Positive Myeloma Research Articles

Sensitivity, specificity, and accuracy of molecular profiling on circulating cell-free DNA in refractory or relapsed multiple myeloma patients, results of MM-EP1 study

As a promising alternative to bone marrow aspiration (BMA), mutational profiling on blood-derived circulating cell-free tumor DNA (cfDNA) is a harmless and simple technique to monitor molecular response and treatment resistance of patients with refractory/relapsed multiple myeloma (R/R MM). We evaluated the sensitivity and specificity of cfDNA compared to BMA CD138 positive myeloma plasma cells (PCs) in a series of 45 R/R MM patients using the 29-gene targeted panel (AmpliSeq) NGS. KRAS, NRAS, FAM46C, DIS3, and TP53 were the most frequently mutated genes. The average sensitivity and specificity of cfDNA detection were 65% and 97%, respectively. The concordance per gene between the two samples was good to excellent according to Cohen’s κ coefficients interpretation. An increased number of mutations detected in cfDNA were associated with a decreased overall survival. In conclusion, we demonstrated cfDNA NGS analysis feasibility and accuracy in R/R MM patients who may benefit from early phase clinical trial.

RAMP up: Risk Adapted Post-Autologous Transplant Maintenance Therapy in MRD Positive Myeloma Patients

Background. Post-autologous stem cell transplant (ASCT) maintenance therapy for patients with newly diagnosed multiple myeloma (MM) continues to evolve. Currently, single agent lenalidomide maintenance remains standard of care but outcomes for patients with high-risk cytogenetics are inferior to those with standard risk disease; optimal maintenance strategies for these patients need to be developed. Minimal residual disease (MRD) has proven to be a powerful prognostic factor for PFS and OS but is not yet used to guide treatment. Isatuximab is a cytolytic CD38-directed, immunoglobulin G1 (IgG1) chimeric monoclonal antibody FDA-approved for the treatment of relapsed or refractory MM that is overall well tolerated and efficacious when used in combination regimens. Our upcoming trial, RAMP UP (NCT05344833), investigates the safety and efficacy of isatuximab combined with lenalidomide as a maintenance regimen in patients who are MRD-positive after autologous transplant. Study Design and Methods. This prospective, multicenter single-arm phase 2 (Figure 1) trial will enroll patients that are MRD-positive (10 -5) by Adaptive next generation sequencing within 90 days (+/- 30 days) post-autologous transplant. We plan to enroll: R-ISS stage 1, 2 or 3; <2 lines of induction therapy prior to transplant; > PR per IMWG criteria post-transplant; and ECOG Performance Status <2. Patients will be treated with isatuximab (10 mg/kg weekly for cycle 1 and every other week thereafter) and lenalidomide (10 mg daily on days 1-21) on 28-day cycles for a total of 3 years. Patients will be monitored for disease response every 3 months and for marrow MRD every 12 months. The primary endpoint is the 1-year rate of CR/MRD negativity (10 -5); secondary endpoints include 1-year rate of CR/MRD negativity (10 -6), IMWG-defined response rates, 3-year overall CR/MRD-negativity, PFS, OS, treatment-related adverse events, and health related quality of life (HRQoL) measured by EORTC-QLQ-C30, EQ-5D-5L, EORTC QLQ-MY20. We propose enrollment of 50 patients to allow for approximately 10% dropout rate in order to have 43 patients analyzed for the primary endpoint; for 50 patients to be treated, we estimate that 75-100 patients may have to be enrolled. Correlative objectives include correlation of isatuximab pharmacokinetics and receptor occupancy with MRD-negative response and time to progression, characterization of differences in the BM and peripheral blood T, B, and myeloid cell compartments using cellular indexing of transcriptomes and epitopes (CITE-Seq) between responders and nonresponders, and concurrent assessment of other disease biomarkers, including peripheral blood circulating multiple myeloma cells (CELLSEARCH), with MRD testing. Discussion. Our study started June 2023. One patient has been enrolled, and two have signed informed consent for enrollment. Ultimately, our study aims to inform MRD risk-adapted approaches for maintenance therapy in myeloma.

Safety and efficacy of letetresgene autoleucel alone or with pembrolizumab for relapsed/refractory multiple myeloma.

This pilot study assessed the safety and efficacy of letetresgene autoleucel (lete-cel; GSK3377794), a genetically modified autologous T-cell therapy targeting New York esophageal squamous cell carcinoma-1 (NY-ESO-1)/L antigen family member 1 isoform A (LAGE-1a)-positive myeloma cells, alone or in combination with pembrolizumab in patients with relapsed/refractory multiple myeloma. Eligible patients expressed NY-ESO-1 and/or LAGE-1a and either HLA-A∗02:01, ∗02:05, or ∗02:06. Patients received lete-cel single infusion alone (arm 1) or with pembrolizumab (arm 2). 127 patients were screened, and 6 patients (3 per arm) were enrolled; patients in arm 1 and 2 received lete-cel alone, or with pembrolizumab, respectively. All patients exhibited grade 3/4 cytopenias, which resolved or improved to grade 1. One patient (arm 1) had grade 3/4 lete-cel-related adverse events (AEs); 2 patients (arm 2) had grade 3/4 AEs related to lete-cel and lymphodepletion. Three patients with grade 1/2 cytokine release syndrome (CRS) exhibited elevated post-lete-cel interleukin-6 levels versus those without CRS. Pooled overall response rate was 50% including 1 patient each with confirmed clinical response, very good clinical response, and partial response, and progression-free survival ranged from 1.3 to 5.2 months. Responders (arm 1: n = 1; arm 2: n = 2) had a time-to-response of 3 weeks, duration of response of 2.1 months. Two responders, but no nonresponders, exhibited elevated cytokine levels after lete-cel infusion. Lete-cel had a manageable safety profile and demonstrated clear but transient antitumor activity in patients with relapsed/refractory multiple myeloma. This trial was registered at www.clinicaltrials.gov as #NCT03168438.

