Clinical Outcome In Multiple Myeloma Patients Research Articles

Inhibition of acyl-CoA synthetase long-chain isozymes decreases multiple myeloma cell proliferation and causes mitochondrial dysfunction.

Multiple myeloma (MM) is an incurable cancer of plasma cells with a 5-year survival rate of 59%. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression; however, the underlying mechanisms remain unclear. Herein, we explore the roles of long-chain fatty acid coenzyme A ligase (ACSL) family members in MM. ACSLs convert free long-chain fatty acids into fatty acyl-CoA esters and play key roles in catabolic and anabolic fatty acid metabolism. Analysis of the Multiple Myeloma Research Foundation (MMRF) CoMMpassSM study showed that high ACSL1 and ACSL4 expression in myeloma cells are both associated with worse clinical outcomes for MM patients. Cancer Dependency Map (DepMap) data showed that all five ACSLs have negative Chronos scores, and ACSL3 and ACSL4 were among the top 25% Hallmark Fatty Acid Metabolism genes that support myeloma cell line fitness. Inhibition of ACSLs in myeloma cell lines in vitro, using the pharmacological inhibitor Triacsin C (TriC), increased apoptosis, decreased proliferation, and decreased cell viability, in a dose- and time-dependent manner. RNA-sequencing analysis of MM.1S cells treated with TriC showed a significant enrichment in apoptosis, ferroptosis, and endoplasmic reticulum (ER) stress, and proteomic analysis of these cells revealed enriched pathways for mitochondrial dysfunction and oxidative phosphorylation. TriC also rewired mitochondrial metabolism by decreasing mitochondrial membrane potential, increasing mitochondrial superoxide levels, decreasing mitochondrial ATP production rates, and impairing cellular respiration. Overall, our data support the hypothesis that suppression of ACSLs in myeloma cells is a novel metabolic target in MM that inhibits their viability, implicating this family as a promising therapeutic target in treating myeloma.

Impact of Visceral Obesity on Clinical Outcome and Quality of Life for Patients with Multiple Myeloma: A Secondary Data Analysis of STaMINA (BMT CTN 0702) Trial

Obesity is a common health problem among multiple myeloma (MM) patients, and it has been linked to poor clinical outcomes and quality of life (QOL). We conducted a secondary analysis of the BMT CTN 0702, a randomized, controlled trial comparing outcomes of three treatment interventions after a single hematopoietic cell transplant (HCT) (N = 758), to investigate the impact of visceral obesity, as measured by waist-to-hip ratio (WHR), on clinical outcomes and QOL in MM patients. A total of 549 MM patients, median age 55.5 years, were enrolled in the study. The majority of patients received triple-drug antimyeloma initial therapy before enrollment, and 29% had high- risk disease according to cytogenetic assessment. The median follow-up time was 6 years. There was no significant association between WHR and progression-free survival (PFS) or overall survival (OS) in MM patients undergoing HCT. Similarly, body mass index (BMI) did not significantly predict PFS or OS. Furthermore, there was no significant correlation between WHR and QOL measures. In conclusion, this study suggests that visceral obesity, as measured by WHR, may not have a significant impact on clinical outcomes in MM patients undergoing HCT. These findings add to the existing literature on the topic and provide valuable information for healthcare professionals and MM patients. Further studies are needed to confirm these results and to investigate other potential factors that may affect clinical outcomes and QOL in this patient population using modern imaging technologies to assess visceral obesity.

The Genetic and Molecular Drivers of Multiple Myeloma: Current Insights, Clinical Implications, and the Path Forward.

Multiple myeloma (MM) is a hematological malignancy characterized by the clonal proliferation of malignant plasma cells within the bone marrow. The disease's complexity is underpinned by a variety of genetic and molecular abnormalities that drive its progression. This review was conducted through a state-of-The-art literature search, primarily utilizing PubMed to gather peer-reviewed articles. We focused on the most comprehensive and cited studies to ensure a thorough understanding of the genetic and molecular landscapes of MM. We detail primary and secondary alterations such as translocations, hyperdiploidy, single nucleotide variants (SNVs), copy number alterations (CNAs), gene fusions, epigenetic modifications, non-coding RNAs, germline predisposing variants, and the influence of the tumor microenvironment (TME). Our analysis highlights the heterogeneity of MM and the challenges it poses in treatment and prognosis, emphasizing the distinction between driver mutations, which actively contribute to oncogenesis, and passenger mutations, which arise due to genomic instability and do not contribute to disease progression. We report key controversies and challenges in defining the genetic drivers of MM, and examine their implications for future therapeutic strategies. We discuss the importance of systems biology approaches in understanding the dependencies and interactions among these alterations, particularly highlighting the impact of double and triple-hit scenarios on disease outcomes. By advancing our understanding of the molecular drivers and their interactions, this review sets the stage for novel therapeutic targets and strategies, ultimately aiming to improve clinical outcomes in MM patients.

