Treatment Of Multiple Myeloma Patients Research Articles

Lenalidomide Increases Human Dendritic Cell Maturation in Multiple Myeloma Modulating Both Monocyte Differentiation and Mesenchymal Stromal Cell Inhibitory Properties through Ikaros and Casein Kinase 1 Degradation, Respectively

The use of the immunomodulatory drugs (IMiDs®), including lenalidomide (LEN), to reverse tumor-mediated immune suppression and amplify multiple myeloma (MM)-specific immunity is currently being explored. Particularly LEN effects on antigen presenting cells are still unknown. In this study we investigated LEN potential effect on Dendritic Cell (DC) differentiation and activity and on the immunosuppressive properties of human mesenchymal stromal cells (hMSCs) on DCs.DCs were obtained either from THP1 (THP1-DC), a human monocytic cell line, or from CD14+ cells, purified by an immunomagnetic method both from peripheral blood (PB) and bone marrow (BM) samples of a total cohort of 19 patients with monoclonal gammopathies including 13 with active MM.Monocyte-derived DC (mo-DC) differentiation was induced by the treatment with GM-CSF and IL-4 for 8 days and TNF-α for the last 24 hours. LEN treatment was performed at concentration ranging from 0.1 to 2 μM. Then, non-adherent cells were analyzed for DC maturation markers (CD83, HLA-DR, CD80, CD86 and CD209) by flow cytometry. The levels of soluble factors involved in the immune response (CCL2, CCL5, CXCL10, IL-6, IL-8, IL-10, IL-12, IFN-γ, TNF-α) were measured in the conditioned media (CM) of mo-DCs by a Bio-Plex® Multiplex System. Moreover, the effect of LEN treatment on DC ability to stimulate T-cell proliferation was investigated through an MTT assay on CD3+ cells, purified from PB of MM patients, co-cultured for 6 days with LEN pretreated autologous mo-DCs, in RPMI medium at 15% AB human serum. Ex vivo studies were also performed in order to evaluate mo-DC differentiation of CD14+ cells purified from PB of 4 MM patients before and after 7 days of LEN treatment at 25 mg/day without Dexamethasone. Moreover, mo-DC differentiation was performed in the presence of CM of human TERT transfected (hTERT) -hMSCs treated for 5 days with LEN (0.1-2 μM). The expression levels of factors with an inhibitory effect on DC development (CCL5, IDO1, IL6, PTGS2, TGFB) were tested in LEN treated hTERT-hMSCs by real time PCR. Finally, the protein levels of Cereblon and its transcription factor targets Aiolos, Ikaros, IRF-4, p62 and Casein kinase (CK) 1 and 2 were evaluated either in THP1-DCs or in hTERT-hMSCs LEN treated cells by Western Blotting.Despite a reduction of both number and % of mature mo-DCs, LEN at the concentration range reached in vivo in MM patients significantly increased the median intensity expression of HLA-DR (LEN vs vehicle, p<0.0001) and CD86 (LEN vs vehicle, p<0.0001) by mo-DCs derived from BM. Similarly, increased HLA-DR (LEN vs vehicle, p=0.047) and CD86 (LEN vs vehicle, p<0.0001) expressions have been seen in mo-DCs derived from PB of both active MM and smoldering MM patients. LEN treatment also enhanced the production of IL-8 in a dose dependent manner (LEN 2 μM vs vehicle, p<0.0001), CCL2 (LEN 2 μM vs vehicle, p=0.0207) and TNF-α levels (LEN 0.1 μM vs vehicle, p=0.0054). Moreover, LEN pretreated mo-DCs showed an increased ability to stimulate CD3+ cell proliferation. Interestingly, even in vivo LEN treatment of MM patients enhances mo-DC differentiation of CD14+ cells in ex-vivo culture (HLA-DR, LEN day7 vs LEN day0 p=0.047; CD209, LEN day7 vs LEN day0 p=0.017). LEN effect on DC differentiation was associated to Ikaros degradation as it was observed a significant down-regulation in THP1-DCs after LEN treatment, with a dose dependent effect. LEN also blunted hTERT-hMSC inhibitory effect on mo-DC differentiation and significantly down-regulated PTGS2 gene expression levels at all concentrations tested (p<0.05). These effects were likely to be not mediated by Aiolos, Ikaros, and IRF-4 because they were not expressed by hMSCs; meanwhile p62, CK1, CK2 were expressed but only CK1 expression was down-regulated after LEN treatment with a dose dependent effect.In conclusion, LEN increased the expression of mature DC markers both in vitro and in ex vivo cultures, enhancingDC ability to stimulate T cell proliferation and to release chemokines involved in the immune response. LEN treatment also reduces the immunosuppressive properties of hMSCs, suggesting new possible effects of IMiDs® on the alloreactivity against MM cells. DisclosuresGiuliani:Celgene: Research Funding; Janssen: Research Funding.

Daratumumab Interferes with Flow Cytometric Evaluation of Multiple Myeloma

The diagnosis of multiple myeloma (MM) requires the demonstration of clonal plasma cells at ≥10% marrow cellularity or a biopsy-proven bony or extra-medullary plasmacytoma, plus one or more myeloma-defining events. Clinical laboratories use multi-parameter flow cytometry (MFC) evaluation of cytoplasmic light chain expression in CD38-bright, CD45-dim or CD138-positive, CD45dim cells to establish plasma cell clonality with a high-degree of sensitivity and specificity.Daratumumab, a humanized IgG1 kappa monoclonal antibody targeting CD38, has been shown to significantly improve outcomes in refractory MM, and daratumumab was granted breakthrough status in 2013. Daratumumab is currently approved for treatment of MM patients who have failed first-line therapies. It has been noted that daratumumab can interfere in blood bank assays for antibody screening, as well as serum protein electrophoresis (SPEP). We describe for the first time daratumumab interference in the assessment of plasma cell neoplasms by MFC; daratumumab interfered with both CD38- and CD138-based gating strategies in three MM patients.Patient A is a 68 year old man with a 10 year history of MM who had failed multiple therapies. He had then been treated with daratumumab for two months, stopping therapy 25 days prior to bone marrow assessment. Patient B is a 53 year old man with a 3 year history MM who had failed numerous treatments. He had been receiving daratumumab monotherapy for two months at the time of his bone marrow studies. On multiple marrow aspirates at times of relapse prior to receiving daratumumab, both patients had demonstrated CD38-bright positive CD45dim/negative plasma cells expressing aberrant CD56, as well as kappa light chain restriction; mature B cells were polyclonal in both. Patient C is a 65 year old man with a four-year history of MM status post autologous stem cell transplantation, who had been receiving carfilzomib and pomalidomide following relapse and continues to have rising lambda light chains and rib pain. He now has abnormal plasma cells in blood worrisome for plasma cell leukemia.Bone marrow aspirates from patients A and B, and blood from patient C demonstrated near absence of CD38-bright events as detected by MFC (Figure 1). Hypothesizing that these results were due to blocking of the CD38 antigen by daratumumab, gating on CD138-positive events was assessed; surprisingly, virtually no CD138-positive events were detected by MFC. All 3 samples demonstrated a CD56-positive CD45dim population; when light chain studies were employed using specific gating on the CD56-positive population, light chain restriction was demonstrated in all patients (Figure 1). Aspirate morphology confirmed numerous abnormal, nucleolated plasma cells (Figure 2A), thus excluding a sampling error. CD138 and CD38 expression was also tested on the marrow biopsy cores from both patients. In contrast to MFC, immunohistochemistry (IHC) showed positive labeling of plasma cells with both CD138 (Figure 2B) and CD38 (Figure 2C). The reason for the labeling discrepancy between MFC and IHC is unknown. The different antibodies in the assays may target different epitopes; alternatively, tissue fixation/decalcification may dissociate the anti-CD38 therapeutic monoclonal from its target.Detection of clonal plasma cell populations is important for assessing response to therapy. Laboratories relying primarily on MFC to assess marrow aspirates without a concomitant biopsy may falsely diagnose remission or significant disease amelioration in daratumumab-treated patients. MFC is generally highly sensitive for monitoring minimal residual disease (MRD) in MM, but daratumumab-treated patients should have their biopsy evaluated to confirm the MRD assessment by MFC. We were able to detect large numbers of plasma cells and also demonstrate clonality in our patients based on an alternative MFC marker, aberrant CD56 expression, an approach that may not be possible in all cases. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Effect of Lenalidomide on Multiple Myeloma Associated Macrophages