Super Enhancer-Mediated Upregulation of HJURP Promotes Growth and Survival of t(4;14)-Positive Multiple Myeloma.

Multiple myeloma is an incurable malignancy with marked clinical and genetic heterogeneity. The cytogenetic abnormality t(4;14) (p16.3;q32.3) confers aggressive behavior in multiple myeloma. Recently, essential oncogenic drivers in a wide range of cancers have been shown to be controlled by super-enhancers (SE). We used chromatin immunoprecipitation sequencing of the active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) to profile unique SEs in t(4;14)-translocated multiple myeloma. The histone chaperone HJURP was aberrantly overexpressed in t(4;14)-positive multiple myeloma due to transcriptional activation by a distal SE induced by the histone lysine methyltransferase NSD2. Silencing of HJURP with short hairpin RNA or CRISPR interference of SE function impaired cell viability and led to apoptosis. Conversely, HJURP overexpression promoted cell proliferation and abrogated apoptosis. Mechanistically, the NSD2/BRD4 complex positively coregulated HJURP transcription by binding the promoter and active elements of its SE. In summary, this study introduces SE profiling as an efficient approach to identify new targets and understand molecular pathogenesis in specific subtypes of cancer. Moreover, HJURP could be a valuable therapeutic target in patients with t(4;14)-positive myeloma. SIGNIFICANCE: A super-enhancer screen in t(4;14) multiple myeloma serves to identify genes that promote growth and survival of myeloma cells, which may be evaluated in future studies as therapeutic targets.

Safety and Efficacy of Letetresgene Autoleucel (lete-cel; GSK3377794) Alone or in Combination with Pembrolizumab in Relapsed and Refractory Multiple Myeloma

Safety and Efficacy of Letetresgene Autoleucel (lete-cel; GSK3377794) Alone or in Combination with Pembrolizumab in Relapsed and Refractory Multiple Myeloma

Preclinical Evaluation of Hdp-101, a Novel Anti-BCMA Antibody-Drug Conjugate, in Multiple Myeloma

Several antibody-drug conjugates (ADCs) are currently being evaluated in clinical trials in a variety of hematologic malignancies. Surprisingly, most ADCs are based on only few toxic compounds, largely limited to microtubuli- or DNA-targeting toxins (auristatins and maytansines or duocarmycins and pyrrolobenzodiazepines, PBDs). These payloads are mainly targeting proliferating cells potentially leading to limited efficacy in diseases with a low proliferation rates such as indolent lymphomas or multiple myeloma. Thus, new compounds with alternative toxicity mechanisms could enhance the therapeutic potential of ADCs. We therefore focus on amanitin based ADCs, so called ATACs (antibody-targeted Amanitin conjugates) comprising a new class of antibody-drug conjugates with amanitin as toxic payload. Amanitin is the most well-known toxin of the amatoxin family and binds to the eukaryotic RNA polymerase II thereby inhibiting the cellular transcription process at very low concentrations irrespective of the proliferation status of the target cell.In the current study, we have assessed in vitro and in vivo specificity andefficacy of HDP-101, an ATAC targeting BCMA (B cell maturation antigen). BCMA (also known as CD269) is expressed on cells of the B cell lineage, predominantly on plasma blasts and plasma cells. It is not expressed on naïve, germinal center, and memory B cells. BCMA is highly expressed on malignant plasma cells and therefore considered an ideal target in multiple myeloma.In vitro cytotoxic potency of HDP-101 was tested on several BCMA-positive myeloma cell lines, as well as on non-proliferating primary CD138+ cells isolated from triple and quadruple refractory myeloma patients. Profound cytotoxic effects were seen at pico- to nanomolar concentrations of HDP-101 even in myeloma cells with low epitope density. Importantly, toxicity was observed neither in non-BCMA expressing control cells nor in myeloma cells exposed to an amanitin-loaded non-target control antibody. In mouse xenograft models of human myeloma, HDP-101 caused dose-dependent tumor regression including complete remissions after a single i.v. dose of 2.0 mg/kg and 4.0 mg/kg in subcutaneous xenografts and after single i.v. doses from 0.1 mg/kg to 2.0 mg/kg in disseminating xenografts.Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index after sequentially applied doses of 0.3 mg/kg, 1.0 mg/kg, and multiple dose applications of 4 x 3.0 mg/kg. Hematology and clinical chemistry parameters were unaffected except for a mild to moderate and transient increase of liver enzymes and lactate dehydrogenase. The half-life of the HDP-101 in serum was ~12 days; the free toxin was detectable at levels close to the lower limit of quantification only.In conclusion, targeted cytotoxic drug delivery to BCMA positive myeloma cell lines was achieved by using HDP-101, an anti-BCMA-ATAC. The mode of action of the payload amanitin led to an efficient anti-tumor response in vitro and in vivo with good tolerability in non-human primate studies yielding a very favorable therapeutic index. Amanitin-based ADCs in the therapy of multiple myeloma is a novel promising approach with a distinct mode of action to overcome drug resistance and improve patient outcome. A First-in-human trial with HDP-101 is expected to start in 2018. DisclosuresBreunig:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Pálfi:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Müller:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Lutz:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Hechler:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Kulke:Heidelberg Pharma GMBH: Employment, Other: Employee Stock Options. Goldschmidt:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Chugai: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Morphosys: Research Funding; Millenium: Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Pahl:WILEX AG: Employment, Membership on an entity's Board of Directors or advisory committees, Other: Employee Stock Options; Heidelberg Pharma GMBH: Employment, Membership on an entity's Board of Directors or advisory committees, Other: Employee Stock Options. Raab:Heidelberg Pharma GMBH: Research Funding.