A Tumor-Bearing Murine Xenograft Model As a Bioassay for Assessing CAR-T Product Potency Shows Positive Predictive Value for Clinical Performance

Potency assays are required for cellular and gene therapy (CGT) final product (FP) lot release, stability testing, and manufacturing updates, as per the FDA draft guidance on Potency Tests for CGT Products (2011), and Manufacturing Changes and Comparability for Human CGT Products (2023). These assays by definition measure the ability of a product to effect a desired result, and the FDA allows for “considerable flexibility” in determining the appropriate potency assay. However, standard in vitro potency assays are limited in their capacity to meaningfully assess manufacturing comparability and consistency, or product quality to provide predictive value for clinical performance. For chimeric antigen receptor (CAR)-T cell products, certificates of analysis typically report key FP attributes such as CAR expression, cell concentration and viability, impurities and target-specific in vitro potency, among others. However, clinical effectiveness of CAR-T is dependent on complex pharmacokinetics (PK)/ pharmacodynamics (PD) including in vivo engraftment, trafficking, drug exposure kinetics, tumor antigen-driven T cell expansion in tumor microenvironment, survival and persistence, and other factors that are not easily recapitulated in short-term in vitro assays. We developed a bioassay for assessing CAR-T FP potency using a tumor-bearing murine NSG xenograft model to evaluate in vivo performance of GMP-manufactured autologous (Auto) or allogeneic (Allo) CAR-T FPs. We hypothesized that this complex in vivo system may better model PK/PD and provide a more meaningful way to assess CAR-T FP potency. Preclinical dose-finding data from both RUO and GMP anti-BCMA CAR-T lots were used to establish a potency specification for RPMI-8226 tumor control at a “stress dose” of 5e6 CAR-T cells at <60% of the tumor volume compared to the untreated control group at 21 days after CAR-T cell infusion. Using these specifications, we evaluated the in vivo potency performance of GMP-manufactured Auto FP samples (P-BCMA-101; NCT03288493) generated from multiple myeloma (MM) patients and administered in the clinic with known responses ranging from poor (no response or stable disease) to significant (very good partial response [VGPR] or stringent complete response [sCR]). In addition, GMP-manufactured Allo FP samples generated from healthy donors (P-BMCA-ALLO1; NCT04960579) were also tested. We report that half of the P-BCMA-101 products (50%) previously demonstrating favorable clinical outcomes in MM patients were also able to control RPMI-8226 tumor in vivo, while none of the P-BCMA-101 products (0%) that resulted in poor clinical responses were capable of tumor control. These data show translational relevance considering the known clinical outcomes in MM patients, albeit at a high level of stringency at current specifications. Interestingly, all the Allo products (100%) performed at least as well as or better than the Auto products capable of controlling tumor, demonstrating a greater potency of Allo CAR-T cells manufactured from healthy donors. There is great demand for potency assays that can reliably assess manufacturing consistency and product quality, and that might provide some level of predictive value for clinical performance, especially considering the current limitations of standard in vitro potency assays. In vivo potency bioassays have been proposed or utilized for other products with complex biological mechanisms of action including vaccines, gene therapies, and other cell therapy products. The potency bioassay described herein demonstrated a level of positive predictive value for clinical performance using Auto FPs of known clinical outcome in MM patients. In the Auto CAR-T setting, this assay could be utilized for correlative studies including possible biomarker identification. In the Allo CAR-T setting, this potency bioassay may inform FP manufacturing consistency and quality for lot release to ensure patients receive only the highest quality products.