Introduction: In multiple myeloma (MM), accessory cells, such as monocytes and macrophages, located in the bone marrow (BM) tumor microenvironment play a crucial role in the fate of malignant cells. Under the influence of the surrounding milieu, monocytes can change their migratory capacity and differentiate into tumor-associated macrophages (TAMs). In the tumor site, TAMs can alter their profile from M1 macrophages with antitumor activities to M2-like macrophages that support tumor growth.Lenalidomide (Len), an immunomodulatory drug, used for MM treatment, is known to target different immune components inducing inflammatory responses; however, its direct influence on monocyte migration, macrophage differentiation and function in the tumor microenvironment is still unclear. The current study has aimed to explore the effect of Len on monocyte recruitment, macrophage polarization and pro-tumor functions.Methods: Monocytes were isolated from peripheral blood mononuclear cells of healthy donors, using anti-CD14 microbeads. To assess their migration capacity, monocytes were allowed to migrate through transwell insert towards the conditioned medium (CM) obtained from the BM of newly diagnosed MM patients or from MM cell lines (RPMI or U266) in the presence or absence of Len (10µM); the percentage of migrating monocytes was determined by FACS. For macrophage generation, monocytes were cultured with M-CSF followed by incubation with IL-4 to obtain M2 macrophages. To generate TAMs, CM obtained from the BM of MM patients was used. Len was added to the culture every 24 hours. The phenotype and functional properties of the generated macrophages were assessed. Endocytosis was evaluated by an antigen uptake assay. Macrophages were incubated with FITC-dextran at 37°C or 4°C, as a control, for 60 minutes, and analyzed by FACS. To test T cell proliferation, autologous lymphocytes labeled with CFSE, were stimulated with PHA and co-cultured with macrophages. T cell (CD3+) division was assessed by FACS. For IFN-γ secretion evaluation, lymphocytes co-cultured with macrophages were stimulated with PMA and ionomycin. The percentage of T cells expressing IFN-γ was quantified by FACS.Results: Monocyte migration towards CM obtained from MM cell lines (RPMI or U266) or from BM of MM patients (80.89%; n=4; p<0.01, 57.17%; n=4; p<0.01 and 42.9%; n=9; p<0.05, respectively) was significantly higher compared to migration towards normal BM CM (25.39%; n=2). Monocytes treated with Len demonstrated significantly decreased migration toward CM of MM cell lines (RPMI or U266) compared to untreated monocytes (45% vs. 80.8%; n=4 and 30.2% vs. 57.1%; n=4, respectively; p<0.01). The effect of Len on monocyte migration toward patient- derived CM was diverse. While 4 samples demonstrated decreased migration compared to untreated cells [51.1% vs. 59.6%; n=4; p<0.01], in 5 samples it increased [39.8% vs. 29.7%; n=5; n=5; p<0.01].To evaluate the effect of Len on macrophage polarization we examined their phenotype and functions. Both M2 macrophages and TAMs treated with Len expressed higher levels of M2 markers CD206, CD163, and cytokine IL-10 compared to untreated macrophages. Functional assays showed that Len increased endocytosis of both M2 macrophages [50% vs. 20%; n=5; P<0.01] and TAMs [47.4% vs. 41.4%; n=6; NS]. Exposure to Len led to suppression of T cell proliferation, when T cells were co-cultured with either autologous M2 macrophages [31% vs. 16%; n=4; p<0.05] or TAMs [39.7% vs. 31.5%; n=3; p<0.01]. In addition, M2 macrophages treated with Len demonstrated a reduction in IFN-γ secretion from T cells compared to untreated M2 macrophages (10.6% vs. 7.1%; n=4; NS).Conclusions: This study has demonstrated that Len has a direct effect on monocyte/macrophage behavior in the microenvironment generated by MM cells. Len is found to reduce monocyte migration, support polarization of macrophages towards the M2 phenotype and promote macrophage immunosuppressive functions, such as endocytosis, reduction of T-cell proliferation and inhibition of IFN-γ production. These findings need to be further investigated in in vivo experiments and could support the benefit of using agents targeting specific pathways associated with TAM development in the treatment of MM patients. DisclosuresNo relevant conflicts of interest to declare.

Next Generation Sequencing of CD138+ Myeloma Cells Could Predict Response Rate and Appropriate Therapy at Diagnosis

