ALBERT

Description: Abstract 5014 Lenalidomide (Rev) is frequently used to treat multiple myeloma (MM). We reported that Rev promotes Dkk expression in MM cells. A recent study reported that resistance to Rev was associated with induction of Wnt/β-catenin signaling by increased β-catenin transcription and its decreased destruction (Bjorklund, JBC 2011). In this study, we evaluated whether these reported effects represent selection of pre-existing cell by exposure to Rev or regulation of the canonical Wnt signaling pathway by Rev, and whether the Wnt signaling pathway is associated with Rev's direct effect on MM cell survival. To test the effect on Rev on proliferation of MM cells lines, the six MM cell lines H929, INA6, MM144, OPM-1, RPMI 8226, and U266 were cultured in growth media containing serial concentrations (0 to 1000 μM) of the drug for 24, 48 and 72 hours, and effect on proliferation measured by MTT assay. Rev diminished proliferation of these cell lines at concentrations between 50 to 1000 μM at 24 hours, and maximal inhibition occurred at 72 hours. Rev had little effect on the proliferation of the five MM cell lines at levels lower than 50 μM. Treatment with ≥5 μM Rev for 24, 48 and 72 hours resulted in increased DKK1 mRNA and Dkk1 protein levels as determined by qRT-PCR and by ELISA, respectively, in a dose dependent fashion, even at concentration that did not inhibit cell proliferation. These data suggest that Rev diminish MM proliferation is independent of its effects on Dkk1. We next examined the effect of Rev on β-catenin protein in cells treated with serial concentrations (0 to 1000 μM) of Rev for 6 hours. Immunoblotting analysis showed increased total β-catenin protein in 8226, OPM-2, H929, MM144 and U266 exposed to ≤100 μM, and no further increase in β-catenin levels when these cells were exposed to Rev concentrations higher than 100 μM. Rev did not affect changes in β-catenin levels in INA6. To determine the effects on Rev concentrations on TCF transcriptional activity, we infected cell lines with lentiviral particles containing the TCF reporter or with empty vector. Rev increased TCF activity at lower concentrations (10–20 μM) in all cells. As Rev concentration increased, TCF transcriptional activity gradually decreased, and was strongly inhibited (over 80%) at concentrations from 125 μM to 1000 μM, depending on the cell line; in this range, Rev suppressed MM proliferation. These results suggest that at cytotoxic concentrations, Rev regulation of TCF transcriptional activity is independent of its effect on total β-catenin levels. It remains to be determined if Rev-mediated inhibition of TCF activity is the cause of the drug's cytotoxic effect, and the mechanism of the concentration dependent effects of Rev on TCF activity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1815 The bone marrow (BM) microenvironment plays critical role in the progression of multiple myeloma (MM) and identification of dysregulated microenvironmental pathways is essential for development of effective interventions for this disease. We and others have shown that bone disease is both a consequence of and a necessity for MM progression and that restoring bone turnover helps control MM. Prooxidant environment has been shown to promote tumorigenesis and bone disease, while circulating levels of antioxidants are reduced in MM patients (Sharma et al., 2009). Heme oxygenase 1 (HMOX1) is an intracellular inducible antioxidant that acts in response to oxidative stress and mediates anti-inflammatory and wound healing processes. The aim of the study was to identify dysregulated microenvironmental factors in myelomatous bone using mesenchymal stem cell (MSC) cytotherapy as an approach previously shown by us to induce bone formation and inhibit osteolysis and MM progression (Li et al., 2011; 2012). Intrabone injection of fetal MSCs into myelomatous bones of SCID-hu mice engrafted with a patient's MM cells resulted in significant upregulation(≥2 folds) of 120 probe sets and downregulation of 44 probe sets 24 hours after MSC cytotherapy, assessed by global gene expression profile (GEP, 9 mice/group). HMOX1, one of top overexpressed genes by MSC cytotherapy (5.2 folds, p