Multiple Myeloma in the Culturally and Linguistically Diverse Southwestern Sydney, Australia

Multiple myeloma (MM) is an incurable haematological cancer with a median overall survival of 5 to 10 years. Multiple American studies have reported significant disparities in the incidence and clinical outcomes in MM patients that may be explained by race or ethnicity. This study sought to characterise the racial/ethnic representation in myeloma patients who presented to the public hospitals in Southwestern Sydney between 2009 and 2020. This retrospective study describes the associations between race/ethnicity and age at diagnosis, cytogenetics, comorbidities, and survival outcomes. Data for analysis was extracted from Mosaiq (electronic medical record system for oncology workflow management) and PathNet® (laboratory information system). Since neither race nor ethnicity are recorded in the NSW Healthcare system, race was defined based on the country of birth while ethnicity was defined as culturally and linguistically diverse (CALD), based on country of birth and language as well as use of an interpreter. Seven regions of birth across 65 countries of birth and five CALD groups were identified in our cohort, with 26% of the cohort speaking a language other than English. Survival outcomes were similar regardless of race or CALD classification. Analysis of the cytogenetics and comorbidities are still in progress. This is the first reported racial study in MM in NSW. The preliminary results presented here demonstrates that automatically classifying patients as Caucasian, solely based on country of birth can be misleading. In a multicultural region like Southwestern Sydney, it is important to consider patients' cultural and linguistic background.

Abstract 18054: Contemporary In-Hospital Outcomes of Congestive Heart Failure With Multiple Myeloma; a Regression Matched Analysis

Introduction: Multiple myeloma (MM) is a disease commonly found in elderly individuals and is associated with a high burden of cardiovascular comorbidity. Various disease manifestations in MM, including amyloidosis, anemia, hyperviscosity, hypercalcemia, intramedullary arteriovenous fistula formations, and chemotherapy-related complications, contribute to an increased risk of congestive heart failure (CHF). The objective of this study was to examine the clinical outcomes of MM patients admitted with CHF. Methods: The National Inpatient Sample database from 2016-2020 was queried to identify patients with a primary diagnosis of CHF and stratified based on the presence of MM. The primary outcome measured was in- hospital mortality. The adjusted odds ratio (aOR) of in-hospital outcomes were calculated using software STAT v.17. Results: Of the 536,700 primary hospitalizations with CHF, 1,965 (0.36%) had MM. On adjusted analysis, the CHF-MM cohort had a higher odds of cardiac arrest (aOR 2.5, 95% CI 1.2-5.5), need for new hemodialysis (aOR 2.4, 95% CI 1.6-3.6) and GI bleeding (aOR 2.1, 95% CI 1.1-3.8) However, there was no statistically significant difference in the odds of in-hospital mortality, stroke, AMI or major bleeding. Furthermore, the presence of MM in CHF related hospitalizations was not associated with higher length of stay and adjusted total cost. Conclusion: Our study reveals that MM patients admitted with CHF have an increased risk of cardiac arrest, new hemodialysis, and GI bleeding. To optimize cardiovascular outcomes in this high-risk patient population, a multidisciplinary approach involving hemato-oncologists and cardiologists is necessary.

Impaired Plasma B Cell Markers in CD138+ Cells Predict Prognosis in Multiple Myeloma

