Poor Prognosis In Multiple Myeloma Research Articles

Small-molecule multi-targeted kinase inhibitor RGB-286638 triggers P53-dependent and -independent anti-multiple myeloma activity through inhibition of transcriptional CDKs

Small molecule multi-targeted CDK inhibitors (CDKIs) are of particular interest due to their potent antitumor activity independent of p53 gene alterations. P53 deletion is associated with a very poor prognosis in multiple myeloma (MM). In this regard, we tested the anti-MM activity of RGB-286638, an indenopyrazole-derived CDKI with Ki-nanomolar activity against transcriptional CDKs. We examined RGB-286638’s mode-of-action in MM cell lines with wild type (wt)-p53 and those expressing mutant p53. RGB-286638 treatment resulted in MM cytotoxicity in vitro associated with inhibition of MM tumor growth and prolonged survival in vivo. RGB-286638 displayed caspase-dependent apoptosis in both wt-p53 and mutant-p53 cells that was closely associated with the downregulation of RNA polymerase II phosphorylation and inhibition of transcription. RGB-286638-triggered p53 accumulation via nucleolar stress and loss of Mdm2, accompanied by induction of p53 DNA binding activity. Additionally, RGB-286638 mediated p53-independent activity, which was confirmed by cytotoxicity in p53-knockdown and p53-mutant cells. We also demonstrated downregulation of oncogenic miR-19, miR-92a-1, and miR-21. Our data provide the rationale for the development of transcriptional CDK inhibitors as therapeutic agents, which activate p53 in competent cells, while circumventing p53 deficiency through alternative p53-independent cell death mechanisms in p53-mutant/deleted cells.

Down-regulation of the cancer/testis antigen 45 (CT45) is associated with altered tumor cell morphology, adhesion and migration

BackgroundDue to their restricted expression in male germ cells and certain tumors, cancer/testis (CT) antigens are regarded as promising targets for tumor therapy. CT45 is a recently identified nuclear CT antigen that was associated with a severe disease score in Hodgkin’s lymphoma and poor prognosis in multiple myeloma. As for many CT antigens, the biological function of CT45 in developing germ cells and in tumor cells is largely unknown.MethodsCT45 expression was down-regulated in CT45-positive Hodgkin’s lymphoma (L428), fibrosarcoma (HT1080) and myeloma (U266B1) cells using RNA interference. An efficient CT45 knock-down was confirmed by immunofluorescence staining and/or Western blotting. These cellular systems allowed us to analyze the impact of CT45 down-regulation on proliferation, cell cycle progression, morphology, adhesion, migration and invasive capacity of tumor cells.ResultsReduced levels of CT45 did not coincide with changes in cell cycle progression or proliferation. However, we observed alterations in cell adherence, morphology and migration/invasion after CT45 down-regulation. Significant changes in the distribution of cytoskeleton-associated proteins were detected by confocal imaging. Changes in cell adherence were recorded in real-time using the xCelligence system with control and siRNA-treated cells. Altered migratory and invasive capacity of CT45 siRNA-treated cells were visualized in 3D migration and invasion assays. Moreover, we found that CT45 down-regulation altered the level of the heterogeneous nuclear ribonucleoprotein syncrip (hnRNP-Q1) which is known to be involved in the control of focal adhesion formation and cell motility.ConclusionsProviding first evidence of a cell biological function of CT45, we suggest that this cancer/testis antigen is involved in the modulation of cell morphology, cell adherence and cell motility. Enhanced motility and/or invasiveness of CT45-positive cells could contribute to the more severe disease progression that is correlated to CT45-positivity in several malignancies.

Carbamazepine and Hematological Malignancies

Received/Gelis tarihi : May 1, 2013 Accepted/Kabul tarihi : June 10, 2013 To the Editor, The letter entitled “Carbamazepine and Multiple Myeloma: Possible Interaction”, written by Gunaldi et al. [1] and published in one of the recent issues of your journal, was quite interesting. We would like to emphasize some points about that letter. In the reported case, the patient’s serum globulin level was 5.44 g/dL, and serum protein electrophoresis (Figure 1 of the cited letter) showed that the M-peak was closest to the beta-fraction. Thus, it becomes important to know the level of light chains and IgA as heavy chains. The serum protein electrophoresis should also be evaluated for biclonal gammopathy due to its pattern shown in Figure 1. Interleukin-6 (IL-6) is the main growth factor for multiple myeloma (MM) and studies of epileptic patients showed elevated levels of IL-6 after carbamazepine therapy [2]. We have no information about the level of IL-6 in the case reported by Gunaldi et al. The specific abnormalities such as t(4;14), t(14;16), and deletion (del) 17p detected by fluorescence in situ hybridization (FISH) analysis were reported as high-risk MM [3]. Tricot et al. [4] reported that partial or complete deletions of chromosome 13 were associated with poor prognosis in MM. Chromosome 13 deletions detected only by FISH independently in the absence of other abnormalities do not carry significantly higher risk, whereas t(11;14) does not predict superior outcome [5]. It is interesting to see cytogenetic abnormalities del17p-t(4;14)-del13 and t(11;14) in same patient. However, the consensus is that the data are not yet adequate to suggest routine use of these FISH markers to predict prognosis [5]. The number of people who had suffered from side effects due to carbamazepine was reported as 14,705. Ten of them (0.07%) had MM [6]. Drug-related (although rarely from carbamazepine) hypogammaglobulinemia is a well-known condition. 9-Acridine carboxaldehyde is one of the carbamazepine metabolites generating activated neutrophils, and presumably monocytes. This metabolite increases lymphocyte proliferations at lower concentrations and decreases them at higher concentrations. The relationship between hypogammaglobulinemia and carbamazepine is thus clearer [7]. There have also been reports of agranulocytosis, leucopenia, pure erythrocyte aplasia, and thrombocytopenia after carbamazepine [8]. Additionally, phenytoin is considered a possible carcinogenic for humans and was reported as a carcinogenic drug in animals [9]. A diagnosis of epilepsy in the same year as a cancer diagnosis carried an increased risk for leukemia, pancreatic cancer, non-Hodgkin’s lymphoma, acute myeloid leukemia, and Hodgkin’s disease, but not for chronic lymphatic leukemia or MM. The issue of cancer incidence in people with epilepsy remains an open question [9,10]. In conclusion, the answer is still unknown as to whether the drugs for epilepsy or epilepsy itself causes MM.

