ALBERT

Description: Gene expression profiling (GEP) reliably predicts overall and progression free survival in multiple myeloma. Driven by the concept that therapy will reveal biology, we applied the GEP70 risk model to 56 patients enrolled in Total Therapy 6 (TT6), a phase 2 trial for previously treated patients. One year survival estimates were 62% vs.97%, p

Description: CS1 is an ideal target for multiple myeloma (MM) therapy as it is highly expressed on MM while having a very limited expression profile in normal tissues.  Elotuzumab (elo), a humanized monoclonal antibody (mAb) targeting CS1, has an acceptable safety profile and clinical activity in relapsed/refractory MM when combined with the immune modulator lenalidomide (len) and low dose dexamethasone (dex).  The primary mechanism of action for elo is NK cell-mediated antibody-dependent cellular cytotoxicity.  Here we report on a patient who was given elo/len via a single patient IND 6 months after receiving therapy with ex vivo activated auto-ENK cells and low dose IL2 as previously described (Szmania et al, Blood ASH Annual Meeting Abstracts 2012;120:1912).  The patient had relapsing GEP70 high-risk MM with cytogenetic abnormalities and had failed multiple lines of prior therapy including 3 auto-PBSC transplants and further salvage treatments including len, bortezomib, pomalidomide and carfilzomib.   Although ENK cell therapy did not induce a response, subsequent disease progression was slow.  IV elo was started 187 days after ENK cell infusion, and given every 14 days at the currently studied dose of 10mg/kg.  Len at 15mg/day was given on days 1-21 of a 28-day cycle.  Dex premedication (p.o. 38mg; IV, 10mg) was added after a grade 2 infusion reaction was observed to elo dose #1. While on the ENK cell protocol this patient had a dramatic increase in circulating NK cell counts peaking 9 days after infusion (6300 NK/µL, a 48-fold increase from baseline). Although still in the high range, NK cell levels at the time of elo treatment had normalized somewhat (539 NK/ml), and the cell surface expression of key activating receptors was consistent with a resting phenotype.  NK cell count remained stable after the first dose of elo (530 NK/ml) but subsequently dipped to 179 NK/µL after elo dose #2.   Since dex has been reported to affect NK cell counts, it is important to note that an additional dose was taken prior to elo dose #2 due to a travel delay (in total 66 mg of dex was taken on this occasion).  Circulating NK cells (collected pre-elo and 11, 25, and 57 days after elo dose #1) had similar low activity against auto-MM collected prior to elo treatment (effector:target ratio 10:1, 0-5% specific lysis) and killing against MM collected after 5 elo doses was only modestly increased (3-12%).  However, the same circulating NK cells exhibited significantly increased cytolytic ability when additional elo (10µg/mL) was added during the in vitro E:T co-incubation (3-11 fold increase in killing over isotype control, p=0.0008) suggesting that the MM targets were not saturated with mAb. Bound mAb may have been reduced in part during target cell isolation and freeze/thaw.  Freshly prepared auto-ENK cells exhibited an activated immunophenotype and induced significantly higher killing of pre-elo MM (45%) compared to non-expanded NK.  ENK killing was higher still against MM collected after 5 doses of elo (61%).  When elo was present during the assay, ENK demonstrated the most effective killing of auto-MM, reaching levels  equivalent to that of the NK sensitive target K562 (85% vs. 82% lysis).  Successful mAb therapy for MM is now moving forward as target antigens with selective, high and homogeneous expression, such as CS1, are identified.  However, the activity of responding effector cells is a critical issue to consider.  Inadequate NK cell count and activity level has been reported in MM and steroids typically given to debulk and preempt mAb-induced infusion reactions may exacerbate this problem.  Immunomodulatory agents given to enhance immune cell activity are not sufficient to reverse the negative effect of steroids.  We have previously shown that large doses of highly activated auto-ENK cells can be safely infused and that these cells expand further after infusion.  In this study we show that ENK cells have significant activity in vitro against auto-MM and that elo further enhances this activity.  Combination therapy incorporating saturating doses of mAb followed by infusion of NK effector cells with optimized activity against auto-MM is an innovative approach that warrants investigation.  Infusing highly activated effector cells after dex/elo may be one way to reap the benefits of combining these modalities while circumventing steroid-induced immune suppression. Disclosures: Barlogie: Celgene: Consultancy, Honoraria, Research Funding; Myeloma Health, LLC: Patents & Royalties.