Description: Abstract 2915 Background: AMG is the most common plasma cell dyscrasia, currently classified as either monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic multiple myeloma (AMM), based on the level of monoclonal immunoglobulin (M-protein), bone marrow plasmacytosis and other criteria defined by the International Myeloma Working Group. While information is available on the impact of clinical variables such as bone marrow plasmacytosis, free light chains, isotype and M-protein on the hazard of progression to symptomatic MM (MM), little is known about the value of the karyotype and DNA content, as determined by DNA/cIg flow cytometry, on the risk of progression from AMG to MM. Methods: Patients from the Myeloma Institute for Research and Therapy (MIRT) with AMG that were enrolled in a prospective observational clinical trial were evaluated. All patients underwent detailed clinical staging at entry and were followed at pre-specified intervals per protocol. Cox proportional hazards regression was used to model univariate and multivariate associations of baseline features with progression to MM. The number of distinct DNA stem lines in the flow cytometry assay, their percentages, respective DNA Indices (DI), cytoplasmic Immunoglobulin Indices (cIgI), and percent of cells in S phase were evaluated alone and in relation to the karyotype report at baseline. A DI between 0.99 and 1.01 referred to diploidy, lesser than 0.99 to hypodiploidy and more than 1.01 hyperdiploidy. Results: Data from 267 eligible MIRT patients with AMG were analyzed. Of these patients 99% (265/267) had performed DNA/cIg flow cytometry and had a karyotype report at diagnosis. Cytogenetic abnormalities were detected in 20 of the 265 patients from whom data were available. From the 265 patients from whom DNA/cIg flow cytometry data were available, no abnormal clones were identified in 14% (37/265), one clone was identified in 95 patients (36%), two clones in 122 patients (46%), three clones in 10 (4%), and in 1 patient 4 clones were identified. Most patients with abnormal DNA content had hyperdiploid clones (132/243 patients). The second most frequent finding was diploid DI, in 39% (104/243) of patients; 3% (7/243) had a hypodiploid DI. The median DI was 1.01 (0.9–2.02) and median cIg was 7 (1–50). Interestingly, the median cIgI value in AMG was more than twice that of its value (3.4, 1–22) in Total Therapy 3 MM patients (p=0.001). In univariate analysis of the parameters in this study, the presence of an abnormal karyotype (p=0.032, HR=2.62), the number of DNA/cIg clones (p=0.016, HR=1.69) and the percentage of the dominant clone (p=0.003, HR=1.03) were significantly related to progression to MM. Ploidy by DNA/cIg analysis, the S-phase fraction, and cIg did not reach statistical significance (p=0.863, p=0.132 and p=0.240, respectively). In multivariate analysis, only the number of abnormal clones (p=0.013, HR=1.78) retained statistical significance, while the percentage of the dominant clone neared significance (p=0.070, HR=1.02). Using running log rank tests we were able to identify optimal cut-points for the percentage of the dominant clone and the number of clones (12% and 2 clones respectively). From these, a risk score was obtained which identifies three distinct groups with 3-yr MM progression probabilities of 12%, 30% and 67% (p

Description: Abstract 2933 Monoclonal gammopathy of undetermined significance (MGUS), a premalignant plasma cell dyscrasia, is differentiated from asymptomatic multiple myeloma (AMM), based on the level of monoclonal immunoglobulin (M spike) and bone marrow plasma cell infiltration (M spike 〉 3 g/dl and/or marrow plasmacytosis 〉10% in AMM). MGUS progresses to active Multiple Myeloma (MM) at a rate of 1–2% per year, thus imparting an average risk of 25% of progression over a lifetime. No single clinical variable, imaging modality or molecular study has yet been identified to predict progression. As a part of SWOG observational trial S0120, 262 patients were enrolled at MIRT and their GEP of purified plasma cells (PC) prospectively obtained. Here we report the results of GEP as predictor of progression. Among those with baseline GEP data available, 127 were non-IgM, AMM or MGUS and were otherwise eligible for the study. Baseline samples were restricted to those samples taken no more than 6 months prior to or 14 days following registration to S0120. If a patient had multiple baseline samples, then the sample taken closest to the patient's registration date was used. GEP studies were performed to assign molecular classification and risk scores, along with GEP-derived amp1q21, delTP53, DKK1 and other variables. With a follow-up of 36 months in the subset with evaluable GEP baseline data in 40 MGUS and 87 AMM cases, GEP characteristics were compared with those of untreated MM patients treated with TT2, TT3, TT4, and TT5 protocols, in the context of normal donor PC signatures. Comparing MGUS with AMM, CD-2 molecular subgroup was over-represented in MGUS and HY in AMM. PSMD4 on chromosome 1q21 was also linked to AMM. In terms of GEP-70 risk model, 36% of AMM and 15% of MGUS had scores exceeding -0.26. Univariate predictors for treatment-requiring MM included the well-known serum-M and bone marrow PC variables, and GEP-70 score 〉-0.26 (HR=8.82, p