Phase II Study of the c-Met Inhibitor ARQ 197 (Tivantinib) in Patients with Relapsed Multiple Myeloma.

Abstract 2976 Background:c-Met receptor tyrosine kinase (RTK) activity has been implicated in establishing the oncogenic phenotype across several human cancers with high levels of the activating c-Met ligand, hepatocyte growth factor (HGF). Malignant plasma cells secrete HGF-activator (HGFA), which converts HGF to its active form, and high HGF levels are correlated with a poor prognosis in multiple myeloma (MM). Syndecan 1 (CD138) on malignant plasma cells binds HGF and potentiates interleukin-6-induced growth and migration. HGF stimulation of myeloma cells also activates autophosphorylation of c-Met and other critical downstream signaling pathways promoting oncogenesis. Finally, pre-clinical studies have shown that suppression of c-Met signaling with a number of small molecules, including ARQ 197, induced myeloma cell apoptosis. Tevantinib-mediated cytotoxic response was observed at concentrations of less than 5 μM, which are achievable in the clinic. These findings supported the hypothesis that suppression of the HGF/c-Met signaling axis could be a rational strategy against relapsed multiple myeloma. Methods:In this phase II study, the efficacy and safety of ARQ 197, a non-competitive and highly selective inhibitor of the c-Met RTK, is being studied in patients with relapsed multiple myeloma. Primary objectives were to determine the overall response rate (ORR) to single-agent tivantinib in patients with relapsed multiple myeloma who had received one to four prior lines of therapy, and to define the toxicities in this population. ARQ 197 was administered at a starting oral dose of 360 mg twice daily with meals for each day of every 4-week treatment cycle. This dose was selected from prior phase I investigations in solid tumors, and at this dose level, steady-state plasma level sof ARQ-197 were 7 μM. Treatment could continue providing that patients did not experience undue toxicities, or disease progression. Tivantinib is provided through the Cancer Therapy Evaluation Program (CTEP), and this study is supported by CTEP, as well as the M. D. Anderson Cancer Center SPORE in Multiple Myeloma. Results:A total of 10 patients have been enrolled and treated to date, all of whom were evaluable for toxicity, with 8 evaluable for response based on having completed two treatment cycles. Patients had received from 1–3 prior lines of therapy for their disease, and 7/10 (70%) had presented with International Staging System stage I disease at diagnosis. All patients on study had an ECOG performance status of 1 or better, and received a median of 3.5 cycles of tivantinib (range 1–7). The most common adverse events (AEs) of any grade seen in at least 30% of patients included diarrhea (30%), dizziness (30%), dry eyes (30%), shortness of breath (30%), memory change (30%), myalgias (40%), fatigue (60%), and neutropenia (60%). Serious AEs (SAEs) occurred in 2 patients, including one patient with grade 3 syncope, and another with grade 4 neutropenia and a grade 3 anal fissure. Stable disease (SD) has been seen as the best response in 5/7 (71%) evaluable patients, which was maintained for up to 7 cycles, while the remaining patients showed evidence of disease progression. Conclusion:Enrollment is continuing to this first study of any c-Met inhibitor in patients with relapsed multiple myeloma to better define the role of single-agent tivantinib in this setting. To date, tivantinib has been tolerated well, and some evidence of activity has been seen, with stable disease in 63% of patients, all of whom were progressing at the time of enrollment. Updated toxicity and efficacy data will be presented at the time of the Annual Meeting. Correlative studies are also underway with the goal of identifying potential predictive biomarkers. Disclosures:Off Label Use: Tivantinib is being evaluated for patients with relapsed myeloma, but is not yet approved in this setting.

The Antimalarial Agent Artesunate Overcomes Bortezomib Resistance in Myeloma Cell Lines Through Non-Caspase Mediated Apoptosis