Introduction2016 NCCN guideline recommended that induction therapy used conventional chemotherapy such as proteasome inhibitors and immunomodulatory drugs for newly diagnosis Multiple Myeloma (MM) patients. Now, HDAC inhibitor, CS-1 Antibody agents, and novel proteasome inhibitor were available for the treatment of MM. Thereby, the outcome of MM was significantly improved regarding response rates and overall survival. However, relapse is inevitable in almost all patients and the cure of myeloma is difficult even now. Recurrence of myeloma is typically more aggressive with each relapse, leading to the development of treatment-refractory disease. For each treatment, we have to choose appropriate new agents. However, we do not know how to choose these new agents Therefore, we try to use next generation sequencer as a tool of drug choosing system. The aim of this study is to identify genetic alterations in MM cells by next generation sequencer for determining the optimal drug and predicting drug resistance in the future.Patients and Methods:At first, we reviewed newly diagnosed 11 patients with MM (Male: Female 6:5) The median age was 62.27 years (range, 37-78 ). Patients received novel agents including bortezomib in Sapporo Medical University Hospital. Patient plasma cell DNA was extracted from magnetic bead-enriched bone marrow CD138-positive fraction.CD138-negative cells and peripheral lymphocyte were used as matched non-tumor cells. Next, forty nanograms of DNA were used for multiplex PCR amplification with an Ion Ampliseq Comprehensive Cancer Panel. This cancer panel offers targeted coverage of all exons in 409 tumor suppressor genes and oncogenes frequently cited and frequently mutated in human cancers. (covered regions: 95.4% of total). We sequenced 15,992 regions which obtained more than 1.5 megabases of target sequence.Result:Each sample underwent on average 8.3 million sequencing reads after quality filtering. The mean read depths were 539x, and >95% of targeted bases were represented by at least 20 reads. The average number of synonymous mutations detected per patient was 5.8 (range 0-11).We also detected copy number variations which segments of genome can be deleted from sequencing data. We found genetic alterations that associated with poor prognosis and refractory to chemotherapy of MM patients.We detected mutationin several patients such as EGFR,IKBKB,ERBB3,MYH11,CYLD,TP53,CDH2. These genomes are mainly involved in several important pathways, including cell cycle regulation, RTK-MAPK-PI3K and NF-kB. And then, we can detect main mutation pathway of cancer cells and choose the pathway blockable agents.Conclusion:We performed targeted next-generation sequencing gene analysis of malignant plasma cells from patients with MM. Next generation sequencing analysis of Myeloma cells can detect mutation and copy number variations. These data predict of drug resistance and facilitate improvements in the treatment of MM patients. We can use targeted next-generation sequencing as tool of drug choosing system. This method is useful for determining the optimal drug for patients with MM in the future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Extending Autologous Hematopoietic Stem Cell Transplantation As First Line Treatment in Multiple Myeloma Patients with Severe Renal Impairment: A Retrospective Study of the Francophone Society of Stem Cell Transplantation and Cellular Therapy (SFGM-TC)

IntroductionRenal impairment occurs in 20-30% of newly diagnosed multiple myeloma patients, requiring dialysis for up to 10% of patients.Recent results ofan IFM (Intergroupe Francophone du Myélome) study confirm the effectiveness of high dose melphalan followed by Autologous Hematopoietic Stem Cell Transplantation (ASCT) as first line therapy in multiple myeloma patients. Most centers exclude renal failure patients due to concerns of toxity, because data are limited for safety and toxicity to consider. This current retrospective study observes toxicity and outcome in 55 multiple myeloma patients with severe renal impairment, undergoing high dose melphalan and ASCT, in the age of bortezomib.MethodsUsing the PROMISE database, 55 multiple myeloma patients French SFGM-TC centers were included from January 2002 to November 2012. Characteristics of the patients presenting with creatinine clearance less than 30ml/min at time of ASCT were included.ResultsMedian age was 61 (40-75) years old. Thirty-nine (71%) presented free light chain myeloma. Twenty-three (42%) patients were on dialysis at the time of ASCT and during hospitalization. Thirty-one (56%) patients received bortezomib as induction therapy. Melphalan dose ranged from 75mg/m² to 200mg/m². Thirty (55%) patients were treated with a dose of 140mg/m². Median delay between diagnosis and ASCT was 5.3 months.Among 50 patients evaluated for toxicity, respectively febrile neutropenia and mucositis occurred in 92% and 90% of patients. Half of the mucositiscases were grade 3-4. The median number of days before neutrophil and platelet recovery was respectively 12 days (10 - 34) and 16 days (7 - 331). Two patients experienced cardiac toxicity (grade 1-2), and 4 patients neurologic toxicity (grade 3 for one patient).Among 51 patients evaluated for response, 12 patients (24%) were at least in Very Good Partial Response (VGPR) before ASCT, including 5 in Complete Response (CR). All of them were treated with bortezomib as induction therapy.After ASCT, 30 patients (58.8%) reached at least VGPR (22 CR and 8 VGPR). Among 30 patients in VGPR or more post ASCT, 22 (73%) were treated with bortezomib before ASCT.Table 1 shows the responses.Fourteen (26%) received two ASCT. One patient died from toxicity (cerebral bleeding during second ASCT). There is no argument for dramatic response improvement with this procedure in comparison to single ASCT.After a median follow-up of 55 months, median progression free survival (PFS) was 55 months, and median overall survival (OS) was 95 months. In multivariate analysis, dose of melphalan at 140mg/m² was significantly correlated with a better PFS (p=0.006), compared to 100mg/m² (or less) and to 200mg/m².During follow-up 29 patients (54%) relapsed and 25 died. The main causes of death were relapse and disease progression (72%). There were only 3 cases of treatment related mortality (TRM). At day 100, the cumulative incidence of TRM was 6%.Among 23 dialysis patients, 6 patients (26%) became dialysis independent, but not during ASCT. Thirteen (56%) were still alive at last follow-up, and 8 (34%) experienced relapse. Median PFS for this subgroup of patients was 73 months.ConclusionWith an acceptable TRM, without major toxicities, high dose melphalan (140mg/m²) following by ASCT appears to be a real benefit for multiple myeloma patients with severe renal failure. This includes patients on dialysis. Response is dramatically improved by this procedure, with median PFS similar to that of patients with normal renal function. Finally, adjunction of bortezomib as induction therapy also improves the efficacy of ASCT. [Display omitted] DisclosuresAugeul-Meunier:gilead: Consultancy; janssen: Consultancy. Karlin:amgen: Consultancy, Honoraria; janssen-cilag: Consultancy, Honoraria; celgene: Consultancy, Honoraria; Bristol: Consultancy; takeda: Consultancy. Benboubker:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Avet-Loiseau:sanofi: Consultancy; amgen: Consultancy; janssen: Consultancy; celgene: Consultancy.

Efficacy and Tolerability of the Histone-Deacetylase Inhibitor Panobinostat in Clinical Practice: a Single Center Experience

Efficacy and Tolerability of the Histone-Deacetylase Inhibitor Panobinostat in Clinical Practice: a Single Center Experience

Extensive Regional Intra-Clonal Heterogeneity in Multiple Myeloma - Implications for Diagnostics, Risk Stratification and Targeted Treatment