Description: Primary human myeloma (MM) cells do not survive in culture while current in vitro and in vivo systems for growing these cells are limited to coculture with specific bone marrow (BM) cell type or growth in immunodeficient animal model. The aim of the study was to determine long-term survival and interaction of primary MM plasma cells with a healthy adult human BM that include immune cells capable of functional activation. This system is different from the autologous BM culture that is already affected by the disease. Whole BM cells from healthy donors were cultured in αMEM medium supplemented with 10% FBS and 10% serum pooled from MM patients. Following 7-9 days the cultures were composed of adherent and nonadherent cellular compartments. The nonadherent compartment contained typical BM hematopoietic cells such as monocytes, B and T lymphocytes and NK and normal plasma cells as assessed by flow cytometry, while the adherent compartment contained cells that morphologically resemble macrophages, osteoclasts, megakaryocytes and fibroblast-like cells. At this culture stage, CD138-selected MM cells from 20 patients were added to the BM cultures (4:1 BM:MM cell ratio) and survival and growth of MM cells were determined after 7 days by assessing proportion of CD45low/intermediate/CD38high MM plasma cells among total number of cells. MM and BM cell viability was constantly high (〉90%) in cocultures. Subsets of primary MM plasma cells, regardless of molecular risk or subtype, were survived and detected in all cases while in 14 of 20 experiments, number of MM plasma cells was increased by 58±12% (p

Description: Introduction Ex vivo activated/expanded natural killer (ENK) cells can induce myeloma cell lysis both in vitro and in murine models and are currently being studied clinically in the setting of high-risk relapsing disease and asymptomatic disease at high risk of progression. This prompted us to study, in myeloma cell lines, whether intrinsic resistance to ENK cell lysis exists, whether repeated challenge with ENK leads to increased resistance, and what the underlying mechanisms of resistance are. Of 11 myeloma cell lines tested in standard 4h chromium release assays, 8 were avidly killed (78-89% lysis, E:T Ratio 10:1) whereas 3 lines were less sensitive (41-65% lysis). Repeated exposure to ENK challenge decreased sensitivity in 4 of 11 lines, that was at least in part due to down-regulation of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Receptors on the myeloma cell surface (Garg et al, Blood 2012, 120:4020). In this study we investigated the resistance issue further via metabolomics, gene expression profiling (GEP) and flow cytometry analysis of OPM2, which was intrinsically resistant and developed further resistance after challenge with ENK cells. Methods Metabolomics was studied using a quantitative proteomic strategy entailing stable isotope labeling with amino acids in cell culture – mass spectrometry (SILAC-MS). Resistant and parental OPM2 cells were grown either in medium with heavy amino acids (13C6 L-Lysine and 13C6 L-Arginine) or with light amino acids (12C6 L-Lysine and 12C6L-Arginine). Reverse labeling with heavy or light amino acids was also done to confirm the results. Cell lysates from heavy and light amino acid labeled cells were pooled, simultaneously resolved on SDS-PAGE, protein bands were excised and analyzed on a mass spectrometer after trypsin digestion. GEP was performed using the Affymetrix U133 Plus 2.0 microarray platform (Santa Clara, CA). The fold change of signal intensity for genes and proteins in resistant vs. parental OPM2 was calculated. The most differentially expressed genes (top 150-fold up or down) and proteins (up or down by 1.3-fold) were compared for commonality. Cell surface protein expression was determined via flow cytometry. The ability of ENK to lyse myeloma cell targets in the presence of isotype control or ICAM-3 blocking antibody was tested in 4h chromium release assays. Results Metabolomics identified 〉3800 proteins and revealed that the abundance of 352 proteins was significantly altered in resistant myeloma cells. These altered proteins were mainly associated with cell cycle, morphology, organization, cellular compromise, immune response, and survival. Further, a comparison of these differentially expressed proteins with GEP data revealed 3 commonly up-regulated molecules: TBC1D8B, HSPA1A and IFI16; and 2 down-regulated molecules: intercellular adhesion molecule (ICAM-3) and BAI3. Of these, ICAM-3, a ligand for leukocyte function-associated antigen-1 (LFA-1) and a potent signaling molecule, was selected for further studies. Flow cytometry confirmed that ICAM-3 cell surface expression was 〉 8-fold lower on resistant versus parental OPM2 cells. Further, blocking of ICAM-3 in cytotoxicity assays resulted in decreased lysis (43% blocked, E:T ratio 5:1), suggesting that this molecule is functionally important and takes part in ENK cell-mediated killing. Conclusion In conclusion, quantitative proteomic analysis demonstrated dynamic changes in the ENK-resistant OPM2 myeloma cells that correlated with GEP and differences in ICAM-3 expression may have functional implications. Studies evaluating the expression of ICAM-3 in myeloma patients at diagnosis and relapse are in progress. Myeloma cells may down-regulate ICAM-3 as a mechanism of escape from immune surveillance and therefore, ICAM-3 may be a useful biomarker to predict sensitivity to ENK cell-mediated killing and aid in the selection of patients most likely to benefit from ENK cell therapy. Disclosures: Barlogie: Celgene: Consultancy, Honoraria, Research Funding; Myeloma Health, LLC: Patents & Royalties.