AbstractAbstract 4015During terminal differentiation of a B-lymphocyte to a mature plasma cell, the protein load increases considerably while the proteasome capacity decreases. The fact that plasma cells operate close to the limit of their proteasomal capacity is being exploited therapeutically and proteasome inhibition has become one of the pillars of myeloma treatment. The importance of the proteasome in Multiple Myeloma (MM) has also been established in prognostic terms (1). De novo or acquired resistance to proteasome inhibitors (PI) confers poor prognosis in MM. In vitro bortezomib resistance is accompanied by cross-resistance to other known PIs, thus highlighting (2), the need for treatment alternatives.Based on reports that various antimalarial drugs exhibit in vitro anti-MM activity through either α-ring proteasome inhibition (mefloquine) or aggresome formation inhibition (quinine), we examined whether Artesunate (ART), a new highly effective antimalarial drug with a known excellent safety profile, could overcome resistance to PI’s currently in clinical use.Bortezomib (BZ)-resistant sublines of the BZ naïve and sensitive JJN3 and U266 cell lines were developed by exposure to progressively increasing BZ concentrations. Eventually the JJN3BR and U266BR sublines were developed with a 20- and 25-fold IC50 increase, respectively, compared to their BZ naïve counterparts. ART was able to inhibit viability in clinically achievable dosages and in a time-dependent manner in IL-6-independent and -dependent MM cell lines (Table 1). JJN3BR and U266BR showed no evidence of cross-resistance compared to their parental cell lines. The RPMI 8226 cell line known to exhibit one of the highest BZ IC50s among MM cell lines proved to be among the most sensitive to ART. The in-vitro antiMM effectiveness of ART was evident also in primary CD-138+ MM cells from six (6) patients with Relapsed/Refractory MM (Table 2). ART in-vitro anti MM activity was also verified by Annexin-V and 7-AAD flow cytometry for its ability to induce both early and late apoptosis.To elucidate ART's mode of action, we measured the sequence and level of caspase activation through the luminescence assay Caspase-Glo. ART exposure initiated the extrinsinc pathway of apoptosis with a brisk increase in activated caspase-8 levels and effector caspase3/7 levels within 3h, followed by caspase 9 activation at 6h. However, actual apoptosis by ART was not accompanied by a marked caspase activity especially when compared to other potent anti-MM agents. The pan-caspase inhibitor Z-VAD-MFK effectively inhibited caspase activation by ART but failed to inhibit ART's effect on viability, as at least 70% of ART's effect was retained in JJN3, U266 and RPMI 8226 cells. Similar results were obtained in flow cytometry-based apoptosis assays. Immunoblotting of the mitochondrial derived non-caspase mediating apoptosis factors AIF, EndoG and HtrA2 in ART treated JJN3 cells revealed that ART-mediated apoptosis was related to the cytoplasmic and subsequently nuclear translocation of AIF. Conclusion:ART shows marked anti-MM activity and overcomes BZ resistance in vitro. Its benign side effect profile support its potential for myeloma therapy. Most anti-cancer agents induce apoptosis in a caspase-dependent manner. ART's mode of action is mainly caspase-independent, indicating that its role in overcoming drug resistance, could also extend to other anti-MM agents. Experiments aimed at further elucidating the mechanism of action of ART and the evaluation of drug combinations including ART are currently under way.Table 1ART IC50 on various MM cell linesCell LineIC50μM (48h)IC50μM (72h)JJN320.716.4JJN3BR19.515.2U26628.218.6U266BR29.719.5KMS-1130.124.3RPMI-82268.76.7INA-62718.9ARH-7736.732.3Table 2ART IC50 on MM primary cellsMM primary cellsIC50μM (48h)Pt165.9Pt256.2Pt345Pt430.4Pt530.4Pt620.1 Disclosures:Tian:University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties.

MiR-15a, Mir-16-1 and Mir-17-92 Cluster Expression Are Linked to Poor Prognosis in Multiple Myeloma

Abstract 5000Multiple myeloma (MM) is characterized by a profound genomic instability of potential prognostic relevance. Loss of chromosome 13, observed in almost half of patients, negatively affects prognosis. MiR-15a, miR16-1 and miR-17-92 cluster, located on 13q, play important roles in the regulation of cell proliferation, differentiation and apoptosis. Therefore, we investigated a possible correlation of miRNA expression with chromosome 13 deletions (del(13)) and prognosis. We measured the expression of miR-15a, miR16-1 in 70 newly diagnosed MM patients and miR-17-92 cluster in 85 newly diagnosed MM patients by quantitative real-time PCR analyses. MiR-15a, miR-16-1 and miR-17 -92 cluster expression levels are independent of the del(13). High levels of miR-15a, miR-16-1, miR-17, miR-20a and miR-92-1 are associated with shorter progression-free survival (PFS), suggesting poor prognosis. Our data suggest that the expression of specific miRNAs may be contributed to MM prognosis. Disclosures:No relevant conflicts of interest to declare.

Decreased LIMD1 Gene Expression Is Associated with HIF-1α Over-Expression and Angiogenesis in Multiple Myeloma

AbstractAbstract 1829 Background:Enhanced angiogenesis in multiple myeloma (MM) is an important step in its tumor growth, invasion, and metastasis into the bone marrow. The bone marrow is a complex and dynamic microenvironment with multiple cell types contributing to niches that support myeloma tumor cell proliferation. One of the important microenvironmental factors, hypoxia-inducible factor 1α (HIF-1α), has been known to be associated with increased angiogenesis and metastatic potential as well as poor prognosis in MM. However, the role of HIF-1α in the etiology, pathogenesis and possible treatment of MM remains unclear. LIM domain-containing protein (LIMD1), a tumor suppressor molecular links prolyl hydroxylases (PHD1, 2 or 3) to von Hippel–Lindau (VHL) as a molecular scaffold, PHDs-LIMD1-VHL. This results in down-regulation of HIF-1α. Experiment design:Following informed consent, ten bone marrow aspirates and five biopsies were obtained from MM patients and healthy subjects, respectively. Bone marrow mononuclear cells (BMMCs) were isolated by using density-gradient centrifugation with Histopaque-1077 (Sigma, St Louis). For gene expression studies, total RNA was isolated from BMMCs. RNA was reverse-transcribed into cDNA and amplified using the Thermo-Script RT-PCR System and PCR performed again using the GeneAmp PCR System 9700. Protein expression in BMMCs from MM and healthy subjects was determined using Western blot analysis and immunohistochemical (IHC) staining. Results:The results showed that LIMD1 gene expression in BMMCs as assessed using RT-PCR in MM patients was reduced compared to healthy subjects. In addition, PHD1 gene expression in BMMCs from MM patients was markedly reduced whereas PHD2 and PHD3 expression was similar to healthy subjects’ BMMCs. The gene expression of VHL1, VHL2, and VHL3 in BMMCs was similar in MM patients compared to healthy subjects. Notably, LIMD1 gene expression in BMMCs from patients with monoclonal gammopathy of undetermined significance and primary amyloidosis patients was lover than among patients with progressive MM. Furthermore, the results of IHC staining showed LIMD1 protein expression in biopsy samples from MM patients with progressive disease was deceased and associated with high expression of HIF-1α. We also determined LIMD1 gene expression in MM patients treated with or without bortezomib. The results showed that this proteasome inhibitor did not affect LIMD1 expression. Conclusion and discussion:LIMD1 expression is reduced with down-regulation of PHD1 and up-regulation of HIF-1α in MM patients. The proteasome inhibitor did not inhibit LIMD1 gene expression in our experiments although it has reported that bortezomib inhibits HIF-1α levels in MM. The results from these studies suggest that LIMD1 may be an important key regulator of HIF1-1a and angiogenesis in MM. It has been reported that knocking out the LIMD1 gene will lead to over-expression of a single gene K-Ras and result in the development of lung adenocarcinoma. We are currently investigating the other pathways that are impacted by the reduction of LIMD1 expression, and how this may lead to the pathogenesis of MM. Disclosures:No relevant conflicts of interest to declare.