INTRODUCTIONIn multiple myeloma (MM) samples for diagnostics, prognostication and response evaluation are most commonly obtained from the patients' posterior iliac crest due to its accessibility and safety, assuming a homogenous spread throughout the bone marrow. However, imaging studies revealed a highly imbalanced distribution of the disease in the majority of the patients, presenting with accumulations of malignant plasma cells (PC) in restricted areas in the bone marrow (BM), so called focal lesions (FL). In line with this pattern, our recently reported preliminary results of paired FL and random BM (RBM) samples strongly indicate an unequal distribution of sub-clones in the BM.Spatial genomic heterogeneity has not been systematically analyzed in MM thus far, although its existence would have a high impact on interpretation of drug resistance studies, risk stratification and personalized treatment based on genomic markers. Here we report on an extended genomic analysis of regional heterogeneity in paired FL and RBM samples including 42 newly diagnosed and 11 extensively treated MM patients with 10 of these patients also being studied longitudinally.MATERIAL & METHODSMM PCs were CD138-enriched. Leukapheresis products were used as controls. For whole exome sequencing (WES) we applied the qXT kit and the SureSelect Clinical Research Exome bait design (Agilent). Paired-End sequencing was performed on an Illumina HiSeq 2500. Sequencing data were aligned to the GRCh37/hg19 reference using BWA. Somatic single nucleotide variants (SNV) were identified using MuTect. Copy number aberrations (CNA) were derived from Illumina HumanOmni 2.5 bead chip data using ASCAT. Subclonal reconstruction was performed using SciClone. Gene expression profiles (GEP) were generated using Affymetrix U133plus2 microarrays. Statistical analyses were carried out using the R software package 3.1.1.RESULTSIn 42 newly diagnosed patients we detected a median number of 86 (34 to 807) mutations per patient with up to 42% (median 5%) of them being unique to a specific site (non-ubiquitous). Among known MM driver genes, BRAF (n=2) and KRAS (n=4) were the genes that most often showed non-ubiquitous mutations at baseline. In treated patients mutations in KRAS, NRAS and RB1 contributed to regional heterogeneity in one patient each. Furthermore, we found temporal heterogeneity in mutations affecting ATRin two patients, aberrations recently associated with poor outcome.Analyzing chromosomal aberrations with known prognostic value we observed three newly diagnosed patients with a site-specific del(1p) affecting CDKN2C and/or FAM46Cwith two of these patients also showing regional heterogeneity in del(17p13). Non-ubiquitous gain(1q) or amp(1q) was seen in two patients at baseline. Of note, in all of these cases the unique event was detected in a FL and one case with a unique gain(1q) at baseline presented with this aberration in subsequent samples. These observations strongly support the concept of FLs being sites of resistant clones able to cause relapse.In four patients a MYC translocation was seen at only one site. In the longitudinal analysis we found one patient in whom a MYC translocation clone was replaced by a clone with a different MYC translocation, indicating that events at the MYC locus are secondary and can be sub-clonal. In contrast, primary IgH translocations were always shared, confirming that they are initiating events.Paired samples from RBM and FLs derived from three newly diagnosed patients showed discordant GEP risk profiles, further supporting the existence of site-specific high risk (HR) clones. To investigate the clinical relevance of this finding we analyzed outcome data of 263 newly diagnosed patients with paired GEP data. The 34 cases with discordant GEP based risk scores showed no significant differences in outcome compared to cases with HR at both sites, suggesting that HR sub-clones drive prognosis even if they are not ubiquitously present.CONCLUSIONSWe show that spatial genomic heterogeneity is common in MM. The existence of site-specific sub-clones highlights the importance of heterogeneity analyses for accurate risk prediction, detection of suitable targets for precision medicine and identification of aberrations contributing to treatment resistance. As a result we strongly recommend to include FL examinations into routine diagnostics and follow-up analyses in MM. DisclosuresAshby:University of Arkansas for Medical Sciences: Employment. Barlogie:Signal Genetics: Patents & Royalties. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria.

Spatial-Temporal Genomic Analyses Reveal a Component of "Big Bang" Kinetics in Multiple Myeloma Evolution

INTRODUCTIONData available to date has been interpreted to show that Multiple myeloma (MM) is the end result of a multistep transformation process from normal to malignant plasma cells (PC) with molecular aberrations being attained through branching pathways. According to this Darwinian model, sequential mutations lead to the outgrowth of fitter clones, which outcompete previously dominant clones. This model does not fully explain the enormous intra-clonal heterogeneity at presentation and the extreme alternating clonal dominance during treatment. Also this model has not be adapted to take Focal Lesions (FL) into account, which are hallmarks of MM seen in imaging studies.An alternative model has been discussed that stresses the importance of early events. In this "Big Bang" model most of the heterogeneity appears at early stages of a malignancy when clonal growth is not environmentally constrained. Transferring this model to the early evolution of MM, ancestor clones containing initiating mutations occupy the available BM niches. Subsequently, sub-clones acquire additional mutations and expand locally with some eventually forming FLs.In order to understand the processes underlying the evolution of MM and the pathologic basis of FLs we analyzed a set of paired FL and random iliac crest samples including 42 newly diagnosed and 11 treated MM patients with 10 of these patients also being studied longitudinally.METHODSPaired-end whole exome sequencing of CD138-enriched MM PCs was performed on an Illumina HiSeq 2500. Somatic single nucleotide variants were called from BWA aligned sequencing reads using MuTect. Copy number aberrations were derived from Illumina HumanOmni 2.5 bead chip data using ASCAT. Subclonal reconstruction was performed using SciClone.RESULTSIn newly diagnosed patients we observed three main sub-clonal patterns. In the first pattern, detected in 18 patients, the clonal composition was very similar between different sites. The second pattern, seen in 5 patients, was associated with site-specific dominant clones. In one of these patients we found four site-specific dominant clones in four different samples with each of them containing private mutations in known cancer driver genes, e.g. BRAF and KRAS. This type of regional clonal dominance strongly supports "Big Bang" dynamics rather than late metastasis in MM.The third pattern, found in 19 patients, was characterized by regionally dominant clones that were also detectable at other sites at low frequencies. In one patient a clone that dominated in two distant FLs infiltrated the third FL and was borderline detectable on the mutational level at the iliac crest. This clone contained a bi-allelic deletion of the tumor suppressor CDKN2C, a gain(1q21) and a del(17p13), a combination of three events that potentially provided a massive fitness advantage, resulting in a selective sweep through the BM.In "Big Bang" type behavior treatment which eradicates clones obviates the advantage of previously dominant clones of being first, restarting clonal competition. As a consequence we would expect to see reduced spatial heterogeneity in treated patients and longitudinally collected samples. Surprisingly, only one patient lacked spatial and temporal heterogeneity. In the majority of patients, however, we observed the opposite with a dramatic spatial heterogeneity which was further enhanced by the site-specific appearance of additional mutations. It is still not clear whether additional site-specific mutations in FLs represent randomly acquired events during treatment contributing to branching evolution or whether treatment led to a regional clonal collapse allowing descendants to catch up with the most-recent common ancestor in FLs.CONCLUSIONSFrequent pronounced spatial heterogeneity supports a component of "Big Bang" kinetics in the evolution of MM. This is an unexpected finding as MM is a BM cancer where easy movement of the MM propagating cells through the BM would be expected. It does, however, explain the massive heterogeneity found at diagnosis. It also highlights the important of FLs in the MM ecosystem as hotspot of evolution which could be the source of units of selection that underlie selective sweeps. It further suggests that aberrations found to be enriched in relapse patients already existed in regionally separated areas at baseline, supporting the recently introduced concept of some tumors being "born bad". DisclosuresMorgan:Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding; Univ of AR for Medical Sciences: Employment; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Effects of INCB052793, a Selective JAK1 Inhibitor, in Combination with Anti-Myeloma Agents on Human Multiple Myeloma (MM) In Vitro and In Vivo

