MM patients treated with Bz eventually relapse, many as non-responders to additional Bz treatments. Chemokine (C-X-C motif) receptor (CXCR4) is a transmembrane, G-protein coupled receptor that is known to be expressed on both normal and malignant plasma cells and is involved in B cell homing to the bone marrow where its ligand SDF-1 is expressed (Nakayama, et al. J. Immunol. (2003) 170: 1136). It was reported that disruption of the MM cell-stroma interaction through chemical inhibition of the cell-surface protein CXCR4 increases Bz sensitivity both in vitro in the presence of bone marrow stromal cells and in vivo(Azab, et al. Blood (2009) 113: 4341). Based on this observation, we hypothesized that genes involved in bone marrow homing would be highly expressed in Bz resistant cells.To determine whether genes involved in bone marrow homing are differentially expressed in Bz sensitive and resistant cells, we utilized isogenic pairs of Bz sensitive and resistant MM cell lines derived from the genetically engineered iMycCα/Bcl-xL mouse model of plasma cell malignancy (Boylan, et al. Cancer Res. (2007) 67: 4069). Initial gene expression profiling of three pairs of mouse cell lines in the absence of Bz revealed significantly decreased expression of Cxcr4 in Bz resistant lines (fold change>2, p<0.05). This observation was validated by flow cytometry in both the Bz resistant mouse lines as well as a derived resistant human MM cell line (MM1.S Bz resistant). Furthermore, when we queried human MM patient GEPs (Zhan, et al. Blood (2006) 108: 2020), we found that low CXCR4 expression was associated with both high-risk and relapsed MM. Based on CXCR4 expression alone we could predict better event-free (p=0.018) and overall survival (p=0.012) in patients with high levels of CXCR4 receiving therapy.To determine whether low CXCR4 expression in the Bz resistant lines correlates with altered bone marrow homing in vivo, isogenic pairs of Bz sensitive and resistant mouse cell lines were transferred back into immunocompetent, syngeneic animals. The Bz resistant cells conferred a more severe phenotype, killing the mice more quickly than sensitive control cells (16 days median survival). Interestingly, FDG-PET and FLT-PET imaging revealed that in contrast to Bz sensitive cells, which showed characteristic homing to the bone marrow, Bz resistant cells resulted primarily in extramedullary disease. These findings strongly suggest that low CXCR4 expression serves as a biomarker for Bz resistance and extramedullary disease in the iMycCα/Bcl-xL mouse model of human myeloma. Disclosures:Stessman:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

The Murine Vk*MYC Myeloma Shares Defining Genetic Lesions with Human Multiple Myeloma.