Expanded Natural Killer (NK) Cells for Immunotherapy: Fresh and Made to Order

AbstractAbstract 1912 IntroductionRemarkable increases in the dose and activity of NK cells can be achieved by co-culture with the HLA class I deficient cell line K562 that has been genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BB-ligand (K562-mb15-41BBL; Fujisaki et al. Cancer Res. 2009;69:4010–4017). We are conducting a clinical trial utilizing these ex-vivo expanded NK cells (ENK) which are produced at the Center for Cell and Gene Therapy (CAGT) at Baylor and then shipped to the University of Arkansas for Medical Sciences (UAMS) for infusion to high-risk relapsed multiple myeloma (MM) patients using the NHLBI-PACT mechanism. Here we report on the characteristics of the ENK cell products sent fresh versus frozen. MethodsApheresis products were collected from MM patients or healthy donors (HD), cryopreserved, and then shipped to CAGT for GMP grade production, as described (Lapteva et al. Cytotherapy 2012; in press). Briefly, mononuclear cells from thawed and ficolled apheresis products were cultured in Stem Cell Growth Medium (CellGenix) supplemented with 10% fetal bovine serum and 10 U/mL IL2 with stimulator cells at a ratio of 1 NK cell to 10 irradiated K562-mb15-41BBL cells (developed at St. Jude Children’s Research Hospital, Memphis, TN). Cells were harvested on day 8–9; products from HD were CD3-depleted. Clinical-grade products were shipped to UAMS overnight either cryopreserved in a dry shipper (n=7) or fresh in 5% human serum albumin on cold packs at 1–11°C (n=4). Cell purity, expression of activating molecules, and viability by 7AAD exclusion was assessed by flow cytometry. Standard 4h chromium-release assays were used to assess potency against K562 cells at a 20:1 ENK: K562 ratio. Student’s t-Test was used to determine significance. ResultsFrom 0.9–1.5×107 starting NK cells, the total number of ENK cells produced was 5.4×109 (range 1.8–24×109). The fold NK-cell expansion was significantly lower for MM patients (n=5, median 22, 12–70 fold) than for HD (n=6, median 95, 31–160 fold; p<0.05). At harvest, median CD3+/CD56+ NK cell purity was 70% (52–88); CD3 depletion of HD products increased CD3+/CD56+ purity to 93% (86–95) resulting in a median CD3+/CD56- T cell content of 0.02% (0.04–1.02). Overall, median viability was 93% (67–98) and potency (defined as lysis of K562 cells at a 20:1 E:T ratio) was 74% (26–92). One product derived from a patient with 21% CD138+ MM cells in the apheresis collection had low expansion (12-fold), viability (66.7%) and potency (26%). For cryopreserved products, viability immediately after thawing was acceptable (median 94%, 75–99) but recovery of viable cells varied from 61% to 100% and thawed ENK failed to lyse K562 cells unless rested overnight. Further, recovery was extremely poor after overnight incubation (median 16%, 10–21). We therefore validated shipment of fresh ENK products. In contrast with frozen NK cells, the median recovery for fresh clinical products post-shipping was 101% (87–151). We confirmed that NK purity, viability, potency and expression of the key activating molecules NKG2D, NKp30, NKp44 and CD226 were retained up to 48h after transfer. ENK further increased by 34% after 72h in vitro incubation in the presence of IL2. Significant in vivo expansion of ENK was observed after infusion of fresh ENK cell products (n=3) but not after infusion of thawed products (n=3, see separate abstract). An additional advantage was that the fresh cells arrived ready to infuse and changes in release criteria relying on rapid and in process testing significantly reduced the time from apheresis collection to ENK infusion, an important consideration when treating high-risk MM patients who can experience rapid disease progression. ConclusionWe conclude that large numbers of clinical grade ENK cells can be generated from both MM patient and HD derived apheresis products by co-culture with IL2 and K562-mb15-41BBL although less vigorous expansion was observed with patient-derived cells. Upon thawing, cryopreserved ENK cells exhibited inferior recovery and potency, and survived poorly during further in vitro culture. In contrast, freshly formulated and shipped ENK cells have excellent recovery and retain cytolytic ability. Robust in vivo expansion was only seen after infusion of fresh ENK cells. Production assistance by CAGT allowed for the rapid implementation of a novel therapy utilizing fresh ENK cells for poor prognosis MM patients. Disclosures:No relevant conflicts of interest to declare.

Bendamustine, Bortezomib and Dexamethasone (BVD) in Elderly Patients with Multiple Myeloma in First Relapse: Updated Results of the Intergroupe Francophone Du Myelome (IFM) 2009-01 Trial