Phenotypic Analysis of NK Cell in Multiple Myeloma Patients

Introduction:The exploitation of host immune function is critical for the development of the novel myeloma therapy. NK cells play an important role in the anti-myeloma immune response, and the investigation of NK cell function provides us a beneficial information. We performed phenotypic analyses of NK cells in patients with multiple myeloma.Methods:Peripheral blood samples were obtained from 30 multiple myeloma patients (10 newly diagnosed cases of myeloma, 10 relapsed, and 10 in complete or partial remission), and 9 healthy volunteers. NK cells were identified as a fraction of CD3 negative and CD56 positive, and NKp46 was further used for NK cell identification in CD56 positive myeloma cell-containing samples. The phenotype of NK cells was characterized by the expression of SALMF7, NKG2D, FcγRIII, PD-1, and CD56bright or dim.Results:At the diagnosis of multiple myeloma, the number of NK cells was significantly low in comparison to that of patients in remission (p=0.013). Regarding immune checkpoint molecule, the expression of PD-1 on the NK cells of myeloma patients was significantly increased in comparison with that of healthy donors (p= 0.003). The expression of SLAMF7 on the NK cell of myeloma patients was decreased significantly (p=0.048), whereas, NKG2D on the NK cells did not differ between myeloma patients and healthy donors. Notably, the expression of FcγRIII, which is a key player in a mechanism of antigen-dependent cytotoxicity, was significantly suppressed in the NK cells of myeloma patients (p=0.001).In patients who were treated with immunomodulatory drugs (IMiDs), although there was no significant change in the number of total NK cells, the CD56dim NK cell fraction was significantly increased(p=0.019), and significant suppression of PD-1 (p=0.035) on the NK cells were observed.Conclusion:The phenotypic analyses suggested that the anti-tumor function of NK cells may be suppressed in myeloma patients. However, IMiDs may increase the activated NK cells. Further investigation is warranted. [Display omitted] DisclosuresHagiwara:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Host and Disease Factors Impacting Presence of Accessory Band during Therapy with Daratumumab in Multiple Myeloma Patients

Achieving and maintaining Complete Response (CR) after autologous hematopoietic cell transplant is the key to improved clinical outcomes in patients (pts) with multiple myeloma (MM). Daratumumab (DARA), an IgG1 Kappa anti-CD38 monoclonal antibody, have shown significant anti-myeloma activity leading to increasing usage in the peri-transplant settings. Frequently, use of DARA may be associated with appearance of low concentration band on serum protein electrophoresis (SPE) impacting CR assessment after transplant. The incidence and factors influencing appearance of this band are largely unknown. This band potentially can be an indirect measurement of the plasma DARA concentration influenced by amount of administered agent and but also with volume of CD38 positive myeloma cells binding to DARA. The objective of this study is to analyze the factors influencing appearance of this band on SPE. Methods: 77 pts who received DARA were retrospectively studied. Pts who did not have available SPE after starting DARA (n: 9) and those who had AL Amyloidosis without Myeloma (n: 3) were excluded. The host and disease characteristics extracted. Pts assigned to two cohorts based on presence (PB) or absence (NB) of the accessory band. The disease response assessment was assessed based on IMWG criteria. Result: Median age for pts in PB and NB was 62 and 64.4 years respectively. All pts received weight-based dosing (16mg/Kg). 31 pts had detectable band assigned to PB cohort and 34 pts without any detectable band assigned to NB. The accessory band appeared over a median of 46 days after starting therapy (range: 7-390 days) and predominantly (74%) during the weekly DARA schedule. No significant difference noted in median weight and serum creatinine between PB and NB cohorts. Median M-spike on SPE at the start of DARA was 1.1 gr/dL (range: 0 - 5.5 gr/dL) in NB cohort vs. 0.3 gr/dL (range: 0 3.8 gr/dL) in PB patients (p-value Conclusion: Based on our data, the frequency of detecting this band is not impacted by type of myeloma (i.e., IgG vs. IgA) but by the magnitude of the M-spike. This may be reflective of larger disease volume in bone marrow with less M protein detected in the peripheral blood. The appearance of accessory band associated with DARA treatment during weekly dosing may be a surrogate early marker for predicting disease response. Further large scale studies are required to confirm this findings.

The Diagnostic Value of Magnetic Resonance Imaging in Identifying Unsuspected Malignancy in Patients Undergoing Percutaneous Vertebral Augmentation for Vertebral Compression Fractures.

The aim of this study was to identify the rate of unsuspected malignancy in vertebral compression fractures (VCFs) treated with percutaneous vertebral augmentation procedures (PVAPs). From 2004-2015, 410 patients with VCFs underwent PVAPs with biopsy in a single tertiary hospital. All patients had preoperative magnetic resonance imaging (MRI) read by consultant radiologists and reviewed by the performing surgeon prior to PVAPs. All procedures were performed by fellowship-trained spine surgeons. A patient was considered to have an unsuspected malignancy if preoperative MRI was negative for malignancy but histology from the operative biopsy was positive. A total of 44 of 45 patients (97.8%) were identified to have malignancy on preoperative MRI. One patient had a negative MRI but positive biopsy (myeloma). This patient also had a positive myeloma panel. A total of 41 of 44 patients with suspicious MRI preoperatively had a history of malignancy with histology consistent with metastatic spread from the known primary. Two patients had a new diagnosis of malignancy (1 breast carcinoma, 1 metastatic cancer likely of breast or gastrointestinal origin). Younger patients were more likely to have a VCF due to malignancy (odds ratio, 28.33 in age < 60 years). Almost 98% of patients with malignancy (44 of 45 patients) could be successfully identified with a preoperative MRI. The addition of a myeloma panel to MRI identified all patients with malignancies prior to PVAP in our study. We recommend MRI and myeloma panel for all patients with VCFs to be treated with PVAPs. For patients who undergo a PVAP, routine biopsy should be performed.