Introduction: Several studies have demonstrated constitutive activation of the JAK-STAT pathway in MM through dysregulated signaling of cytokines such as IL-6. In addition to its crucial role in promoting the growth, proliferation and survival of myeloma cells, IL-6 is also a potent stimulator of osteoclastogenesis and influences the tumor microenvironment in the bone marrow (BM) of MM patients by promoting an immunosuppressive milieu. Since JAK1 has been shown to be important for IL-6 signaling in MM, studies to assess the effect of JAK1 inhibition alone and in combination with other anti-MM agents were undertaken.Methods: The human MM cell lines RPMI8226 and U266 were obtained from ATCC and MM1S was kindly supplied by Steven Rosen, MD (Northwestern University, Chicago, IL). BM aspirates were obtained from patients with MM as approved by the Institutional Review Board (Western IRB BIO 001) and informed consent was obtained in accordance with the Declaration of Helsinki. BM mononuclear cells (MCs) were isolated using density-gradient centrifugation with Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). All cells were maintained in RPMI1640 (Omega Scientific, Tarzana, CA) supplemented with 10% fetal bovine serum (FBS), 2mM l-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin, in an atmosphere of 5% carbon dioxide at 37°C. Primary MM BMMCs were cultured in the presence of the JAK1 selective inhibitor INCB052793 plus a panel of anti-MM agents including the alkylating agents cyclophosphamide (CY), melphalan (MEL), and bendamustine (BEN), the proteasome inhibitor carfilzomib (CAR), dexamethasone (DEX) or the immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM). Cells from RPMI8226 or U266 MM cell lines were cultured in the presence of INCB052793 plus CY, MEL, BEN, CAR, DEX, LEN, or POM. After 48 hours, cell viability was assessed using the MTS assay. For the in vivo studies, mice were implanted with a piece of the human MM tumor LAGk-1A. Seven days post-implantation, mice were randomized into treatment groups, and tumor size was measured on a weekly basis. All in vivo studies were approved by the institutional animal care and use committee.Results: In vitro studies demonstrated that combinations of INCB052793 with a broad spectrum of anti-MM agents synergistically inhibited the viability of BMMCs from MM patients. INCB052793 plus the three alkylating agents or CAR synergistically inhibited the viability of these cells. INCB052793 plus CY or MEL also significantly decreased the viability of the MM1 cell line. In vivo, LAGk-1A-bearing mice had significantly smaller tumors when treated with INCB052793 alone when compared to vehicle control at day 35 post implantation. This was in contrast to mice treated with single agent DEX, LEN or POM. Although the combination of INCB052793 with DEX, LEN or POM did not synergistically inhibit MM cell line growth in vitro, mice receiving the doublets of INCB052793 and DEX, LEN or POM demonstrated an effect on tumor growth that was superior to the doublets of DEX with LEN or POM. Mice receiving the triple combination of INCB052793 + DEX with LEN or POM demonstrated the most significant reduction in tumor growth compared with all other combinations tested. The inhibition of tumor growth with these combinations was observed throughout the study (through day 70) and all combinations were well tolerated. Concomitant with effects on tumor growth, a significant reduction in serum human IgG levels was also observed. In a separate study also using the LAGk-1A model, we evaluated the combination of INCB052793 with CAR or bortezomib (BOR). Combinations of INCB052793 + CAR or BOR were superior at inhibiting tumor growth when compared to single agent INCB052793.Conclusion: These in vitro and in vivo preclinical studies demonstrate that the combination of the JAK1 inhibitor INCB052793 with a broad spectrum of anti-MM agents are effective, and provide further support for the clinical evaluation of these drug combinations for treating MM patients. Studies to further understand the mechanistic effects of these combinations on MM signaling and the tumor microenvironment are ongoing. DisclosuresBerenson:Amgen Inc: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Ultra-Deep Targeted Sequencing Does Not Identify MM Patients with Different Prognosis: Results from a Randomized Phase II Clinical Trial

Next-generation sequencing (NGS) has revealed new insight into the complexity of clonal and subclonal architecture of multiple myeloma (MM). The introduction of targeted studies allows the detection of mutations with very low allele frequencies at affordable price. However, there is few information about the prognostic impact of this mutational profile in series of homogeneously treated MM patients.In this study, we analyzed the most frequently mutated genes in MM from patients included in a randomized clinical trial applying the highest read depth to date. We performed a correlation study between our results and cytogenetics aberrations and clinical outcomes.We used Ampliseq Library Kit 2.0 to target 77 genes (M3Pv2.0 panel) related to critical pathophysiological pathways, associated to drug resistance or targetable with molecular drugs in MM. We sequenced DNA (15ng) from CD138+ purified plasma cells from 80 MM patients at diagnosis included in GEM2010MAS65 clinical trial. We sequenced at an average read depth of 1500X and using Ion Proton sequencer (Thermo Fisher, USA, PaloAlto, CA). We used Ion Reporter software applying in-house modifications in call variants process. We also sequenced DNA from CD138 negative population in 30% of patients in order to filter out potential artifacts and stablish conditions for excluding germline variants. We first fitted a cox-proportional hazard model to predict survival and a second approach using a penalized regression LASSO.We identified 137 gene mutations in the 80 patients analyzed, 54 mutations were predicted pathogenic by Shif and Poliphen and 65 have been described in COSMIC database. 85% of patients harbored at least 1 mutation. The most frequently mutated genes were KRAS (16%), DIS3 (15%), NRAS (13%), BRAF (6%), and TP53 (5%), accounting for the 54% of the total number of mutations. The most frequently mutated pathways were RAS and NFKB in 34% and 28% of patients respectively. Mutational allele frequency for KRAS, BRAF and TP53 was, in all cases, lower than 50% while DIS3 showed mutations in a broad range (from 2 to 85%). Only one patient presented one mutation in NRAS at 73 % of allele frequency. For patients with more than one mutation, two different scenarios have been found. Some patients showed several genes mutated in a similar allele frequency values. On the contrary, a complex subclonal structure was confirmed in 3 patients, with mutations at very different clonal frequency. CD138 negative fraction was sequenced to confirm that these mutations were exclusive of plasma cells. The first of them had two mutations in DIS3 at 8 and 53% of allele frequency, second in KRAS and DIS3 at 13 and 63% respectively and the third in NRAS and IFNGR2 at 5 and 37% respectively. We found no differences in the survival based on pathway, mutational allele frequency or number of mutations. We also investigated pairwise associations between events and we not found significant correlations (Figure 1). Cox-proportional hazard model only showed a negative impact in survival of patients with TP53 mutations.This ultra-deep targeted sequencing strategy is able to detect mutations in most of patients, at very low allele frequency and using a minimal amount of DNA. Despite of its huge capacity to detect mutations, we have not identified new prognostic factors in MM patients treated with highly efficient therapies. However, this study again confirms the complexity of the genomic landscape of MM and the great heterogeneity between patients. The role of theses mutations at relapse as well as in the subsequent treatment effectiveness remains unknown and should be preferably investigated in larger cohorts of homogeneously treated MM patients. [Display omitted] DisclosuresMartínez-López:Novartis: Honoraria, Speakers Bureau. Oriol:Janssen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Stewart:celgene: Consultancy.