AbstractAbstract 4044 Background:Prognosis of relapse is severe in elderly multiple myeloma (MM). In recent studies, median survival at progression after 1st line therapy was between 9 and 13 months (T. Facon, Lancet 2007; C. Hulin, J Clin Oncol 2009). Bortezomib (V) plus dexamethasone (D) is a major regimen in the treatment of relapses. Bendamustine (B) demonstrated to be highly active in advanced MM. The IFM 2009-01 trial evaluates the combination of B, V and D in elderly patients with progressive MM on or after 1st line treatment. Methods:Phase 2 IFM 2009-01 trial was dedicated to patients older than 65 years in 1st relapse or refractory to 1st line therapy. Inclusion criteria were measurable disease, PS ECOG 0–2, ANC > 1.5×109/l, platelets > 100×109/l, serum creatinine level < 250 mcmol/l, AST and ALT < 3xULN. Pts with prior exposure to bortezomib were excluded. Treatment regimen was B 70 mg/m2 D1–8, V 1.3 mg/m2 D1–8-15–22 and D 20 mg D1–8-15–22 every 28 days. 6 cycles were administered. Responders were assigned to receive maintenance treatment with 6 additional cycles administered 1 month out of 2. Response was evaluated according to IMWG criteria. Response rate was the primary objective. Progression-free survival (PFS), overall survival (OS) and toxicity were secondary end-points. Results:The present analysis is restricted to the 6 monthly cycles. From 03/2010 to 07/2011, 73 pts were included. Median age was 75.8 years (range 66–86). Median time from diagnosis to inclusion was 29 months. All pts received only 1 prior line of therapy: melphalan-prednisone (MP) in 12, MP-Thalidomide in 44, Lenalidomide-Dexamethasone (LD) in 14, other IMiD-based regimen in 3. 49 pts (67.1%) achieved at least partial response [best response CR: 9 pts (12.3%), VGPR: 12 pts (16.5%), PR: 28 pts (38.3%), MR: 6 pts (8.2%), SD: 4 pts (5.5%), progression: 13 pts (17.8%), early discontinuation: 1 pt (1.3%)]. Adverse prognostic factors for response were 1st line regimen with IMiD (p=0.006) and del17p (p=0.036). At 6 months, PFS was 67.1% and OS 80.8%. Cause of death was MM in 9 pts, sepsis in 4 pts and renal failure in 1 pt. Grade 3–4 adverse events were neutropenia: 16 pts (21.9%), thrombocytopenia: 7 pts (9.6%), sepsis: 14 pts (19.2%), gastro-intestinal: 9 pts (12.3%), anaphylaxis: 1 pt (1.3%). Grade 2 peripheral neuropathy occurred in 10 pts (13.6%) and grade 3 in 2 pts (2.7%). At 6 months, treatment was discontinued in 33 pts (45.2%). Cause of discontinuation was progressive MM in 18 pts (24.6%), failure to achieve PR in 5 pts (6.8%), adverse event in 8 pts (10.9%), patient refusal or lost to follow-up 1 pt (1.3%) each. Conclusion:In this elderly population with poor prognosis MM, BVD combination provides a high overall response rate and manageable toxicity. These results compare favorably with those achieved with VD or LD. Disclosures:Hulin:celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; janssen: Membership on an entity's Board of Directors or advisory committees. Kolb:janssen: Honoraria; celgene: Honoraria. Roussel:celgene: Honoraria; janssen: Honoraria.

MiR-15a, miR-16-1 and miR-17-92 cluster expression are linked to poor prognosis in multiple myeloma

Multiple myeloma (MM) is characterized by a profound genomic instability of potential prognostic relevance. Loss of chromosome 13, observed in almost half of patients, negatively affects prognosis. MiR-15a, miR16-1 and miR-17-92 cluster, located on 13q, play important roles in the regulation of cell proliferation, differentiation and apoptosis. Therefore, we investigated a possible correlation of miRNA expression with chromosome 13 deletions (del(13)) and prognosis. We measured the expression of miR-15a, miR16-1 in 70 newly diagnosed MM patients and miR-17-92 cluster in 85 newly diagnosed MM patients by quantitative real-time PCR analyses. MiR-15a, miR-16-1 and miR-17-92 cluster expression levels are independent of the del(13). High levels of miR-15a, miR-16-1, miR-17, miR-20a and miR-92-1 are associated with shorter progression-free survival (PFS), suggesting poor prognosis. Our data suggest that the expression of specific miRNAs may be contributing to MM prognosis.

1088P - Effect of 13Q Deletion on IL-6 Production in Patients with Multiple Myeloma

ABSTRACT Background Numerous studies have shown a correlation between 13q deletion and poor prognosis in multiple myeloma (MM), but the mechanisms are not fully understood. Earlier studies suggest that this lesion involves large segments or the entire long arm involving the retinoblastoma (Rb) gene. In myeloma, Rb gene is believed to downregulate interleukin -6 (IL-6) which plays a central role in the pathogenesis of MM. Therefore, it has been hypothesized that loss of Rb gene might be associated with very high expression of IL-6 and subsequent bad prognosis. Materials and methods Forty MM patients and 20 matched controls were included in this study. Interphase fluorescence in situ hybridization (FISH) analysis was performed using LSI 13q14-specific probe. Serum levels of IL-6 were determined by ELISA. All patients received conventional chemotherapy. Refractory patients received other therapeutic regimen of Thalidomide or Bortezomib. Results Significant increase (p Conclusion This study suggests that 13q deletions are associated with increased production of IL-6 in MM and this could be a possible cause of the associated bad prognosis. In addition, the results also show the potential to improve responses in patients with refractory MM with the introduction of novel therapies. Disclosure All authors have declared no conflicts of interest.

Identification of pluripotent and adult stem cell genes unrelated to cell cycle and associated with poor prognosis in multiple myeloma.

Gene expression-based scores used to predict risk in cancer frequently include genes coding for DNA replication, repair or recombination. Using two independent cohorts of 206 and 345 previously-untreated patients with Multiple Myeloma (MM), we identified 50 cell cycle-unrelated genes overexpressed in multiple myeloma cells (MMCs) compared to normal human proliferating plasmablasts and non-proliferating bone marrow plasma cells and which have prognostic value for overall survival. Thirty-seven of these 50 myeloma genes (74%) were enriched in genes overexpressed in one of 3 normal human stem cell populations – pluripotent (18), hematopoietic (10) or mesenchymal stem cells (9) - and only three genes were enriched in one of 5 populations of differentiated cells (memory B lymphocytes, T lymphocytes, polymorphonuclear cells, monocytes, osteoclasts). These 37 genes shared by MMCs and adult or pluripotent stem cells were used to build a stem cell score (SCscore), which proved to be strongly prognostic in the 2 independent cohorts of patients compared to other gene expression-based risk scores or usual clinical scores using multivariate Cox analysis. This finding highlights cell cycle-unrelated prognostic genes shared by myeloma cells and normal stem cells, whose products might be important for normal and malignant stem cell biology.