PF573 SINGLE CELL RNA-SEQ REVEALED A RARE ITGA2 EXPRESSING CELL SUBPOPULATION WITH DISTINCT CANCER STEM CELL MARKER SIGNATURE IN MULTIPLE MYELOMA

Background: Multiple myeloma (MM) stem cells (MMSC) are a rare cell population capable of self-renewal and differentiation into the predominating mature myeloma cells. Specific targeting this cell population could effectively eradicate the tumors in the patients. Previous studies reported CD138 non-expressing myeloma cells as putative MMSC. However, conflicting results by subsequent studies demonstrated the tumor-initiating and clonogenic cells expressed CD138 and apoptotic features in CD138 non-expressing cells. Aims: In this study, we aimed at identification of MMSC by examination of the transcriptome of myeloma cells at single cell level. Methods: Single cell RNA-Seq with flow-based SMART-Seq2 protocol was employed to determine the transcriptome of 768 myeloma cells from MM1 s and NCIH929 cell lines (384 cells each line) and 68 cells from a clinical MM bone marrow sample. The percentages of myeloma cells expressing the candidate biomarker(s) protein were determined by flow cytometry. Results: Results of quality control analyses indicated ∼400K read counts per cell with 15–20% of multiple mapping representing ribosomal RNAs and 12–15% mitochondrial RNAs and with 6700–8600 variable genes for transcriptome clustering analyses. By unsupervised analysis using t-distributed stochastic neighbor embedding (t-SNE), myeloma cells were clustered according to their cell cycle statuses. After controlling the cell cycle effects by regression, two clusters with similar cell numbers were observed in each cell line. Interestingly, the results of supervised analysis with t-SNE on 24 cancer stem cell markers expression revealed a rare subpopulation of myeloma cells (MM1 s: 2.6%; NCIH929: 3.6%) that were signified by Integrin alpha 2 (ITGA2) expression but no expression of OCT4 and NANOG stem cell markers. Flow cytometry using anti-ITGA2 antibody confirmed the presence of this rare cell population (∼0.3%) in MM cells. For the cells from the clinical sample, 3 clusters were observed by unsupervised analysis after controlling cell cycle effects. Studies of the signature genes for each cluster indicated two clusters were derived from erythroid cells and lymphocytes; while the 3rd cluster (n = 49) from myeloma cells, within which one ITGA2-expressing cell was idenitifed. ITGA2 encodes a cell surface protein CD49b integrin receptor as a marker for primitive human hematopoietic stem cells. In leiomyoma, CD49b marked the side population cells and the stemness of CD49b-expressing cells was also demonstrated. Recently, increased NOTCH1 and its downstream targets expression implying high activity of NOTCH signaling, which are involved in stem cell self-renewal and differentiation, were reported in CD49b-expressing trophoblast progenitor cells in human placenta. All these findings suggested the link of ITGA2 and stem cell properties. Functional studies of the ITGA2 positive myeloma cells are currently in progress regarding their clonogenic potentials and self-renewal capacity. Summary/Conclusion: Our study revealed a rare subpopulation of ITGA2-expressing cells with distinct cancer stem cell markers signature in MM cell lines and a clinical sample, potentially representing MMSC.

The snoRNA target of t(4;14) in multiple myeloma regulates ribosome biogenesis.

The orphan small nucleolar RNA (snoRNA) ACA11 is overexpressed as a result of the t(4;14) chromosomal translocation in multiple myeloma (MM), increases reactive oxygen species, and drives cell proliferation. Like other snoRNAs, ACA11 is predominantly localized to a sub‐nuclear organelle, the nucleolus. We hypothesized that increased ACA11 expression would increase ribosome biogenesis and protein synthesis. We found that ACA11 overexpression in MM cells increased nucleolar area and number as well as argyrophilic nuclear organizing regions (AgNORs). Supporting these data, samples from t(4;14)‐positive patients had higher AgNORs scores than t(4;14)‐negative samples. ACA11 also upregulated ribosome production, pre‐47S rRNA synthesis, and protein synthesis in a ROS‐dependent manner. Lastly, ACA11 overexpression enhanced the response to proteasome inhibitor in MM cells, while no effect was found in response to high doses of melphalan. Together, these data demonstrate that ACA11 stimulates ribosome biogenesis and influences responses to chemotherapy. ACA11 may be a useful target to individualize the treatment for t(4;14)‐positive myeloma patients.

IKZF1/3 Protein Expressions Are Associated with a Better Survival in Relapsed/Refractory Multiple Myeloma Patients Treated with Lenalidomide

It has been demonstrated that lenalidomide causes selective degradation of IKZF1 (Ikaros) and IKZF3 (Aiolos) which are two essential transcription factors for proliferation of multiple myeloma cells. Consequently, the drug sets up a molecular sequence of events that lead to programmed cell death in the tumoral cells. This anti-proliferative effect is mediated by down-regulation of c-Myc and interferon regulatory factor 4 (IRF4). However, it is not clear whether IKZF1/IKZF3 protein expression in myeloma cells is predictive of clinical outcome. Thus, we evaluated bone marrow samples of 50 relapsed/refractory multiple myeloma (MM) patients regarding IKZF1/3 protein expression before starting lenalidomide. There were 31 males and 19 females with median age of 59 years (range 41-75). They all had received lenalidomide-based therapy after relapse following autologous stem cell transplantation (ASCT), thalidomide, or bortezomib. The median follow-up was 86.4 months. By immunohistochemistry (IHC), CD138 positive myeloma cell aggregates were examined for IKZF1 and IKZF3 protein expression. We used H-score method (range 0-300) based on the intensity and percentage of the stained myeloma cells. Cases were considered positive for IKZF1 or IKZF3 if H-score is equal or over 150 or 200, respectively. IKZF1 showed nuclear staining but IKZF3 showed both nuclear and cytoplasmic staining.IKZF1 and IKZF3 were expressed in 72% and 58% of the bone marrow specimens, respectively. IKZF1 and IKZF3 expressions were strongly correlated (p<0.0001). Both IKZF1 & IKZF3 expressions were associated with longer progression free (p=0.0029 & p<0.0001, respectively) and overall survivals (p=0.0014 & p<0.0001, respectively). IKZF3 (p=0.0025) expression but not IKZF1 (p=0.094) was correlated with the clinical response to lenalidomide. There was no significant association between IKZF1/3 protein expression and other clinical or biological risk factors including age, gender, International staging system (ISS), hemoglobin, calcium, creatinine, bone lytic lesions, β2 micro-globulin, albumin or cytogenetic risk factors such as del (13q), del (17p), t(4;14), and amp (1q21).Our study demonstrates that expressions of the IKZF1/3 proteins detected by IHC are correlated with superior survival outcome in refractory MM patients treated with lenalidomide. IHC is routinely available, robust and inexpensive method. Thus, if confirmed in a larger prospective study, IKZF1/3 immunostaining can be readily adopted in clinical practice for prediction of drug response and clinical outcomes in MM patients receiving lenalidomide therapy. DisclosuresReece:Celgene: Consultancy, Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Merck: Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Abstract 3791: TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents

Abstract Background: Multiple myeloma and its associated bone disease are generally incurable. Hence, better therapies are needed, ideally targeting biomolecules implicated in the aberrant biology of the disease. Overexpression of transient receptor potential cation channel TRPV6, a highly selective calcium channel has been observed in breast, colon, thyroid, ovary and prostate cancer tissues, and in several tumour cell lines. TRPV6 expression has also been seen in osteoclasts, however its role in bone metabolism remains unclear. The reciprocal interaction between osteoclasts and myeloma cells is pivotal to the generation of bone lesions that characterize myeloma. While myeloma cells secrete factors promoting osteoclast activity, osteoclasts in turn are known to induce myeloma cell growth and survival. The TRPV6 peptide antagonist SOR-C13 is currently in phase I clinical trials as an anti-cancer therapy for advanced cancers. We investigated the expression and potential therapeutic significance of TRPV6 in human osteoclasts and myeloma cells using shrew venom derived peptide antagonists. Methods: High affinity TRPV6 peptide antagonists SOR-C13 and SOR-C27 derived from the C-terminus of venom from the short-tailed shrew, Blarina brevicauda were used. Human primary osteoclasts were generated in vitro from human bone marrow (BM) aspirates; characterized by Hoechst-phalloidin staining, Tartrate resistant acid phosphatase (TRAP) staining, TRAP enzyme activity and Cathepsin K expression. CD138 positive myeloma cells were isolated from patient bone marrow specimens by EasySep immunomagnetic separation, or examined in tissue microarrays of patient BM core biopsies. TRPV6 expression in primary human osteoclasts, myeloma cell lines and myeloma patient BM microarray was checked by qPCR, immunohistochemistry or immunoblotting. Anti-resorptive potential of SOR peptides using human osteoclasts was evaluated in Osteoassay plates that mimic bone, and myeloma cell growth inhibition was determined by prestoblue cell viability assays. Results: We found strong expression of TRPV6 protein in human myeloma cells and osteoclasts by immunohistochemical staining on myeloma patient bone marrow sections. Human osteoclasts generated in vitro and CD138 positive myeloma patient bone marrow plasma cells were found to express TRPV6. We saw dose-dependent inhibition of osteoclast activity in vitro by SOR-C13 and SOR-C27, including markedly reduced osteoclast formation, decreased TRAP activity and reduced osteoassay surface resorption. TRPV6 peptide antagonists were also found to inhibit the growth of human myeloma cell lines U266 and KMM-1. Conclusion: Anti-myeloma and anti-osteoclast activity of human TRPV6 antagonist peptides was seen in the current study. Taken together, our findings suggest a novel therapeutic approach for multiple myeloma involving TRPV6 inhibition to target both myeloma cells and osteoclasts. Citation Format: Alli Murugesan, Philippe Tremblay, Ming Han, Bithika Ray, Tyler Lutes, Tony Reiman. TRPV6 calcium channel peptide antagonists as novel antimyeloma and antiresorptive agents. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3791.

Interfering with Arginine Metabolism As a New Treatment Strategy for Multiple Myeloma

IntroductionStarvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far.MethodsHuman myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry.Results5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group.ConclusionMyeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. DisclosuresBomalaski:Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

CD26 Is a Novel Target for the Treatment of Tumor Progression and Its Related Osteolytic Bone Disease in Multiple Myeloma