Abstract 1808Poster Board I-834We have developed the first mouse model of multiple myeloma (MM), Vk*MYC, in an immune competent mouse, with indolent plasma cell (PC) growth confined to the bone marrow, and in which the major biological and clinical features of the human disease are recapitulated, including the post germinal center origin of the tumor cells, the presence of a M-spike in the serum, anemia, diffuse bone disease and drug sensitivity.To further characterize karyotypically and phenotypically the Vk*MYC tumors, we performed gene expression profile (GEP) and aCGH analysis on CD138+ purified MM cells from Vk*MYC mice. For GEP, we analyzed 33 individual tumors on the Affymetrix 430_2 array for which publicly available datasets can be used to compare levels of gene expression. For aCGH, we used the Agilent 244K array to analyze 19 individual tumors.By GEP we first confirmed the PC origin of the Vk*MYC tumors, that clearly express the typical PC markers, including CD138, IRF4, BLIMP, XBP1, IgG or IgA, but not B cell markers like IgM, IgD, PAX5, or BCL6. Next, we analyzed the expression of the three cyclin D, that are almost universally dysregulated in human myeloma patients and cell lines, but are not expressed in normal human or murine PCs. We found that the Vk*MYC tumors express high levels of cyclin D2, and occasionally cyclin D1, but never cyclin D3 that, instead, is commonly expressed in B cells. Interestingly, in one sample lacking any D type cyclin expression we detected loss of Rb expression (and homozygous deletion of the gene, see below), as in the “TC none” group of human MM patients, highlighting once again the importance of this pathway in both human and mouse myeloma.aGCH analysis indicated that the genomic complexity of Vk*MYC tumors is low, and complex rearrangements were rarely observed. In contrast with the recurrent trisomy of odd number chromosomes present in approximately 50% of human MM patients, we found no evidences of hyperdiploidy in Vk*MYC mice, except perhaps in one tumor with trisomy 15 and 17. Instead, we detected monosomy 5 in 10/19, monosomy 14 in 4/19 and trisomy 16 in 4/19 tumors. Interestingly a large portion of the murine chr.14 is syntenic to the human chr.13, frequently lost in MM patients. After monosomy 13, the most frequent chromosomal change in human MM is loss of a sex chromosome. In the mouse, the most common numerical abnormality identified was loss of a sex chromosome: 10/11 male had loss of Y and 5/8 female had loss of X.From the analysis of aCGH data publicly available on the MMRC portal, we have generated a list of the most common bi-allelic deletions present in human myeloma patients and cell lines. Interestingly, several of these lesions are also present in Vk*MYC tumors. The Rb1 gene, frequently affected by monosomy 13 in human MM, is bi-allelically lost in 3/19 Vk*MYC tumors. In one case, we also detected a focal monoallelic deletion encompassing Rb1, in addition to the four tumors with monosomy 14. Other focal bi-allelic deletion identified are: Cdkn2a (p16 and p19ARF), FHIT and PTEN. The latter was found in a Vk*MYC tumor selected in-vivo to be bortezomib resistant. On chr.X we identified recurrent (5/19 tumors) bi-allelic deletions of UTX, the histone demethylase gene recently reported to be deleted in various cancers, including MM tumors and human myeloma cell lines. While we are in the process of performing functional studies in human cell lines to identify a role for UTX in myeloma pathogenesis, we took advantage of the relative genetic uniformity of the Vk*MYC mice to identify by GEP a signature associated with UTX lost. We separated our cohort of Vk*MYC tumors for which GEP data are available in two groups based on expression of UTX and generated by volcano plot a list of genes that distinguished the two groups. Strikingly, loss of UTX was associated with loss of expression of a subset of lymphoid genes and acquisition of a “macrophage signature”, suggesting a role for UTX in lineage commitment. These data provide a genetic basis for tumor plasticity similar to epithelial mesenchymal transition observed in some solid tumors, in which the tumor gradually acquires supporting characteristics of its microenvironment.We conclude that the Vk*MYC provide a remarkably faithful model of MM not only phenotypically but also genetically, that provides an excellent framework for functional characterization of the molecular pathogenesis of MM DisclosuresBergsagel:Genentech: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding.

Detection of Interleukin-1β and Interleukin-6 in Human Multiple Myeloma by Fluorescent in situ Hybridization.

Using fluorescent in situ hybridization together with cell surface marker staining, we studied the expression of mRNA of IL-6 and mRNA of IL-1β in bone marrow samples from human multiple myeloma patients. It is known that IL-6 can stimulate B cell growth and differentiation and recently it has been suggested that IL-6 is responsible for autocrine growth stimulation of myeloma cells and that IL-1 may play a role in bone resorption. These interleukins have previously been detected in the supernatants of cultured myeloma cells. Here we report the expression of IL-1β mRNA by plasma cells, T cells and macrophages according to morphology and immunologic marker analysis, suggesting that not only myeloma cells but other cell types can also contribute to the production of IL-1β and thus to bone-resorption. IL-6 mRNA could not be detected in plasma cells from bone marrow aspirates but were present in monocytes and T cells, suggesting that in vivo IL-6 stimulates the growth of myeloma cells in a paracrine instead of an autocrine way.

Multiple myeloma: an immunologic profile. Cytotoxic and suppressive effects of the EA rosette-forming cell.

Lymphoid cells bearing receptors for human IgG anti-Rh antibody but not otherwise identifiable as T or B cells were isolated from the peripheral blood of human multiple myeloma patients. These cells do not demonstrate surface immunoglobulin, do not secrete immunoglobulin in culture after stimulation with pokeweed mitogen, and do not possess receptors for the third component of complement. They do not form rosettes with sheep erythrocytes, and do not respond to known T cell mitogens. These cells are not phagocytic and do not possess demonstrable nonspecific esterase. Thus, they must be considered at present, members of the "third population" lymphoid cells. These cells are active in the cytotoxic response by myeloma peripheral blood lymphocytes to myeloma plasma cells accounting for approximately 60% of such activity. They are also responsible for part of the suppression of pokeweed mitogen-stimulated immunoglobulin production, and at equivalent suppressor to target cell ratios are more active than immunoregulatory monocytes. Thus, these cells play an important, not previously widely appreciated role in the pathophysiology of the human myeloma patient.