Extramedullary disease portends poor prognosis in multiple myeloma and is over-represented in high-risk disease even in the era of novel agents

Extramedullary disease is an uncommon manifestation in multiple myeloma and can either accompany newly diagnosed disease or develop with disease progression or relapse. We evaluated the impact of this disease feature on patients' outcome in the context of novel agents. We analyzed clinical and biological features of extramedullary disease in 936 patients with multiple myeloma enrolled in Total Therapy protocols, 240 patients in non-Total Therapy protocols, and 789 non-protocol patients, all of whom had baseline positron emission tomography scans to document extramedullary disease at diagnosis and its subsequent development at the time of disease progression or relapse. The most common sites for extramedullary disease at diagnosis were skin and soft tissue whereas liver involvement was the striking feature in extramedullary disease at disease relapse or progression. Regardless of therapy, extramedullary disease was associated with shorter progression-free and overall survival, as well as the presence of anemia, thrombocytopenia, elevated serum lactate dehydrogenase, cytogenetic abnormalities, and high-risk features in 70-and 80-gene risk models in univariate analysis. Multivariate analysis with logistic regression revealed that this disease feature was more prevalent in patients with an elevated centrosome index, as determined by gene expression profiling, as well as in myeloma molecular subtypes that are more prone to relapse. These include the MF subtype (also called the "MAF" subtype, associated with over-expression of the MAF gene seen with chromosome translocation 14;16 or 14;20) and the PR subtype (also called the "Proliferation" subtype, associated with overexpression of pro-proliferative genes). These data show that extramedullary disease is more prevalent in genomically defined high-risk multiple myeloma and is associated with shorter progression-free survival and overall survival, even in the era of novel agents. All clinical trials included in the analyses were registered with www.clinicaltrials.gov (NCT00083551, NCT00083876, NCT00081939, NCT00572169, NCT00644228,NCT00002548,NCT00734877).

Effect of 13q deletion on IL-6 production in patients with multiple myeloma.

e18570 Background: Numerous studies have shown a correlation between 13q deletion and poor prognosis in multiple myeloma (MM), but the mechanisms are not fully understood. Earlier studies suggest that this lesion involves large segments or the entire long arm involving the retinoblastoma (Rb) gene. In myeloma, Rb gene is believed to down regulate interleukin -6 (IL-6) which plays a central role in the pathogenesis of MM. Therefore, it has been hypothesized that loss of Rb gene might be associated with very high expression of IL-6 and subsequent bad prognosis. Methods: Forty MM patients and 20 matched controls were included in this study. Interphase fluorescence in situ hybridization (FISH) analysis was performed using LSI 13q14-specific probe. Serum levels of IL-6 were determined by ELISA. All patients received conventional chemotherapy. Refractory patients received other therapeutic regimen of thalidomide or bortezomib. Results: Significant increase (p &lt; 0.001) of IL-6 production was recorded in patients with 13q deletion compared to patients with normal chromosome 13q status. These patients were also refractory to conventional chemotherapy but showed striking response to thalidomide or bortezomib. Conclusions: This study suggests that 13q deletions are associated with increased production of IL-6 in MM and this could be a possible cause of the associated bad prognosis. In addition, the results also show the potential to improve responses inpatients with refractory MM with the introduction of novel therapies.

New antimyeloma drugs in spinal cord infiltration for multiple myeloma. Determination of lenalidomide in cerebrospinal fluid with ultrasensitive high-performance liquid chromatography

A 64-year-old woman with IgA kappa multiple myeloma was treated with thalidomide-dexa- methasone. Due to progression of the disease, bortezomib, doxorubicin and dexamethasone were administered, followed by autologous stem cell transplantation. Although near-complete remission was achieved, 5 months later, neuro- logical symptoms appeared and the patient was diagnosed with multiple myeloma with cells in- filtrating the spinal cord. Bis-chloronitrosourea, bortezomib and lenalidomide were then admin- istered and although the patient remained neu- rologically asymptomatic, she died 3 months later becausee of disease progression. Lena- lidomide entered into the cerebrospinal fluid (confirmed by ultrasensitive high-performance liquid chromatography), although did not im- prove the poor prognosis of multiple myeloma involving the central nervous system.

Aerobic Glycolysis: A Possible Target for Treating Multiple Myeloma (MM) with High Serum LDH Levels