Description: Introduction Monoclonal gammopathy of undetermined significance (MGUS) progresses to asymptomatic (AMM) or symptomatic myeloma (MM) at an annual rate of 1%. Primary cytogenetic aberrations of chromosome 14q32 translocations with one of the partner genes (MMSET, FGFR3, CCND3, CCND1, MAF, and MAFB), and secondary mutations, such as gain of 1q21 and deletion of 17p13 (TP53 locus), have been detected at diagnosis and during disease progression of MGUS or AMM (Lopez-Corral et al, Clin Cancer Res 2011). The goals of this study were to understand the underlying genetic features of MGUS and AMM, and define significant biomarkers for MGUS and AMM in disease progression. Methods Bone marrow aspirates were obtained from 221 patients with MGUS or AMM. Of 118 patients initially diagnosed with MGUS, 13 (11%) progressed to AMM or MM, while 29 of 103 (28%) patients initially diagnosed with AMM progressed to MM. In an attempt to characterize the immunoglobulin heavy chain (IGH) gene in MGUS and AMM, bone marrow clonal plasma cells (PCs) were analyzed using interphase fluorescence in situ hybridization (FISH) with two probes at 14q32 for the IGH regions. Specially, the IGH constant region (IGHC) and variable region (IGHV) were simultaneously probed to investigate the overall patterns of rearrangement. The IGHC probe is comprised of 240kb DNA sequences from IgG4 to IgM. The IGHV probe is comprised of 150kb DNA sequences from IgV3-20 to IgV7-34. Clonal PCs in the bone marrow aspirates were identified using kappa (κ) and lambda (λ) light chain restrictions. 14q32 translocations were determined based on gene expression levels of each of the partner genes combined with FISH of fusions of either the IGHC or the IGHV probe and a probe of the partner gene. Gain of 1q21 and deletion of TP53 locus were routinely performed on newly diagnosed patients. Results We found highly-diversified IGHC and IGHV signal patterns (Table) in MGUS in comparison to the predominance of a single FISH pattern in AMM/MM. In MGUS, the heterogeneous IGHC/V patterns were observed in both the κ and λ light chain restricted populations; however, none of the PC clones comprised a dominant population (〉50%), also reflected in low-risk serum free light chain (sFLC) ratios (

Description: Key Points MDS-CAs were observed in 11% of 1080 patients and often preceded clinical MDS/acute leukemia. Risk factors for MDS-type cytogenetic abnormalities included immuno-modulatory drugs, older age, male gender, and low CD34 dose (

Description: Multiple myeloma (MM) is a malignancy of terminally differentiated clonal plasma cells displaying significant molecular heterogeneity with 7 subgroups defined by gene expression profiling (GEP). Our previous work showed that MS and MF subgroups have been associated with inferior survival (Zhan et al, blood 2006). Furthermore, clinical studies have demonstrated that the addition of the proteasome inhibitor (PI) bortezomib (Bzb) to high dose melphalan based regimens provided a major advantage to patients in MS subgroup (Barlogie NJE 2006, Blood 2008; Pineda-Roman et al BJH 2008), while patients in the MF subgroups did not benefit from Bzb (Nair, Blood 2010). These findings led us to hypothesize that overexpression of MAF protein confers innate resistance to Bzb. In the present study, we assessed the ability of MAF to influence the innate resistance to Bzb and identify the molecular mechanism underlying the resistance of Bzb in high MAF-expressing patients. To investigate association of the limited therapeutic effect of Bzb with molecular subgroup of myeloma, we established the IC50 of Bzb in 24 myeloma cell lines (MMCL) belonging to different GEP-based molecular subgroups. IC50 concentration were higher (〉25nM) in 7 of 9 MAF and in all 5 MAFb MMCL, and 〉40 nM in 5 MAF and one MAFb MMCL, which expressed the highest levels of MAF protein, as determined by immunoblotting analysis. In contrast, Bzb IC50 levels were lower (7.5-20 nM) for the MMCL belonging to the other molecular subgroups. These results indicate that high MAF expression in myeloma cells may contribute to primary resistance to Bzb. Mechanistically, immunoblotting analysis demonstrated that exposure to Bzb resulted in increased MAF protein levels. These results suggested that Bzb prevents the degradation of MAF protein in myeloma cells. To further confirm that Bzb-induced stabilization of MAF protein confers resistance to Bzb, we overexpressed MAF cDNA in myeloma cells lacking MAF expression, and silenced MAF expression in myeloma cells expressing high level of MAF mRNA and protein. MM cells were infected with Lentiviral vector containing MAF cDNA or with empty vector, and stable clones selected with puromycin, designated as MMmaf and MMEV, respectively. qPCR and immunoblotting analysis showed that expression of MAF mRNA and protein in MMmaf cells were significantly higher (1.8x105-fold) than in MMEV cells. The functionality of ectopic MAF protein was confirmed by qRT-PCR analysis of downstream target genes; the mRNA level of E-cadherin was higher in MMmaf cells than those of MMEV (1.42-fold, p