Description: Abstract 2910 Background: 18-fluorodeoxy-glucose positron emission tomography (18-FDG PET) scanning is increasingly viewed as an important novel imaging tool in the initial workup of multiple myeloma (MM). Recent work from Italian investigators validated our previous report on the prognostic implications of PET scanning in terms of poorer outcome in the presence of higher focal lesion (FL) number, greater FDG uptake intensity, expressed as maximum standardized uptake value (SUVmax) and presence of extramedullary disease. Here, for the first time, we are reporting the prognostic implication of PET-FL and PET-SUVmax suppression after induction chemotherapy as they pertain to survival of patients enrolled in TT3A protocol employing VTD (bortezomib, thalidomide, dexamethasone) and TT3B protocol employing VRD(bortezomib, lenalidomide, dexamethasone), with median follow-up of 78 months and 48 months, respectively. Methods: Imaging parameters included standard metastatic skeletal survey (MBS) delineating the number of osteolytic lesions, STIR-derived MRI-based foal lesion (FL) number (limited to the axial skeleton bone marrow of head, spine, and pelvis). In the absence of FL, diffuse hyper-intense marrow (DHIM) involvement was also annotated1. As for PET scanning, FL number, SUVmax and EMD parameters were captured. PET studies were repeated on day 7 after initiation of the DT-PACE portion of the first induction cycle, prior to first transplant, and prior to maintenance initiation. Imaging data were complete for 429 patients at baseline including 394 with additional information on gene expression profiling (GEP)-derived high-risk. Day-7 PET data were available on 308 and 277 patients, respectively. Cox regression modeling was employed for overall survival (OS), progression free survival (PFS) and complete response duration (CRD). Results: Patient characteristics were fairly comparable among the TT3A and TT3B patients except higher proportions of patients with high-risk features (high beta-2-microglobulin, low albumin, high GEP-derived centrosome index) in TT3B. The number of MBS-OL (〉2) adversely affected OS (p=0.0001) and PFS (p=0.005) in TT3A (p=0.0001), PFS (p=0.03) in TT3B with a trend apparent for OS (p=0.06). For CRD, trends were apparent for shorter CRD in the presence of OL 〉2 in TT3A. In case of baseline PET, results were quite consistent for the 3 endpoints examined and between protocols applied. Thus, patients with 0 or 1–3 FL had equally favorable OS, PFS and, less pronounced in TT3B, CRD. Those with FL 〉3 fared poorly. Adverse consequences of FL-associated SUVmax 〉3.9 are apparent for OS, PFS and CRD in TT3A, whereas with TT3B only trends were apparent. Among follow-up imaging, Day-7 PET was the earliest measure. As is apparent in Figure 1, the presence of more than 3 FL imparted shorter OS and PFS in both TT3A and TT3B, with comparable outcomes noted for patients presenting with 0 or 1–3 FL at that time. Similar trends were apparent for CRD with both TT3A and TT3B. Cox regression modeling that considered additional information on day-7 PET results revealed an independent negative impact of day-7 FL〉3 in the absence and presence of GEP data for both OS and PFS. Importantly, such information made baseline PET-FL irrelevant for OS and PFS, with MBS-OL 〉2 remaining in the model for OS, even when GEP data were also analyzed. The other surviving baseline variables included GEP-70-defined high risk for all 3 endpoints examined including CRD, where no imaging parameter emerged as significant. In the case of day-7 PET-SUV max, prognostic significance only applied to OS and PFS in TT3A. Conclusions: The data presented herein, demonstrate that failure of FDG suppression as early as post-induction chemotherapy carries adverse outcome and can serve as prognostic indicators of long-term survival. We have thus established that early PET follow-up scanning can identify further risk discrimination that can be exploited toward therapy change. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2944 Multiple myeloma (MM) bone disease is characterized by increased activity of osteoclasts and reduced osteoblast numbers. We recently reported that cytotherapy with mesenchymal stem cells (MSCs) promotes bone formation, inhibits bone disease and reduces MM growth (Yaccoby et al., 2006; Li et al., 2011) and that MSCs secrete bone remodeling associated factors such as decorin that directly inhibits osteoclastogenesis and promotes osteoblastogenesis (Li et al., 2012). To shed light on molecular mechanisms associated with the cytotherapeutic effects of MSCs we exploited the SCID-hu model engrafted with MM cells from various patients and cytotherapeutically treated with fetal or healthy donors MSCs. Human global gene expression profile was performed in nonmyelomatous implanted bones (n=5) and in myelomatous implanted bones injected with human MSCs (1×106 cells/bone) and analyzed immediately (control group, n=17) or 24 hours later (n=16). We indentified heme oxygenase 1 (HMOX1) as one of the top significant upregulated genes consistently induced by 24 hours cytotherapy using fetal MSCs or MSCs from 3 different donors. HMOX1 induction was confirmed by qRT-PCR and immunohistochemistry and was also found to be consistently induced by MSCs cytotherapy in myelomatous bones engrafted with MM cells from different patients (n=4). Further analysis revealed lower expression of HMOX1 in myelomatous versus nonmyelomatous bones. HMOX1, an inducible antioxidant, degrades intracellular heme into carbon monoxide, free iron and biliverdin and is involved in oxidative stress response and intracellular iron homeostasis both of which are known to regulate osteoclastogenesis. Culture of human blood mononucleated cells or committed osteoclast precursors with MSCs (non-contact co-culture) in osteoclastogenic medium supplemented with RANKL and M-CSF inhibited multinucleated osteoclast formation by 70% (p

Description: Abstract 1814 Gene expression profiling (GEP) provides valuable information on molecular subclass and prognostic risk of multiple myeloma (MM). In addition to subclass designation, GEP also predicts many cytogenetic aberrations in MM (Zhou, Blood 119:e148). We re-examined GEP derived assignments of translocation-related molecular subgroups (CD1, CD2 MF, and MS) in correlation to FISH data. Focusing on 14q32 translocation, we used FISH probes to MMSET/FGFR3, CCND3, CCND1, MAF, and MAFB in combinations with probes specific to the immunoglobulin heavy chain (IGH) constant and viable regions (IGHC and IGHV) to identify translocations in myeloma cells from Total Therapy (TT) 4 and TT5 patients at baseline. Combining our FISH data with GEP expression of probe sets specific to the IgH translocation partners, we identified threshold expression levels; above this threshold the expression level was clearly indicative of genomic translocations of t(4;14)(p16;q32), t(6;14)(p21;q32), t(11;14)(q13;32), t(14;16)(q32;q23), or t(14;20)(q32;q11). Below these thresholds, increased signals reflected gene copy numbers. Overall, 42.2% (113/268) of patients were predicted and confirmed to have 14q translocations: 14.9% had t(4;14), 3.4% t(6;14), 19.4% t(11;14), 3.4% t(14;16), and 1.1% t(14;20). In cases of GEP indicated simultaneous spikes of several 14q translocation partners, we discovered that 14q translocation were indeed mutually exclusive - one 14q translocation per clone. The spikes of the other 14q partner gene(s) shown by GEP, reflected copy number driven increases, or in a few cases, the co-existence of distinct clones, each with a unique 14q translocation. Using threshold expression levels equivalent to those developed in the TT4 and TT5 training set, we predicted that 42.3% (335/792) of patients treated on TT2 and TT3 protocols had 14q translocations at baseline, as follows; 14.9% t(4;14), 1.4% t(6;14), 19.8% t(11;14), 4.4% (14;16), and 1.8% t(14;20). Next, we correlated the effects of 14q translocations with overall survival of patients treated with TT2 and TT3. Patients whose GEP spikes predicted MMSET/FGFR3 and CCND3 translocations significantly benefited from the inclusion of Bortezomib in TT3; and patients with CCND1 and MAFB translocations had improved median survivals comparing 8 years follow-ups of TT2 and TT3. In contrast, patients with high MAF expression clearly did not benefit from the inclusion of the proteasome inhibitor in TT3. In conclusion, by combining interphase FISH and GEP we established predictive threshold expression levels of 14q translocations in MM and identified a group of patients who do not benefit from Bortezomib. This single-gene approach to define molecular subgroups is expedient and provides important information of prognostic significance. Disclosures: Tian: University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties. Sawyer:University of Arkansas for Medical Sciences: Employment, under panding process, under panding process Patents & Royalties. Epstein:University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties. Barlogie:University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties.