Abstract 1799 Introduction:A number of studies have shown that the high level of serum lactate dehydrogenase (LDH) serves as an indicator for poor prognosis in multiple myeloma (MM). LDH is a key enzyme for glycolysis converting pyruvate to lactate, which is eventually utilized as an energy source particularly in tumor cells. It has been reported that cancer cells utilize this glycolysis pathway even in the presence of adequate oxygen to provide cancer cells with energy, called the Warburg effect (aerobic glycolysis). Myc is known to regulate LDH and pyruvate dehydrogenase kinase 1 (PDK1), which are master regulators of glycolysis (Figure 1). Although myc is a well known gene expressed in MM cells, there has been no report analyzing its association with the glycolysis-regulating genetic system, which is located downstream to the myc gene, in MM cells. In the present study, we examined if the glycolysis system is directly or indirectly associated with the survival of MM cells. Methods:MM cells were purified from primary bone marrow samples from 54 patients using CD138-magnetic beads. Written informed consent was obtained from all cases. Seven MM cell lines, RPMI8226, U266, KMS12BM, KMS12PE, KHM11, KMM1 and KMS11, were employed. Five genes associated with glycolysis, i.e., c-MYC, GLUT1 (glucose transporter 1), LDHA (LDH-encoding gene), hypoxia induced factor-1 alpha (HIF1a) and PDK1, were examined using real time PCR analysis. Glucose consumption and lactate production in culture supernatants of MM cell lines were analyzed. Oxamate, a competitive inhibitor of LDHA, was utilized to quantify cytotoxic effects on MM cells. Cytotoxicity was evaluated with AnnexinV/PI staining. Results:Heterogeneous expression of LDHA gene was observed (Figure 2A). High LDHA mRNA expression levels significantly correlated with poor survival (Figure 2B, p<0.01). A significant correlation between serum LDH levels and the mRNA expression levels of LDHA, was also found (p<0.01). Moreover, LDHA mRNA expression was significantly higher in MM cells than in plasma cells from patients with monoclonal gammopathy of undetermined significance (MGUS) (p<0.01). LDHA expression levels correlated with the expression levels of (i) c-MYC (p<0.0001) (ii) PDK1 (p<0.0023), a key enzyme regulating the Warburg effect, and (iii) GLUT1 (p<0.0003), while it did not correlate with HIF1a expression. It was also found that the greater glucose consumption, the greater lactate production as well as LDH activity in MM cell lines with higher LDHA mRNA expression. Finally, we found that an LDH-inhibitor, oxamate, activated caspase-3 (Figure 3) and induced apoptosis in MM cell lines as well as primary MM cells. [Display omitted] [Display omitted] [Display omitted] Conclusion:Our results suggest that aerobic glycolysis (the Warburg effect) is up-regulated in MM cells of patients with high serum LDH levels and that the aberrant expression of LDHA, PDK1 and GLUT1 is critical for the survival of MM cells with high serum LDH levels. Thus, aerobic glycolysis itself could serve as a novel therapeutic target in MM patients. Since MM with high serum LDH is with poor prognosis even after the advent of new agents, the present data might have a clinical relevance and might open a new avenue to develop novel therapeutic modalities for treating MM with high serum LDH levels. Disclosures:No relevant conflicts of interest to declare.

Gain of 1q21 Is a Cytogenetic Independent Risk Factor in Light Chain Amyloidosis Patients Treated with Melphalan / Dexamethasone As First-Line Therapy,

Abstract 3946▪▪This icon denotes a clinically relevant abstractThe aim of the current study was to identify cytogenetic risk factors as detected by iFISH in light chain (AL) amyloidosis. We analyzed a cohort of 91 consecutive patients receiving first-line chemotherapy according to the melphalan / dexamethasone protocol (M-Dex) (Palladini et al., Blood 2004, Dietrich et al., Blood 2010). Our study population consisted of patients not eligible for high dose chemotherapy due to age, reduced general health and the severity of heart involvement. Accordingly, the median age was 67 years (range 40 – 77 years), the median Karnofsky index 80% (50% – 90%), the median number of involved organs 2 (range: 1 – 6). 71/91 (78%) patients had cardiac involvement, among them 45 with NYHA stage III/IV. The median NTproBNP values were 4004 ng/l (range 71–48.292) ng/l, the median dFLC – designating the differential between the involved minus the uninvolved serum free light chain – was 204 mg/l (range 1 – 3.888 mg/l). The estimated median follow-up since start of M-Dex was 38 months.Translocation t(4;14) and deletion del17p13, which have been established as indicators of poor prognosis in multiple myeloma, are very rarely detected in AL amyloidosis (Bochtler et al., Blood 2008). In this study cohort we could only detect 4 out of 91 patients (4%) with at least one of these two cytogenetic aberrations. We therefore focused on gain of 1q21, which has also been described as an adverse prognostic factor in multiple myeloma and is more prevalent in AL amyloidosis (Bochtler et al., Blood 2011). Accordingly, 21/91 patients (23%) in our study cohort tested positive for gain of 1q21. The patient groups with and without gain of 1q21 were comparable regarding age, Karnofsky index, number of involved organs and the NTproBNP values to account for cardiac involvement. However, dFLC was higher in gain of 1q21 positive patients (table 1).Table 1Factor (median)Gain 1q21No gain 1q21pAge69660.17Karnofsky Index75800.70Number of involved organs230.30NT-proBNP425140040.81dFLC2801540.01In univariate analysis, gain of 1q21 was associated with a statistically significant inferior overall survival (OS, p=0.001, figure 1). The median OS was 12.5 months in patients with gain of 1q21 as compared to 38.3 months in those without. Statistical significance was also reached for hematologic event free survival (EFS, 2.8 months versus 4.1 months, p=0.04, figure 2) Hereby, failure to achieve at least a partial hematologic remission to the first 3 M-Dex cycles was counted as an event along with death, hematologic relapse or progression and initiation of an alternative chemotherapy regimen in order to account for patients initially unresponsive to M-Dex. [Display omitted] [Display omitted] In multivariate analysis we tested the prognostic significance of gain of 1q21 in comparison with established hematologic and clinical parameters including age, gender, kappa versus lambda light chain restriction, the detection of an intact immunoglobulin, dFLC values, Karnofsky index, number of involved organs and NTproBNP values using the proportional hazards model of Cox. Markedly, gain of 1q21 was confirmed as a strong predictor of poor outcome for OS (p<0.001), along with Karnofsky index, NTproBNP and age (p=0.03, p<0.001 and p=0.05, respectively). Again, statistical significance for gain of 1q21 and NTproBNP was also observed for EFS (p=0.04 and p=0.03, resp.).In conclusion, we have identified gain of 1q21 for the first time as an independent adverse risk factor in AL amyloidosis in a patient cohort with advanced disease uniformly treated with the standard therapy M-Dex. The poor outcome in these patients seems to be attributable to a worse responsiveness of the plasma cell clone to M-Dex. Further studies are required to test whether the poor prognosis conferred by gain of 1q21 is inherent with this cytogenetic aberration irrespective of the chosen therapy regimen or whether the poor prognosis can be overcome by alternative treatment strategies in these high risk patients. Disclosures:No relevant conflicts of interest to declare.