Background: Multiple myeloma (MM), characterized by the proliferation of clonal plasma B-cell in the bone marrow, is the second most common hematologic malignancy in the US. MM represents the final stage in a continuum of plasma cell dyscrasias, arising from the premalignant conditions monoclonal gammopathy of undetermined significance (MGUS) and smoldering MM (SMM). Despite therapeutic advances, MM remains incurable with the prognostic improvement observed only in a subset of patients. The variations in clinical course are largely caused by the underlying intertumoral heterogeneity between different patients. To further improve the clinical outcome of MM patients, biomarkers that can effectively stratified patients are critically needed for directing personalized treatment. In this study, we hypothesize that the magnitude of plasma B cell malignancy is a primary factor that determines the clinical phenotypes of MM patients, which can be reflected by gene expression in CD138+ cells. Methods: Using single-cell RNA sequencing (scRNA-seq) data, we develop a plasma B gene signature and apply it to gene expression profiles of CD138+ cells from MM patients (Figure 1 A). The resulting score, denoted as plasma B cell malignancy (PBM) score, indicates the degree of plasma B cell malignancy of MM samples with a higher value indicating higher malignancy status. Genomic and/or transcriptomic data from five MM studies (i.e., MMRF CoMMpass, UAMS, MAQC-II and APEX phase 3 trial and Chng study) were used to examine the associations between PBM score and known MM driver genes, and its clinical impact on MM progression and prognosis. Univariate and multivariate Cox regression models were constructed to evaluate the prognostic value of patient specific PBM scores when used alone or combined with established clinical factors. Results: Survival analysis indicates that the PBM score defined based on plasma B cell markers are significantly associated with patient overall survival. In contrast, gene signatures charactering other immune cells are less (other three B cell types) or not prognostic (non-B immune cell types) (Figure 1 B). Then we investigated the effects of PBM score on MM development and prognosis. Overall, a higher PBM score was significantly associated with a more advanced stage in the spectrum of plasma cell dyscrasias (all P < 0.05, Figure 1C). Higher PBM score was also associated with shorter overall survival (OS) in patients with MM (HR = 1.72, P < 0.001) (Figure 1D) and its prognostic effect was independent of the currently available clinical prognostic model, such as the International Staging System (ISS) (Figure 1E). Mutation status of TP53 gene has been found to be associated with patient prognosis. Investigations on the relationships between PBM score and MM driver genes found that the PBM score was significantly increased in patients carrying TP53 mutations compared to those with mild-type alleles. Of note, PBM scores could predict patients’ survival even among those who don't carry TP53 mutations, with log-rank P values of 0.002 (Figure 1F). The model based on the PBM score alone had a c-index of 0.62 to predict the OS of MM. The clinical model based on age, sex, race, and (ISS) stage had a c-index of 0.67 to predict OS. Incorporating PBM score into the models based on clinical factors significantly improved the prognostic performance of the models, with the c-index increased to 0.70 for the model that combined PBM and clinical factors (P < 0.001) (Figure 1G).Conclusions: There may be a clinical utility of the PBM score to predict MM outcomes in combination with more standard clinical parameters. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic value of B-cell maturation antigen, CD56 expression and neutrophil lymphocyte ratio in patients with multiple myeloma

Introduction: B-cell maturation antigen (BCMA) plays a critical role in regulating B-cell proliferation and differentiation into plasma cells. CD56 is involved in the adhesion of myeloma cells to the bone marrow matrix. Thus, lack of CD56 expression is associated with a higher incidence of extramedullary disease. An elevated neutrophil-lymphocyte ratio (NLR) has been recognized as a poor prognostic factor in various hematological malignancies. The aim of this study was to evaluate the role of BCMA, CD56 and NLR as novel prognostic markers in multiple myeloma (MM). Material and methods: The study included 80 subjects, 40 MM patients, and 40 normal healthy age- and sex-matched controls. BCMA was analyzed by enzyme-linked immune sorbent assay. Flow cytometry was used for the determination of CD56 expression. NLR was calculated from the complete blood count differential count. All patients received six cycles of bortezomib, cyclophosphamide and dexamethasone (VCD). Treatment outcome was assessed and progression-free survival (PFS) was estimated using Kaplan-Meier survival analysis. Results: Patients who achieved complete remission showed lower BCMA levels, positive CD56 expression, and lower NLR. Moreover, higher BCMA levels and CD56 negative expression were significantly associated with shorter PFS. Conclusions: Our study emphasizes the importance of BCMA, CD56 and NLR to predict the clinical outcome in MM patients. This could help in better risk stratification and tailored clinical management of MM patients.