Description: Abstract 2918 Background: Genetic heterogeneity is a well-known characteristic of MM. Two broad subtypes, Hyperdiploid MM (〉46) and Non Hyperdiploid MM (≤46), are defined by chromosome number and are each associated with a distinct prognosis. However, karyotype in MM is informative in no more than one-third of newly diagnosed cases. DNA flow cytometry has given excellent results in the quantification of the cell DNA content. Furthermore, the combined DNA and cytoplasmic Immunoglobulin (DNA/cIg) flow cytometric analysis offers a number of distinct advantages in the study of MM compared to other assays. The relative quantification of cIg is feasible even for cases of non-secreting MM, the characterization of the ploidy of that clone, or even the identification of more than one abnormal clone in the same monoclonal population based on differential DNA content. Methods: 480 patients that met the criteria for symptomatic or progressive MM were enrolled to the Total Therapy (TT) 3A & B protocols as previously published. The number of distinct DNA ploidy clones (NC) found on DNA/cIg analysis, their percentages and their respective DNA Index (DI) and cIg Index (cIgI), and percentage of cells in S phase, were evaluated alone and in relevance with known powerful prognostic parameters as previously published (Table 1). A DI between 0.99 and 1.01 referred to diploidy, lesser than 0.99 to hypodiploidy and more than 1.01 to hyperdiploidy. Cox proportional hazards regression was used to perform univariate and multivariate analysis of the various prognostic factors for overall survival (OS) and progression-free survival (PFS). Results: Of the total cohort of TT3 patients, 98% had DNA/cIg flow cytometry done on initial diagnosis. In 28%, no clone was identified; in 22% one clone, in 37% two clones, in 13% three clones and in 0.5% four clones were identified. Of 342 patients with at least one cell clone, hyperdiploidy was the feature of the dominant clone in 49% (169/342), diploidy in 42% (147/342) and hypodiploidy in 8% (26/342). The median values for DI and cIgI were 1.01 (0.72–2.02) and 3.4 (1.0–22), respectively. In the univariate analysis for OS, NC was highly significant (p1 was statistically significant (p=0.021, HR=0.65). The same was also true for the cIgI of the dominant clone (p=0.019, HR=0.74). Regarding PFS, NC (p=0.005, HR=1.23), diploidy of the dominant clone (p=0.042, HR=1.40), the DI〉1 of the dominant (p=0.006, HR=0.63) and the secondary (p=0.031, HR=1.58) clone as well as the cIgI of the dominant clone (p=0.037, HR=0.81) were statistically significant. In the multivariate analysis regarding OS, the NC was able to enter both models with and without GEP (p=0.007, HR=1.26 and p=0.002, HR=1.29 respectively). Regarding PFS, NC yielded similar results (p=0.054, HR=1.15 and p=0.013, HR=1.19 for the GEP including and non-including model respectively). Implementation of GEP and DNA/cIg was able to identify 3 distinct prognostic groups in hyperdiploidy both for OS and PFS (Figure 1). Conclusion: Hyperdiploidy is the most common ploidy status in MM. The level of cIg production has a prognostic impact in MM. Clonal heterogeneity as portrayed by the NC in DNA/cIg is a major prognostic factor in MM. When DNA/cIg is combined with GEP in hyperdiploid cases, it is able to stratify them into three distinct prognostic groups. Disclosures: No relevant conflicts of interest to declare.