Description: Introduction Non-Secretory Multiple Myeloma (NSMM) is a well described entity with an estimated prevalence of 3% characterized by typical morphological and pathological MM characteristics and the absence of an M-protein on immunofixation electrophoresis (BJH, 2003, 121:749-757). The serum free light chain (sFLC) assay can detect an abnormal serum free light chain level in up to two-thirds of cases, suggesting that many cases of NSMM are at least minimally secretory. Among the true NSMM cases there is a subset in which no cytoplasmic Immunoglobulin synthesis is detected. This entity of ‘’Non Producing’’ Multiple Myeloma (NPMM) though recognized three decades ago (J Clin Invest. 1985;76(2):765-769) has only been described in a small number of case reports. The purpose of this study is to confirm the existence of and subsequently systemically describe NPMM, through the interrogation of MIRT’s MM Data Base (MMDB). Methods MMDB was interrogated from January 2000 until January 2013. Clinical and laboratory data were systemically reviewed for the MM isotype classification. Results 210 MM patients were identified as NSMM. Flow cytometry data on cytoplasmic immunoglobulin (cIg), performed at initial diagnosis, was available for 197 out of 210 patients. 19 cases revealed no cIg by flow cytometry thus identifying them as NPMM. Review of sFLC assay results revealed that in all cases but one, the free immunoglobulin light chain levels were low and the sFLC ratio was normal, confirming that the negative result of flow cytometry was not due to a technical failure. In one single case elevated sFCL levels were measured, which could be traced to grade 3 renal failure resulting in accumulation of FLC in the serum. The MM diagnoses were confirmed by chart review for each of the 19 cases, which revealed that all cases had symptomatic MM. The basic clinical/laboratory characteristics of the 19 NPMM cases are shown in table 1. Sixteen of the 19 cases had results on immunoglobulin In Situ Hybridization (ISH) and 5 were found to be positive for κ or λ light chains. In two cases a mixed population of light chain positive and negative plasma cells by ISH was identified. α and γ heavy chain positivity was found in 4 and 1 cases respectively. Thus in pathological terms, two categories of “minimally producing” and “ totally non producing” MM can be identified. There was no overlap between the cases with cytoplasmic heavy or light chain positivity by ISH. Osteolytic bone disease by X-rays was evident in 12/19 cases and active focal lesions were seen in 11/19 patients by PET and 16/19 by MRI. Metaphase cytogenetic analysis at initial diagnosis was abnormal in 10 out of 19 available samples, with the t(11;14) translocation found in 3 of them. Baseline Gene Expression Profiling (GEP) was available for 16 cases. 13 (81%) of them belonged to the CD1/CD2 subgroup, and all of them were characterized by markedly high cyclin D1 expression, the hallmark of the t(11;14) translocation in MM. In comparison, only 21% of patients enrolled in the Total Therapy 3 trial belonged to the CD1 or CD2 molecular subgroups (94/441, p