IMPACT OF BONE MARROW FIBROSIS IN MYELOMA PATIENTS UNDERGONE AUTOLOGOUS STEM CELL TRANSPLANTATION

Autologous hematopoetic stem cell transplantation (aHSCT) after high dose chemotherapy is a standard treatment for multiple myeloma (MM) patients. The successful aHSCT depends on collection of sufficient numbers of hematopoietic progenitor stem cells and sustained engraftment following infusion. The aim of the present study is to determine the the impact of bone marrow fibrosis (BMF) on the clinical outcomes of MM patients who underwent aHSCT. Retrospectively, bone marrow trephine biopsy analyzed in 73 MM patients who were treated with hematopoietic stem cell transplantation (aHSCT) following bortezomib based induction regimen. The BM biopsy samples of all patients were re-evaluated by a single pathologists The patients divided into 4 groups according to fibrosis degree and the correlations in initial characteristic features, therapeutic response, survival, mobilization and engraftment outcomes were reviewed between the groups. Comparative analyses revealed that the median apheresis number was found statistically different according to groups (p=0.04). No significance was detected between the fibrozis grade and the number of peripheral blood CD34+ cell collection results and recovery time of neutrophils and platelets. Overall survival and progression free survival were found similar in groups, however relapse of disease was statistically different in patients with fibrosis (p=0.01). After induction treatment, a regression was observed in fibrosis grade of patients who had fibrosis at the time of diagnosis. Therefore we suggest to evaluate fibrosis status in all MM patients during each histopathological examination. Difficulties may be experienced during stem cell collection in transplant eligible MM patients with fibrosis at diagnosis. Therefore, we recommend that clinicians should be more careful in these patients during the induction treatment and stem cell mobilization.

NK Cell Reconstitution After Autologous Hematopoietic Stem Cell Transplantation: Association Between NK Cell Maturation Stage and Outcome in Multiple Myeloma

Autologous hematopoietic stem cell transplantation (autoHSCT) is a standard of care for transplant-eligible patients with multiple myeloma (MM). Among factors that influence outcome after autoHSCT, it has been suggested that the number of natural killer (NK) cells plays an important role. However, the impact that different NK cell subsets and their phenotype could have in disease progression after autoHSCT are less clear. For this reason, we have phenotypically and functionally characterized NK cells during immune system reconstitution after autoHSCT in 54 MM patients. Shortly after leukocyte recovery, an extensive redistribution of NK cell subsets occurs in these patients. In addition, NK cells undergo a profound phenotypic change characterized, among others, by their increased proliferative capacity and immature phenotype. Importantly, MM patients who showed lower frequencies of the mature highly differentiated NKG2A-CD57+ NK cell subset at +30 and +100 days after autoHSCT experienced superior progression-free survival and had a longer time to the next treatment than those with higher frequencies. Our results provide significant insights into NK cell reconstitution after autoHSCT and suggest that the degree of NK cell maturation after autoHSCT affects the clinical outcome of MM patients treated with this therapeutic strategy.

From VAD to VRD: Is Transplant Still Needed in the Upfront Setting of Myeloma?

High-dose therapy followed by autologous stem cell transplantation (ASCT) is considered the standard of care for transplant-eligible patients with newly diagnosed multiple myeloma (MM). With new treatment combinations offering the advantage of improved clinical outcomes of MM patients, the utilization of ASCT is again being addressed in the evolving treatment landscape. In this article, we review the role of frontline ASCT in the management of patients with MM.

Determinants of response and mechanisms of resistance of CAR T-cell therapy in multiple myeloma.

BCMA-specific CAR T-cells have substantial therapeutic potential in multiple myeloma (MM), but most patients eventually relapse. Determinants of response and mechanisms of resistance are most likely multifactorial and include MM-related factors, premanufacturing T-cell characteristics, CAR T-cell-related features, and several components of the immunosuppressive microenvironment. Efforts to improve the potency and safety of CAR T-cell therapy include optimizing CAR design, combinatorial approaches to enhance persistence and activity, treatment of less heavily pretreated patients, and dual-antigen targeting to prevent antigen escape. We expect that these rationally designed strategies will contribute to further improvement in the clinical outcome of MM patients.