Background: CD26, a 110-kDa transmembrane glycoprotein with DPPIV activity, has been implicated in tumorigenesis and shown to be expressed in several tumor cells including malignant lymphoma, whereas its role has not been characterized in plasma cell malignancies, yet. We have recently shown that CD26 is intensely expressed in activated osteoclasts (OCs) in osteolytic bone tumors including multiple myeloma (MM). CD26 is not expressed in hematopoietic stem cells, but M-CSF and sRANKL induced human OC differentiation with the upregulation of CD26 expression in monocyte-macrophage lineage cells, OC precursor cells and OCs. Blockade of CD26 signaling with humanized anti-CD26 monoclonal antibody (YS110) impaired OC differentiation via inhibiting RANKL-induced MKK3/6-p38MAPK-mi/Mitf phosphorylation in OC precursor cells (Nishida et al., 2014). In the present study, we characterize the biological function of CD26 in MM cells in the bone marrow (BM) and investigate the therapeutic potential of YS110 to treat MM cell growth and reduce MM-induced osteolytic lesions.Methods and Results: Although CD26 expression was not detected any of 11 MM cell lines in mono-culture, CD26 expression levels were upregulated in all MM cell lines when co-cultured with OCs for 72 hours by flow cytometry and immunohistochemistry analysis. CD26 protein levels in MM cell lines were also enhanced in co-culture with OCs by immunoblotting. To determine the factors responsible for CD26 upregulation in MM cells, we examined CD26 expression in MM cell lines under transwell co-culture conditions with OCs or under stimulation with anti-apoptotic cytokines produced by OCs. CD26 expression in MM cell lines was reduced under transwell co-culture conditions with OCs. TNFα, BAFF, APRIL and SDF-1 slightly upregulated CD26 expression in MM cell lines. To further explore CD26 expression in BM of MM patients, we performed immunohistochemical stainig on decalcified bone sections of biopsy specimens of MM cases. CD26/CD138 positive plasma cells were detected around CD26 positive OCs and certain endothelial vascular cells in several cases. Next, to clarify the role of CD26 in MM cell survival, we examined the effects of YS110 on MM cell growth and related osteolytic bone lesions in vitro and in vivo. YS110 had no significant effects on viability of MM cell lines in mono-culture, but dose-dependently inhibited growth of MM cell lines in co-culture with OCs. YS110 immediately inhibited p38 activation and its downstream Hsp27 phosphorylation in MM cells. Contitnued treatment of MM cells with YS110 also led to the reduction of total Hsp27 protein at 12 hours and 16 hours after the treatment of YS110, with commensurate decrease of phospho-Hsp27 and to increased MM apoptosis correlated with the induction of p53, increased activation of caspases and PARP, as well as the reduction of Mcl-1 and c-Myc. In parallel, we constructed a xenograft of murine model of human MM. A total of 5x106 CD26 positive MM cell lines by co-culture with OCs were inoculated to mice (NOD/SCID-s.c. mice) or directly injected into the implanted human bone chips in mice (NOD/SCID-hu mice). Mice treated with YS110 (500μg/dose) showed a significant effect in inhibiting MM tumor weight compared with control mice 4 weeks after tumor cell inoculation. To further investigate whether YS110 could reduce MM-induced osteolysis as well as suppress MM cell growth, we used NOD/SCID-hu mice model. Mice were treated with YS110 after the first detection of tumor growth. Continuous YS110 treatment significantly suppressed MM cell growth after 4 weeks. Immunohistochemistry for CD138 and TRAP staining showed decreased numbers of MM cells and TRAP positive OCs, with decreased bone resorption activity in human bones from YS110-treated versus control mice. Lastly, we examined the effects of targeting CD26 with YS110 in CD26 positive myeloma stem-like cells (SP cells). CD26 expression was demonstrated in SP cells of several MM cell lines in co-culture with OCs. YS110 dose-dependently inhibited cell viability of CD26 positive SP cells co-cultured with OCs, indicating targeting CD26 might reduce chemoresistance in MM.Conclusions: Theses results suggest that CD26 may be a promising novel target in MM, strongly supporting YS110 to inhibit MM cell growth in the BM as well as its related osteolytic bone lesions. Our results provide the framework for clinical development of YS110 as a novel therapeutic option in MM. DisclosuresMorimoto:Y's Therapeutics: Consultancy, Research Funding. Yamada:Y's Therapeutics: Consultancy, Research Funding.

Differential ICAM3 Gene Expression Correlates with Susceptibility to Natural Killer Cell-Mediated Lysis in Multiple Myeloma

Introduction We previously reported on the generation of highly activated/expanded natural killer cells (ENKs) after coculture with K562 cells modified to express membrane bound IL15 and 41BB-ligand. These cells have potent antimyeloma properties in vitro , in a NGS mouse model, and are safe when given to advanced multiple myeloma (MM) patients. (Szmania et al , J Immunother 2015) A potential obstacle to the effectiveness of ENK-based immunotherapy of MM is the evasion of immune recognition. We have generated 4 MM cell lines (OPM2, JJN3, ANBL6, and INA-6) which are resistant to ENK-mediated lysis to study mechanisms of resistance. These lines were derived from parental lines by repeated challenge with ENKs and maintained resistance long term when cultured without further exposure to ENKs.(Garg et al , Blood 2012, 120:4020) We have shown by stable isotope labeling with amino acids in cell culture-mass spectrometry, gene expression profiling (GEP), and flow cytometry that ICAM3 is downregulated in the ENK-resistant version of OPM2 (OPM2-R) compared to the parental OPM2. (OPM2-P; Garg et al , Blood 2013, 122:3105) We investigated OPM2-P and OPM2-R by whole exome sequencing (WES) and RNA sequencing (RNAseq) with a focus on ICAM3, evaluated ICAM3 cell surface expression on patient myeloma cells, and studied the importance of ICAM3 expression on ENK functionality. Methods DNA and RNA were extracted from OPM2-P and OPM2-R cells using the Qiagen AllPrep kit. WES libraries were prepared with the Agilent qXT and Agilent SureSelect Clinical Research Exome kits with additional baits covering the Ig and MYC loci. RNAseq libraries were prepared using the Illumina TruSeq stranded mRNA kit. Samples were sequenced 100bp PE on an Illumina HiSeq2500. Samples for WES were sequenced to a mean coverage of >120x and RNAseq to a target of >100M reads. WES data were aligned to the Ensembl GRCh37/hg19 human reference using BWA mem. Somatic variants were called MuTect. RNAseq data were analyzed using Tuxedo Suite. Data were aligned to the Ensembl GRCh37/hg19 human reference using TopHat with Bowtie2. Transcriptome reconstruction, quantification and differential analysis was performed using CuffLinks. ENK-mediated lysis of myeloma cells was measured by 4 hour chromium release assay in the presence of isotype or ICAM3 blocking antibody. Bone marrow aspirates were obtained from MM patients after informed consent in accordance with the Declaration of Helsinki. Primary myeloma cells were selected with CD138-coated immunomagnetic beads and ICAM3 expression was assessed by flow cytometry gated on viable CD138 positive cells. Results There was no mutation in ICAM3 in OPM2-R by WES, but RNAseq found a significant reduction in ICAM3 RNA in OPM2-R compared to OPM2-P ( p Conclusion Our findings demonstrate that MM patients harbor ICAM3-negative myeloma populations in varying frequencies, and we hypothesize that these cells may be similarly resistant to ENK-mediated lysis. Functional assays exploring this question are in progress. By understanding the mechanisms of ENK resistance and immune escape in MM, we hope to elucidate a surrogate biomarker which will allow us to select subjects who are most likely to benefit from cellular immunotherapeutic strategies for enrollment in future ENK-based clinical trials. Additionally, the ICAM3/LFA-1 interaction is also important for adhesion of T cells to their targets; therefore, down-regulation of ICAM3 may also have functional implications in the efficacy of T cell-based therapies for MM. Disclosures Garg: University of Arkansas for Medical Sciences: Employment. Chavan: University of Arkansas for Medical Sciences: Employment. Stone: University of Arkansas for Medical Sciences: Employment. Stivers: University of Arkansas for Medical Sciences: Employment. Warden: University of Arkansas for Medical Sciences: Employment. Skinner: University of Arkansas for Medical Sciences: Employment. Lingo: University of Arkansas for Medical Sciences: Employment. Greenway: University of Arkansas for Medical Sciences: Employment. Khan: University of Arkansas for Medical Sciences: Employment. Johann: University of Arkansas for Medical Sciences: Employment. Heuck: Millenium: Other: Advisory Board; Celgene: Consultancy; Janssen: Other: Advisory Board; University of Arkansas for Medical Sciences: Employment; Foundation Medicine: Honoraria. Barlogie: University of Arkansas for Medical Sciences: Employment. Morgan: University of Arkansas for Medical Sciences: Employment; Weismann Institute: Honoraria; MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity9s Board of Directors or advisory committees; CancerNet: Honoraria; Takeda: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Epstein: University of Arkansas for Medical Sciences: Employment. van Rhee: University of Arkansa for Medical Sciences: Employment.