Outcome according to cytogenetic abnormalities and DNA ploidy in myeloma patients receiving short induction with weekly bortezomib followed by maintenance

AbstractCytogenetic abnormalities (CAs) such as t(4;14), t(14;16) or del(17p), and nonhyperdiploidy are associated with poor prognosis in multiple myeloma. We evaluated the influence of CAs by FISH and DNA ploidy by flow cytometry on response and survival in 232 elderly, newly diagnosed multiple myeloma patients receiving an induction with weekly bortezomib followed by maintenance therapy with bortezomib-based combinations. Response was similar in the high-risk and standard-risk CA groups, both after induction (21% vs 27% complete responses [CRs]) and maintenance (39% vs 45% CR). However, high-risk patients showed shorter progression-free survival (PFS) than standard-risk patients, both from the first (24 vs 33 months; P = .04) and second randomization (17 vs 27 months; P = .01). This also translated into shorter overall survival (OS) for high-risk patients (3-year OS: 55% vs 77%; P = .001). This adverse prognosis applied to either t(4;14) or del(17p). Concerning DNA ploidy, hyperdiploid patients showed longer OS than nonhyperdiploid patients (77% vs 63% at 3 years; P = .04), and this was more evident in patients treated with bortezomib, thalidomide, and prednisone (77% vs 53% at 3 years; P = .02). The present schema does not overcome the negative prognosis of high-risk CAs and nonhyperdiploidy. This trial was registered with www.ClinicalTrials.gov as NCT00443235.

MiR-17-92 cluster microRNAs confers tumorigenicity in multiple myeloma

miRNAs play important roles in the regulation of cell proliferation, differentiation and apoptosis. The deregulation of miRNAs expression contributes to tumorigenesis by modulating oncogenic and tumor suppressor signaling pathways. Oncogenic transcription factor Myc can control expression of a large set of microRNAs (miRNAs). Previous studies have shown that the expression of miR-17-92 cluster, a polycistron encoding six microRNAs (miRNA), has close relationship with the expression of Myc. In current study, silencing Myc in multiple myeloma (MM)cells induced cell death and growth inhibition, and downregulated expression of miR-17-92 cluster. Overexpression of miR-17 or miR-18 could partly abrogated Myc-knockdown-induced MM cell apoptosis. One of the mechanism of Myc inhibiting MM cell apoptosis is through Myc activates miR-17-92 cluster and subsequently down-modulates proapoptotic protein Bim. Although miR-17-92 cluster are located at 13q31.3, the expression of miR-18, miR-19 and miR-20 (especially miR-19) in patients with del(13q14) was higher than those without del(13q14). Patients with miR-17, miR-20 and miR-92 high-expression had shorter PFS compared to those with miR-17, miR-20 and miR-92 low-expression. These results suggest the Myc-inducible miR-17-92 cluster miRNAs contribute to tumorigenesis and poor prognosis in multiple myeloma.

UDP Glucuronosyltransferase 2B17 Double Deletion Genotype Is An Independent Marker for Poor Prognosis In Multiple Myeloma

AbstractAbstract 2955The enzyme uridine diphospho glucuronosyltransferase 2B17 (UGT2B17), localized on chromosome band 4q13, glucuronidates various endogenous compounds and xenobiotics, in particular androgens. The gene UGT2B17 shows a remarkable copy number variation (CNV) and, of all genes, by far the highest difference in mRNA expression between the Asian and Caucasian normal populations. Several reports indicate a role of UGT2B17 in cancer progression and moreover, in drug response. Furthermore, UGT2B17 has immunologic effects as a minor histocompatibility antigen. However, its role in multiple myeloma has not yet been investigated.We analyzed the UGT2B17 CNV by specific PCR in a retrospective series of 142 Caucasian patients diagnosed with multiple myeloma. Median patient age was 57.3 years at diagnosis, 40.1% were female and 59.9% male. Paraprotein was IgG in 44.4% and IgA in 22.5%. Cytogenetic aberrations (data available in 115 patients) were del13q in 40.5%, t(11;14) in 26.1%, t(4;14) in 5.3% and del17p in 7.7%. Treatment consisted of autologous or allogeneic stem cell transplantation in 72 (50.7%) patients. The remaining patients received various conventional dose regimens. We compared UGT2B17 CNV with relevant clinical and molecular markers for prognosis and with a sample of 449 healthy donors.Distribution of the CNV in multiple myeloma was 15.5% double deletion (del/del), 43.0% heterozygous (ins/del) and 41.5% wild type (ins/ins). This did not differ significantly from 10.7% del/del, 46.3% ins/del and 43.0% ins/ins within healthy donors. The UGT2B17 del/del genotype was significantly associated with poor clinical outcome. Median progression free survival (PFS) was 14.3 months for del/del patients and 25.8 months for patients having one or two UGT2B17 alleles (HR 1.70; 95%CI 1.23–2.31; p=0.001) (Figure 1). Median overall survival (OS) was 43.9 months for del/del compared to 72.5 months in patients with ins/ins or ins/del (not significant). There was no significant difference in PFS or OS between wild type and heterozygous genotype. We did not observe an association of UGT2B17 CNV with any other prognostic molecular or cytogenetic markers in multiple myeloma. In subset analysis, the impact of UGT2B17 del/del was more pronounced in male patients and in patients receiving conventional dose therapy. Importantly, the impact of the deletion genotype on PFS was independent from ISS stage, sex, chromosomal aberrations, paraprotein, and stem cell therapy in multivariable analysis (HR (del/del) 2.87; 95%CI 1.62, 5.07; p<0.001).We conclude that UGT2B17 double deletion is a novel independent prognostic marker for multiple myeloma. The enzyme has various roles in androgen and drug metabolism, cancer progression and histocompatibility. Further analyses are warranted to investigate the functional impact and potential therapeutic consequences. [Display omitted] Disclosures:No relevant conflicts of interest to declare.