Bortezomib-based treatment for multiple myeloma patients with renal impairment: A systematic review and meta-analysis of observational studies.

Background:Renal insufficiency is a common and severe complication of patients with multiple myeloma. The aim of this study was to evaluate bortezomib-based treatment for multiple myeloma patients with renal insufficiency.Methods:The Cochrane Library, Embase, PubMed, ISI, China National Knowledge Infrastructure, Chinese Biomedical Literature Service System, Chongqing VIP Database, and Wan Fang Data were systematically searched to identify observational studies from January 1, 2001, to December 31, 2015. Myeloma response rate and renal remission rate were pooled by using risk ratio and 95% confidence interval (CI). The Cochran Q and I statistics were used to assess heterogeneity. Sensitivity analysis was performed to test the feasibility of pooled results. Publication bias was conducted when included studies were ≥9. Furthermore, grades of evidence were performed to evaluate study quality.Results:Eleven retrospective cohort studies were included in the final analysis. The number of available studies and risk ratios (95% CI) were, respectively, 10 and 1.48 (95% CI: 1.28–1.71) for myeloma overall response, 6 and 3.69 (95% CI: 2.22–6.13) for myeloma complete response, 9 and 1.47 (95% CI: 1.28–1.69) for renal overall remission, and 8 and 1.49 (95% CI: 1.26–1.75) for renal complete remission. No significant publication bias was observed and sensitivity analysis confirmed the stability of results. The overall qualities of evidence were high for myeloma complete response and medium for the other 3 outcomes based on the Grading of Recommendations, Assessment, Development and Evaluation system.Conclusion:Current evidence indicated that bortezomib-based treatment could improve myeloma overall response (especially myeloma complete response) and renal overall remission (including renal complete remission).

Treatment of multiple myeloma with monoclonal antibodies and the dilemma of false positive M-spikes in peripheral blood

ObjectivesTo characterize the effect of three humanized IgG κ monoclonal antibodies (daratumumab, isatuximab, and elotuzumab) on the interpretation of results generated by protein electrophoresis, immunofixation, free light chain, and heavy/light chain assays performed on human serum. MethodsHealthy volunteer serum and serum from multiple myeloma patients were supplemented with clinically relevant concentrations of each of the three monoclonal antibodies. These specimens then underwent analysis via serum protein electrophoresis, immunofixation, serum free light chain quantification, heavy/light chain quantification, total IgG, and total protein. In addition, serum specimens from patients who had undergone treatment with elotuzumab for multiple myeloma underwent similar analysis. ResultsAddition of the study drugs to serum from both the healthy donor as well as multiple myeloma patients resulted in a visible and quantifiable M-protein on SPEP and a visible IgGκ band by IFE. Increases were also noted in total IgG, IgGκ, and IgGκ/IgGλ-ratios. Analysis of serum from multiple myeloma patients receiving study drug showed similar findings with an additional IgGκ band and quantifiable M-protein with similar migration patterns in specimens drawn after administration. ConclusionThe treatment of multiple myeloma patients with monoclonal antibodies results in a visible and quantifiable M-protein that has the potential to falsely indicate poor response to therapy.

Increase of CIK cell efficacy by upregulating cell surface MICA and inhibition of NKG2D ligand shedding in multiple myeloma.

Multiple myeloma, which is a monoclonal plasma cell malignancy, still remains incurable despite recent progress in our understanding of this disorder. Adoptive immunotherapy of multiple myeloma using cytokine-induced killer cells is yielding promising results in clinical trials; however, some myeloma cells still evade immune surveillance by various unknown molecular mechanisms. This study aims at increasing the efficacy of cytokine-induced killer cells in targeting this tumor, using selective small-molecule inhibitors which increase and stabilize surface expression of the natural killer group 2, member D ligand, major histocompatibility complex class I polypeptide-related sequence A (MICA) on myeloma cells. We treated 2 multiple myeloma cell lines-U266 and KMS-12-PE-with 3 drugs. One of these drugs (sodium butyrate) is a histone deacetylase inhibitor. Another drug which was used (matrix metalloproteinase inhibitor III) blocks ligand shedding while the third drug (phenylarsine oxide) obstructs surface ligand internalization. The effect of these drugs on cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, surface ligand expression was examined using flow cytometry, and ligand shedding was assessed using enzyme-linked immunosorbent assay. We demonstrated that cytokine-induced killer cells have increased cytotoxicity against multiple myeloma cells after combined drug treatment than without drug pretreatment. We also established that this increased cytotoxicity was due to potent upregulation and stabilization of surface MICA on the surface of these tumor cell lines. Our study thus highlights further therapeutic options which could be used for the treatment of multiple myeloma patients.

Rare SNPs in receptor tyrosine kinases are negative outcome predictors in multiple myeloma.

Multiple myeloma (MM) is a plasma cell disorder that is characterized by a great genetic heterogeneity. Recent next generation sequencing studies revealed an accumulation of tumor-associated mutations in receptor tyrosine kinases (RTKs) which may also contribute to the activation of survival pathways in MM. To investigate the clinical role of RTK-mutations in MM, we deep-sequenced the coding DNA-sequence of EGFR, EPHA2, ERBB3, IGF1R, NTRK1 and NTRK2 which were previously found to be mutated in MM, in 75 uniformly treated MM patients of the “Deutsche Studiengruppe Multiples Myelom”. Subsequently, we correlated the detected mutations with common cytogenetic alterations and clinical parameters. We identified 11 novel non-synonymous SNVs or rare patient-specific SNPs, not listed in the SNP databases 1000 genomes and dbSNP, in 10 primary MM cases. The mutations predominantly affected the tyrosine-kinase and ligand-binding domains and no correlation with cytogenetic parameters was found. Interestingly, however, patients with RTK-mutations, specifically those with rare patient-specific SNPs, showed a significantly lower overall, event-free and progression-free survival. This indicates that RTK SNVs and rare patient-specific RTK SNPs are of prognostic relevance and suggests that MM patients with RTK-mutations could potentially profit from treatment with RTK-inhibitors.

Patient reported outcomes (PROs) of multiple myeloma (MM) patients treated with panobinostat (PAN) after ≥2 lines of therapy based on the international phase 3, randomized, double-blind, placebo-controlled, PANORAMA-1 trial.