The clinical significance of decreasing CD138 positive myeloma cells in bone marrow

CD138, called syndecan-1, is a heparan sulphate proteoglycan which is known as one of cell adhesion molecules. It has been considered that CD138 is strongly expressed on multiple myeloma cells. However expression of CD138 is reduced in some patients during their clinical courses. To clarify the clinical significance of this phenomenon, we evaluated correlation between CD138 expression levels by flow cytometry and data in our institution.

Identification of an Epithelial-to-Mesenchymal Transition (EMT)-like Programme in t(4;14)-Positive Multiple Myeloma Reveals Novel Targets for Therapeutic Intervention

Introduction: Prognosis for multiple myeloma (MM) patients has improved dramatically since the introduction of novel therapies. However, prognosis in the t(4;14) subgroup, characterized by expression of the histone methyltransferase MMSET, remains relatively poor due to the acquisition of a highly aggressive, motile and invasive phenotype and a generally modest response to therapy. We have previously demonstrated that MMSET promotes an epithelial-to-mesenchymal transition (EMT) in prostate cancer1. While the term EMT is not commonly used to describe MM, we hypothesise that an EMT-like process plays a critical role in t(4;14)­-positive MM disease pathogenesis. In this study, we conducted a comprehensive evaluation of the association between t(4;14) and an EMT-related gene expression signature in MM and identify N-cadherin as a therapeutically-targetable EMT-related gene in t(4;14)-positive MM.Methods and results: Expression of a core EMT-related signature, comprising 169 mesenchymal genes and 49 epithelial genes, was assessed in CD138-selected MM plasma cells from newly-diagnosed MM patients in four independent microarray datasets (E-GEOD-19784 [n = 327 MM patients], E-GEOD-26863 [n = 304], E-MTAB-317 [n = 226] and E-MTAB-363 [n = 155]), accessed through ArrayExpress (EMBL-EBI). In each dataset, gene expression was compared in t(4;14)-positive and t(4;14)-negative patients using linear models for microarray data (LIMMA). Twenty six mesenchymal genes were found to be upregulated in t(4;14)-positive patients across the 4 datasets (p < 0.05, Fisher’s method). A general loss of epithelial genes was not observed, likely due to the haematopoietic origins of MM. Upregulated genes included key EMT drivers (TWIST1, SOX9, TCF4) and genes associated with the cytoskeleton (VIM), adhesion and migration (CDH2, ITGB1, NCAM1) and signalling pathways involved in EMT (BMPR1A, IL6R, TGFB2). The t(4;14)-mediated regulation of EMT-related genes including TWIST1, CDH2, BMPR1A and ITGB1 was confirmed by assessing the effects of MMSET knockdown, knockout and add-back in the t(4;14)-positive human myeloma cell line KMS-11.We have previously demonstrated that N-cadherin (CDH2) expression is elevated in plasma cells from approximately 50% of newly diagnosed multiple myeloma (MM) patients and that elevated serum N-cadherin is associated with poor prognosis2. In order to identify whether N-cadherin is a potential therapeutic target in t(4;14)-positive myeloma, the effects of an N-cadherin peptide inhibitor ADH-1 (Exherin™) or shRNA-mediated N-cadherin knock-down were assessed on MM PC adhesion and proliferation in vivo and in vitro. The role of N-cadherin in MM tumour establishment and intramedullary growth was investigated using the C57BL/KalwRijHsd mouse model of MM. In this model, intravenously injected luciferase-expressing mouse MM PC cells (5TGM1-SFG) home to the bone marrow and initiate systemic MM disease. C57BL/KalwRijHsd mice bearing 5TGM1-SFG N-cadherin knock-down cells had significantly reduced tumour burden, as assessed by bioluminescent imaging and serum paraprotein levels, after 4 weeks compared with mice bearing control 5TGM1-SFG cells. Furthermore, daily intraperitoneal ADH-1 administration (100 mg/kg/day) to 5TGM1-SFG cell-bearing mice significantly significantly decreased tumour burden when administered at the time of tumour inoculation, but had no effect on established tumours. These results suggest that N-cadherin plays a role in extravasation, homing and/or tumour establishment in vivo.Conclusion: This study identified an extensive EMT-like gene expression signature driven by MMSET in t(4;14)-positive MM patients. This provides insight as to how the t(4;14) translocation leads poor prognostic outcomes in up to 20% of MM patients. Furthermore, these studies demonstrate a potential role for N-cadherin in MM tumour dissemination in t(4;14)-positive MM and suggest that N-cadherin represents a novel candidate for therapeutic targeting in these patients.1Ezponda et al. Oncogene, 20132Vandyke et al. Br J Haematol, 2013 DisclosuresNo relevant conflicts of interest to declare.