Description: MGUS and Multiple Myeloma (MM) are two to three times more common in African Americans (AA) compared to Caucasians (CAU). They also differ in many disease characteristics, most notably the better disease specific survival of AA compared to CAU. Recent improvements in outcomes have helped the CAU population, not benefitting the AA to the same degree. Although this disparity is well appreciated, little is know regarding the associated molecular differences. Availability of large sample sizes of GEPs from CD138+ selected PC offered the opportunity to investigate whether race related differences in PC-GEP exist. The training set included 467 Caucasians and 43 African Americans, and the test set included 209 CAU and 13 AA. A model was developed based on the 27 most differentially expressed probesets (by q-value) in the training set (ROC-AUC, 0.999), and was validated in the test set (ROC-AUC, 0.979). These data were further corroborated in a validation set of asymptomatic MM (AMM) (63CAU/7AA, ROC-AUC, 0.941). Heatmaps of the 27-gene model for the training, test and validation sets are shown in Figure 1a -c. Performance analysis of this 27-gene model revealed a sensitivity of 69.2% and 90.9%, a specificity of 99.1% and 98.1% and a positive predictive value of 99.2% and 96.4% in the MM test set (table 1) and AMM validation set (table 2) respectively.Figure 1aHeatmap for 27-gene model, training set(Key: blue = African-American, red=Caucasian)Figure 1a. Heatmap for 27-gene model, training set. / (Key: blue = African-American, red=Caucasian)Figure 1bHeatmap for 27-gene model, test set(Key: blue = African-American, red=Caucasian)Figure 1b. Heatmap for 27-gene model, test set. / (Key: blue = African-American, red=Caucasian)Figure 1cHeatmap, 27-gene model, AMM validation set(Key: blue = African-American, red=Caucasian)Validation set: 63 Caucasian, 7 African-American with AMM baseline GEP samplesFigure 1c. Heatmap, 27-gene model, AMM validation set. / (Key: blue = African-American, red=Caucasian). / Validation set: 63 Caucasian, 7 African-American with AMM baseline GEP samplesTable 1Summary measures of the 27-gene model, test setMeasureValueSensitivity9/13 (69.2%)Specificity205/209 (98.1%)Positive Predictive Value214/222 (96.4%)Table 2Summary measures of the 27-gene model, AMM validation setMeasureValueSensitivity5/7 (71.4%)Specificity62/63 (98.4%)Positive Predictive Value67/70 (95.7%) The bone marrow microenvironment (ME) has a significant impact on the behavior of myeloma cells. We therefore also sought to investigate the differences in the bone marrow ME between AA and CAU. Using a set of 783 probesets that are unique to the bone marrow microenvironment, we analyzed GEPs of whole bone marrow biopsies in a training set of 250 CAU and 22 AA patients, identifying only 7 probesets with a false discovery rate

Description: Background Gene expression profiling (GEP) via microarray analysis enables the measurement of expression levels for tens of thousands of genes in a single experiment, and it has been widely used in clinical practice for cancer classification, risk stratification, and treatment selection. However, results of GEP-based clinical diagnostic/prognostic tests can be highly affected by batch effects when clinical samples are processed differently (e.g. on different days, by different technicians, or using different sample protocols). Yet, this problem is rarely discussed in the literature. Understanding the role of batch effects on GEP-based conclusions is vital because GEP-based-risk treatment assignment has been used for personalized treatment to improve patients' survival: patients with low risk and favorable clinical and biological features can be treated with a less intensive, lower toxicity treatment, while patients with high risk and unfavorable features can be treated with a more aggressive, potentially more toxic therapeutic approach. Here, we investigate how sample processing discrepancies influence various GEP-based prognostic models in multiple myeloma (MM) and how to adjust for such effects during data analysis. Methods/Results In 2009, Affymetrix discontinued their One-Cycle and Two-Cycle Target Labeling and Control Reagents (hereon referred to as the 'old' kit) and replaced it with a 3' IVT Express Kit (hereon referred to as the 'new' kit). To examine the impact of the replacement kit on GEP results, we set out to process eleven CD138-enriched patient plasma cell samples using both the new and old kits side-by-side before hybridizing them separately to the Affymetrix HG-U133 Plus 2.0 arrays. Various GEP-based MM prognostic scores, including UAMS-70, UAMS-80, UAMS-17, EMC-92, IFM-15, MRC-IX-6, and MILLENNIUM-100, were calculated and compared between the matched GEP pairs with either MAS5 or RMA normalization, with and without batch effect adjustment by ComBat (Combating Batch Effects When Combining Batches of Gene Expression Microarray Data). Both the UAMS-70 and UAMS-80 scores are based on log2 ratios between unfavorable and favorable genes regarding survival, which are self-normalized. However, with MAS5 alone, the UAMS-70 score was similar between the two kits (p-value=0.37 from paired t-test) but not for UAMS-80 score, which was significantly higher under the new kit (p-value