8054Background: PAN, in combination with bortezomib (BTZ) and dexamethasone (DEX), is FDA-approved for the treatment of MM patients who have received ≥ 2 prior regimens, including BTZ and an immuno...

Risk of Second Primary Cancers in Multiple Myeloma Survivors in German and Swedish Cancer Registries.

We aimed at investigating the distribution and risk of second primary cancers (SPCs) in multiple myeloma (MM) survivors in Germany and Sweden to provide etiological understanding of SPCs and insight into their incidence rates and recording practices. MM patients diagnosed in 1997–2010 at age ≥15 years were selected from the Swedish (nationwide) and 12 German cancer registries. Standardized incidence ratios (SIRs) were used to assess risk of a specific SPC compared to risk of the same first cancer in the corresponding background population. Among 18,735 survivors of first MM in Germany and 7,560 in Sweden, overall 752 and 349 SPCs were recorded, respectively. Significantly elevated SIRs of specific SPCs were observed for acute myeloid leukemia (AML; SIR = 4.9) in Germany and for kidney cancer (2.3), AML (2.3) and nervous system cancer (1.9) in Sweden. Elevated risk for AML was more pronounced in the earlier diagnosis period compared to the later, i.e., 9.7 (4.2–19) for 1997–2003 period versus 3.5 (1.5–6.9) for 2004–2010 in Germany; 3.8 (1.4–8.3) for 1997–2003 versus 2.2 (0.3–7.8) for 2004–2010 in Sweden. We found elevated risk for AML for overall, early diagnosis periods and longer follow-up times in both populations, suggesting possible side effects of treatment for MM patients.

Novel Patient Derived Multiple Myeloma Model Reflects Sensitivity Towards Anticancer Treatment in Multiple Myeloma Patients

Background Appropriate animal models for hematological malignancies are highly attractive, because they allow the study of the tumor biology and underlying disease mechanisms. They also constitute a major prerequisite for rapid bench-to-bedside translation of investigational anticancer therapies. To validate our multiple myeloma patient (pt)-derived xenograft (MM PDX) model (Schueler et al, Expert Opin Biol Ther, 2013), we systematically analyzed a panel of MM PDX with regard to their sensitivity towards standard of care treatment and compared these data with the pts' clinical outcome.Methods Bone marrow (BM) cells of 11 MM pts were implanted intratibialy (i.t.) into 103 NOD/Shi-scid/IL-2Rγnull (NOG) mice (n= 6-18 / pt sample). Mice were treated according to pts' therapy with VCD (Bortezomib, Cyclophosphamide, Dexamethasone), or to evaluate additional treatment options with Rd (Lenalidomide, Dexamethasone). Tumor growth and antitumoral activity in mice were assessed in tumor-bearing mice and compared to untreated control mice as well as to pts' response. Tumor growth in the mouse model was monitored by whole-body fluorescence-based in-vivo-imaging (IVI) using CF750-labeled α-HLA ABC antibody before and during treatment as well as 24h after last treatment cycle as described (Schueler J. PLOSone 2013). Mock-injected animals served as negative controls. Engraftment of human MM cells in murine organs was confirmed by flow cytometry and patho-histological analyses (immunostaining) at the end of the study.Results The pt cohort included a typical MM clientele for referral centers, with a median age of 75 years (range 56-85), median BM infiltration of 80% (20-90), and high- and standard-risk cytogenetics in 5 and 6 pts, respectively. All pts had advanced disease with Durie&Salmon stage III and active/symptomatic MM. All pts received VCD after diagnosis and BM sampling.MM cell engraftment could reliably be determined from experimental day 10 on in all 11 MM PDX models, at all assessed sites, namely within the BM, spleen and peripheral blood (PB) of recipient mice. Individual pt samples displayed distinct tumor growth patterns in vivo. Fluorescence intensity of engrafted murine organs ranged from 2- to 15-fold compared to mock injected control mice. Mean IVI signals in BM of recipient mice were 10-fold higher as compared to spleen signals, qualifying the BM niche as the preferred homing localization of pts' MM cells. Of note, both injected and non-injected BM sites were infiltrated by MM cells 10 days after tumor cell injection. Engraftment of human MM cells in the respective murine organs was confirmed by flow cytometry (HLA ABC, CD138, CD38) and histology and verified MM engraftment via both methods, confirming prior reports (Schüler PLOSone 2013; Groen Blood 2012;120:e9-16, Overdijk MAbs. 2015;7:311-21). The murine engraftment capacity was independent of MM type, disease stage, BM infiltration and cytogenetics of the donor pt.VCD was applied to 9 different MM PDX models and induced partial remission (PR; defined as at least 50% reduction of murine tumor load in BM, spleen and/or PB) in 5 out of 9 tested MM PDX models, whereas 2 cases each showed stable disease (SD) or progression (PD). The response rates in the mouse avatars mirrored the clinical outcome of the respective MM pts in 8/9 cases; only one MM pt showed serological and clinical PR, whereas the corresponding mice displayed SD. Rd induced PR in 1 and PD in a second MM PDX model, underlining the feasibility of MM PDX for drug screening approaches.Conclusions Due to the complex tumor biology, murine models of MM are still challenging. Our data support the preclinical rationale to use i.t.-injected NOG mice, since they closely resemble clinical MM with respect to symptoms, disseminated disease sites and response to anticancer treatment. Possible applications for the MM mouse avatars include development of new anticancer drugs as well as definition of biomarker strategies and selection of treatment options for individual pts with relapsed/refractory MM. The data of our preclinical study may serve as a useful future strategy to guide treatment decisions in refractory pts. The suitability as a drug development tool will be additionally determined performing treatment experiments with novel agents, e.g. elotuzumab or daratumumab. DisclosuresSchueler:Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment.

CAR-Engineered T Cells Specific for the Elotuzumab Target SLAMF7 Eliminate Primary Myeloma Cells and Confer Selective Fratricide of SLAMF7+ Normal Lymphocyte Subsets