Description: IL-6 signaling can be enhanced through transsignaling by the soluble IL-6 receptor (sIL-6r), allowing for the pleiotropic cytokine to affect cells it would not ordinarily have an effect on. Serum levels of sIL-6r can be used as an independent prognostic indicator and further stratify the GEP 70-gene low-risk group to identify an intermediate-risk group in multiple myeloma (MM). By analyzing more than 600 MM patients with ELISA, genotyping, and gene expression profiling tools, we show how the combination of 2 independent molecular genetic events is related to synergistic increases in sIL-6r levels. We also show that the rs2228145 minor allele is related to increased expression levels of an IL-6r splice variant that purportedly codes exclusively for a sIL-6r isoform. Together, the SNP rs2228145 minor allele C and amplification of chromosome 1q21 are significantly correlated to an increase in sIL-6r levels, which are associated with lower overall survival in 70-gene low-risk disease, and aid in identification of the intermediate-risk MM group.

Description: Abstract 324 Background: There has been recent evidence that lenalidomide, while used in the treatment of myelodysplastic syndromes (MDS), also has been associated with an enhanced risk of secondary malignancies including MDS. Here we are examining the impact of maintenance on the development of metaphase-based MDS-associated cytogenetic abnormalities (MDS-CA), and subsequent development of therapy related MDS (t-MDS) and acute leukemia (t-AL) in the two arms of Total Therapy 2 (TT2) and two successive Total Therapy 3 trials (TT3A, TT3B). Methods: The details of TT2/TT3A/TT3B trials and patient outcomes have been reported previously. TT2 (median follow-up 118 months) patients were then randomized to receive thalidomide maintenance (TT2+Thal) for 3 years versus no thalidomide (TT2-Thal). Maintenance in TT3A (median follow-up 78 moths) consisted of VTD in the first year and TD for years 2 and 3 in TT3A, while TT3B (median follow-up 48 months) employed VRD for all 3 years. The patient populations were as follows: TT2-Thal arm (n=283), TT2+Thal arm (n=270), TT3A (n=279) and TT3B (n=162). Bone marrow examination with conventional cytogenetics was performed at baseline, post-induction, post-transplant and then at least semi-annually from maintenance in all patients prospectively. Chi-square and Fisher's exact tests were used to compare baseline characteristics between protocols. Univariate and multivariate Cox proportional hazard regression were used to model associations between baseline covariates and onset of MDS-CA and t-MDS/t-AL. Kaplan and Meier method was used to model cumulative incidence of MDS-CA and t-MDS/t-AL. Results: Baseline characteristics were fairly comparable between the two arms of TT2 and the two TT3 studies, except a higher proportion of GEP70 high risk patients in TT3B. Cumulative incidence of MDS-CA was significantly higher in TT3B protocol from time of first transplant (Figure 1), with the most common cytogenetic abnormality being deletion of chromosome 20 (del20, 35%), followed by monosomy/deletion 7 (27%) and monosomy/deletion 5 (17%) across all protocols. Of the 71 patients with an MDS-CA, 29 were also diagnosed with clinical MDS or AML; for these 29 patients, the median time to this diagnosis from first transplant was 58 months. The cumulative incidence of MDS-CA was higher and achieved more statistical significance (Figure 2) in TT3B compared to TT3A when landmarked from the start of maintenance. In univariate analysis for all patients, TT3B emerged as a significant variable in the univariate Cox regression model for MDS-CA, and in both the univariate and multivariate models for del20. Univariate and multivariate modeling within TT3B patient population did not reveal significance with cumulative dosing of different maintenance drugs. Discussion: Three randomized phase III trials (IFM-2005-02, CALGB 101104 and MM-015) have demonstrated an increase incidence of second malignancies in patients receiving lenalidomide maintenance after having been exposed to alkylating agent melphalan. In our present study, we are reporting an increased incidence of MDS-CA in the TT3B trial that employed VRD maintenance when compared with predecessor TT3A and TT2 trials. The data suggests that the risk is associated with lenalidomide exposure rather than its cumulative dosing in patients with prior exposure to therapy with alkylating agents. Pairwise log-rank comparisons: Disclosures: Sawyer: University of Arkansas for Medical Sciences: Employment, under panding process, under panding process Patents & Royalties.