Mafb Protein Confers Primary Resistance of Myeloma to Proteasome Inhibitors

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 both the MS and MF subgroups 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 while patients in the MF subgroups (including C-MAF and MAFb) did not benefit from Bzb (Nair, Blood 2010). We have previously demonstrated that Bzb prevents c-MAF protein degradation leading to primary drug resistance (Abstract # 281, ASH 2013). In the present study, we assessed the ability of MAFb, another MAF family member, to influence the innate resistance to proteasome inhibitors (PI) and identify the molecular mechanism underlying the resistance of proteasome inhibitors in high MAFb-expressing patients.To investigate the association of the limited therapeutic effect of proteasome inhibitors in the different molecular subgroup of myeloma, we compared the IC50 of Bzb and carfilzomib (CFZ) in 29 myeloma cell lines (MMCL) belonging to different GEP-based molecular subgroups. IC50 concentrations of Bzb were higher (>25 nM) in all 4 MAFb MMCL and >60 nM in one MAFb MMCL, which expressed the highest level of MAFb protein, as determined by immunoblot analysis. In contrast, Bzb IC50 levels were lower (7.5-20 nM) for the MMCL belonging to the other molecular subgroups. For CFZ, IC50 concentrations were higher (>30nM) in all 11 c-MAF cell lines, while the IC50 levels were lower (2-20 nM) for the MMCL belonging to other molecular subgroup. One MMCL harboring t (14; 20) with high IC50 of Bzb (35 nM) showed low IC50 (10 nM) for CFZ. These results indicate that high MAFb expression in myeloma cells may contribute to primary resistance to Bzb and MM cells that express high MAFb protein although resistant to Bzb are sensitive to CFZ. Mechanistically, immunoblotting analysis demonstrated that exposure to Bzb resulted in increased MAFb protein levels in a dose-dependent manner, suggesting that Bzb prevents the degradation of MAFb protein in myeloma cells.To further confirm that drug-induced stabilization of MAFb protein confers resistance to Bzb and partially to CFZ, we generated loss of functional MAFb cells by silencing MAFb expression in a t (14;20) positive myeloma cell line using lentiviral shRNA specific to MAFb mRNA (shMAFb). shMAFb infected myeloma cells had 85% lower levels of MAFb mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, significantly decreased ITGB (9.1 fold), E-cadherin (2.5-fold), CCND2 (5.5-fold), and CCR1 (25-fold) levels were observed in these cells, compared with the cells infected with control viral vector. Silencing MAFb expression significantly decreased proliferation of myeloma cells (65.1% decrease, p=2.1E-6). Moreover, Bzb treatment of the cells infected with shMAFb led to 50.2%, inhibition (P=2.9E-8) of proliferation compared with control cells. Similarly, CFZ treatment of cells with silenced MAFb resulted in 54.3% (P=3.5E-8) inhibition of proliferation compared with control cells. Taken together, our results indicate that high expression of MAFb protein, similar to c-MAF, confers primary resistance to Bzb as well as to CFZ. In addition Bzb induces stabilization of MAFb protein, further increasing resistance to Bzb. These data provide the molecular rational for adopting an alternative therapeutic strategy for high-MAFb expressing myeloma patients. DisclosuresMorgan:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Janssen: Membership on an entity’s Board of Directors or advisory committees; Myeloma UK: Membership on an entity’s Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity’s Board of Directors or advisory committees; The Binding Site: Membership on an entity’s Board of Directors or advisory committees; MMRF: Membership on an entity’s Board of Directors or advisory committees.

To maintain or not to maintain: treatment forever in myeloma?

Achieving a first complete remission in myeloma has become feasible with novel agent-based combination therapies followed by autologous stem cell transplantation (ASCT), leading to CR rates of 40%. But continuously occurring relapses in these patients have lead to the revival of maintenance (MT) concepts aiming to eliminate or control minimal residual disease when myeloma burden is low and not too many clonal tidings have been induced. On the other side, the clinical effectiveness of MT has to be balanced against its considerable cost, toxicity and effects on quality of life. Owing to low effectiveness and side effects of chemotherapy, steroids as monotherapy and α-interferon are obsolete options for maintenance concepts, while low-dose thalidomide should be considered in low-risk patients that do not achieve at least very good partial remission after ASCT, when other MT options are not available. There is limited data on the effectiveness of Bortezomib (BTZ) MT in high-risk patients with respect to both progression-free survival (PFS) and overall survival (OS). Lenalidomide (LEN) MT after ASCT shows a clear PFS benefit, but data on OS and the influence on the outcome of subsequent therapies are conflicting. Toxicity includes haematotoxicity, venous thromboembolism and the induction of secondary primary malignancies. LEN-MT cannot be considered a standard approach after ASCT yet, but should be discussed on a case by case basis with every patient, as well as BTZ-MT in t(4;14) positive myelomas. In non-transplant eligible patients keeping patients on prolonged therapies has been shown to be beneficial and the distinction to MT is often purely semantic.