Background: SLAMF7 (CS1, CD319) is uniformly and highly expressed in multiple myeloma (MM) where it promotes adhesion and survival of malignant plasma cells (mPCs) in the bone marrow niche. It is absent on normal solid organ tissues but known to be expressed on lymphocyte subsets (T, B and NK cells). Clinical evaluation of the anti-SLAMF7 monoclonal antibody (mAb) Elotuzumab (huLuc63) has resulted in marked reversible lymphodepletion and conferred potent anti-MM efficacy in combination therapy. Here, we evaluated the potential to generate SLAMF7-directed chimeric antigen receptor (CAR) modified T cells from previously treated MM patients and analyzed their potency against autologous mPCs as well as fratricidal activity against normal lymphocyte subsets.Methods: Flow cytometric analyses for SLAMF7 expression on mPCs and normal lymphocyte subsets of MM patients (n=67) and healthy donors (n=20) was performed using specific mAbs and matched isotype controls. A SLAMF7-specific CAR was constructed using the VH/VL targeting domains of mAb huLuc63, fused to an Ig-Fc spacer and a signaling module of CD3ζ and CD28. Lentiviral gene transfer was performed into CD3/CD28-bead stimulated bulk CD4+ and CD8+ T cells of MM patients (n=7). CAR transgene positive T cells were enriched using an EGFRt transduction marker and expanded for functional analyses.Results: We confirmed high SLAMF7 expression levels on mPCs in all analyzed samples and detected SLAMF7 expression on a fraction of normal lymphocytes obtained from peripheral blood of MM patients, including naïve and memory CD4+ (95% CI: 33-59%) and CD8+ T cells (75-95%), B cells (25-35%) and NK cells (94-98%). Remarkably, the proportion of SLAMF7+ cells was significantly higher in MM patients compared to healthy donors in all corresponding lymphocyte subsets (p<0.05). Despite high level SLAMF7 expression on the input T cell population, functional CD4+ and CD8+ T cells expressing the SLAMF7-CAR could be readily generated in all 7 MM patients, and expanded to therapeutically relevant doses in a single expansion cycle following enrichment (>107 cells). We analyzed the kinetics of SLAMF7 expression on CD4+ and CD8+ CAR T cells during the manufacturing process and detected rapid disappearance of SLAMF7+ T cells in T cell lines modified with the SLAMF7-CAR. By contrast unmodified T cells and T cell lines expressing a CD19-CAR retained a significant proportion of SLAMF7+ T cells, suggesting that expression of the SLAMF7-CAR induced killing of SLAMF7+ T cells. In vitro functional testing of SLAMF7-CAR CD4+ and CD8+ T-cell lines confirmed potent specific lysis of SLAMF7+ MM cell lines including MM1.S and OPM-2 and stable SLAMF7-transfectants of K562, as well as antigen specific IFNγ secretion and productive proliferation. In a flow cytometry based cytotoxicity assay, co-incubation of mPCs with autologous (or allogeneic) SLAMF7-CAR T cells resulted in elimination of >90% of mPCs after a 4-hour incubation period, whereas CD19-CAR or unmodified T cells had no discernible effects. Moreover, in an in vivo xenograft MM model (NSG/MM1.S) a single administration of SLAMF7-CAR T cells resulted in complete MM clearance and long-term survival, whereas mice treated with CD19-CAR or unmodified T cells rapidly expired from progressive disease. Finally, we analyzed the fratricidal potential of SLAMF7-CAR T cells to predict hematologic toxicities that might occur in a clinical setting. Co-incubation of purified CD4+ and CD8+ primary T cells, B cells and NK cells with SLAMF7-CAR T cells resulted in rapid and specific elimination of only SLAMF7+ subsets, whereas SLAMF7- cells remained viable and functional as confirmed for CD4+ and CD8+ T cells by inducible IFNγ secretion.Conclusion: Our data demonstrate that SLAMF7-specific CAR T cells can be reproducibly generated from MM patients and exert remarkable anti-myeloma efficacy in pre-clinical models in vitro and in vivo. Lymphocytic fratricide does not preclude the manufacture of SLAMF7-CAR T cells but might be associated with acute (cytokine storm) or chronic (viral infections) side effects in a clinical setting. However, such toxicities may be prevented e.g. by preparative lymphodepletion and antiviral prophylaxis and enable the implementation of SLAMF7-CAR T cell therapy as a safe and effective modality in the treatment of MM. DisclosuresKnop:Celgene Corporation: Consultancy. Einsele:Novartis: Consultancy, Honoraria, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Induction therapy alters plasma fibrin clot properties in multiple myeloma patients: association with thromboembolic complications.

Induction therapy in patients with multiple myeloma increases the risk of thromboembolism. We have recently shown that multiple myeloma patients tend to form denser fibrin clots displaying poor lysability. We investigated the effect of induction therapy on fibrin clot properties in multiple myeloma patients. Ex-vivo plasma fibrin clot permeability, turbidity, susceptibility to lysis, thrombin generation, factor VIII and fibrinolytic proteins were compared in 48 multiple myeloma patients prior to and following 3 months of induction therapy, mainly with cyclophosphamide-thalidomide-dexamethasone regimen. Patients on thromboprophylaxis with aspirin or heparins were eligible. A 3-month induction therapy resulted in improved clot properties, that is higher clot permeability, compaction, shorter lag phase and higher final turbidity, along with shorter clot lysis time and higher rate of D-dimer release from fibrin clots than the baseline values. The therapy also resulted in lower thrombin generation, antiplasmin and thrombin-activatable fibrinolysis inhibitor (TAFI), but elevated factor VIII. Progressive disease was associated with lower posttreatment clot permeability and lysability. Despite thromboprophylaxis, two patients developed ischemic stroke and 10 had venous thromboembolism. They were characterized by pretreatment lower clot permeability, prolonged clot lysis time, longer lag phase, higher peak thrombin generation, TAFI and plasminogen activator inhibitor -1. Formation of denser plasma fibrin clots with reduced lysability and increased thrombin generation at baseline could predispose to thrombotic complications during induction treatment in multiple myeloma patients. We observed improved fibrin clot properties and thrombin generation in multiple myeloma patients except those with progressive disease.

Lipopolysaccharide-Induced CXCL10 mRNA Level and Six Stimulant-mRNA Combinations in Whole Blood: Novel Biomarkers for Bortezomib Responses Obtained from a Prospective Multicenter Trial for Patients with Multiple Myeloma.

To identify predictive biomarkers for clinical responses to bortezomib treatment, 0.06 mL of each whole blood without any cell separation procedures was stimulated ex vivo using five agents, and eight mRNAs were quantified. In six centers, heparinized peripheral blood was prospectively obtained from 80 previously treated or untreated, symptomatic multiple myeloma (MM) patients with measurable levels of M-proteins. The blood sample was procured prior to treatment as well as 2-3 days and 1-3 weeks after the first dose of bortezomib, which was intravenously administered biweekly or weekly, during the first cycle. Six stimulant-mRNA combinations; that is, lipopolysaccharide (LPS)-granulocyte-macrophage colony-stimulating factor (GM-CSF), LPS-CXCL chemokine 10 (CXCL10), LPS-CCL chemokine 4 (CCL4), phytohemagglutinin-CCL4, zymosan A (ZA)-GMCSF and ZA-CCL4 showed significantly higher induction in the complete and very good partial response group than in the stable and progressive disease group, as determined by both parametric (t-test) and non-parametric (unpaired Mann-Whitney test) tests. Moreover, LPS-induced CXCL10 mRNA expression was significantly suppressed 2-3 days after the first dose of bortezomib in all patients, as determined by both parametric (t-test) and non-parametric (paired Wilcoxon test) tests, whereas the complete and very good partial response group showed sustained suppression 1-3 weeks after the first dose. Thus, pretreatment LPS-CXCL10 mRNA and/or the six combinations may serve as potential biomarkers for the response to bortezomib treatment in MM patients.