The Translational Switch of MYC Protein Aliases in Myeloma Tumor Cells

Our systematic investigation on MYC (C-Myc) - a crucial pro-regeneration transcription factor - has determined that genetic aberrations (chromosomal rearrangements) and overexpression of the gene are highly prevalent in multiple myeloma (MM). At diagnosis, the rearrangements of MYC locus (8q24) were detected in 51% of MM patients using karyotyping and interphase FISH with DNA probe sets to MYC and the flank genomic sequences. The abnormalities of 8q24 in MM include monosomy (5%), amplification (≥3 copies; 13%), break-apart (14%), jumping (11%), and others (8%). Such aberrations were present in 49% of low-risk MM (183/376) and in 62% of high-risk MM (51/82) defined by the gene expression profiling cancer-risk model (GEP-70). The GEP also indicated that 74% of MM patients had excessively high levels of MYC overexpression. Nonetheless, there was no significant correlation between the MYC cytogenetic abnormalities and the levels of MYC expression in myeloma tumor cells - unlike the primary IGH translocations that drive transcriptional spikes of its partner genes. MYC expression by GEP was unable to precisely predict the presence of MYC chromosomal rearrangements in MM. In fact, the clinical outcomes of MM patients with MYC aberrations vs. the patients without detectable 8q24 events showed that neither the numerical variations nor the structural rearrangements of 8q24 had significant impact on the overall survival of patients who were treated on Total Therapy protocols. Further, we have shown that chromosomal rearrangements and the levels of MYC overexpression were not intrinsically relevant to the translation of MYC protein in myeloma. Importantly, the fate of myeloma cells was associated with the presence of two endogenous polypeptide-isoforms of MYC - the canonical MYC protein alias (P01106; 48.8kD) and an alternative MYC protein alias (alt-MYC; &gt; 50kD). In newly diagnosed MM, P01106 was absent in most of the patients; whereas, it was a constitutive alias well maintained across the myeloma tumor cells from the patients with relapsed and progressive diseases and in all of the self-renewable myeloma cell lines. In contrast, alt-MYC alias was present as a stand-alone alias or co-existing with P01106 alias in newly diagnosed myeloma. Most importantly, both MYC aliases were absent in some cell lines and primary tumor cells suggesting cancer cell growth may not be dependent on any of MYC aliases despite of high levels of MYC transcription. Since the MYC gene originated from a viral integration, MYC mRNA has dual-translation features due to an internal ribosome entry segment (IRES) at 5'UTR. Previous studies have determined that the translations can be alternatively initiated from an in-frame CUG (Leucine) codon to generate NP_002458 alias or at a downstream canonical cap-dependent AUG (Methionine) codon to generate P01106 alias. Although these two aliases share identical C-termini, the translational switch can extend the N-terminal in NP_002458 alias. In vitro, the switch of P01106 to alt-MYC has resulted in cell growth arrest. Such alternation was induced by a transient gene transfection of P53 tumor suppressor (TP53); seemingly, the alternative MYC alias acts as an antagonist to P01106 in cell proliferation. Overall, it was generally unpredictable which of the MYC aliases were the dominant driver; therefore, MYC cytogenetic rearrangements and overexpression may have unknown indirect impacts on the outcomes. We are currently examining the precise amino acid sequences on both N-terminal and C-terminal ends of MYC aliases to study the regulatory mechanisms of the translational switching. This may indicate a novel anti-cancer strategy to cease cancer proliferation by deliberately inducing alternative translation of the antagonistic alias of an oncogene, and eventually overcome cooperation of the chromosomal rearrangements and dysregulated oncogene transcriptions in progressive cancer cells. Disclosures van Rhee: Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; EUSA: Consultancy; Castleman Disease Collaborative Network: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Kite Pharma: Consultancy.

Loss-of-Function of Gabarap Impairs Bortezomib-Induced Anti-Tumor Immunity in Multiple Myeloma: Clinical Application

Immune escape underlies progression of disease and resistance to therapy in multiple myeloma (MM). Conversely, restoration of anti-tumor immunity primed by immunogenic cancer cells and intratumoral dendritic cells (DCs) may be exploited to convert the immunologically "cold" MM into a "hot" MM; and may lead to long-term clinical benefit, even in patient subgroups with high-risk (HR) cytogenetics and poor survival. Here, we investigated the mechanisms, biologic sequelae, and clinical benefits of bortezomib (BTZ)-induced immunogenic cell death (ICD), the immunogenic consequence of apoptosis resulting in specific anti-MM immunity via T-cell priming by DCs. We first show that BTZ can induce hallmarks of ICD in human and murine MM cell lines (n=5), including exposure of endoplasmic reticulum protein calreticulin (CALR) that functions as an "eat me signal". Specifically, our data show that co-culture with BTZ-treated MM cells can induce phenotypic and functional changes in immature DCs including higher expression of CD86/CD83 on cell surface and enhanced uptake of BTZ-treated MM cells, as assessed by flow cytometry- and confocal-based phagocytosis assay, respectively. Notably, we show that CALR has a key role in BTZ-induced immunogenicity, since these functional sequelae were abrogated in vitro when DCs were co-cultured with CALRKO MM cells. We then validated these findings in 2 different in vivo syngeneic models. First, we observed that anti-MM activity of BTZ resulted in more potent 5TGM1 tumor cell shrinkage in immunocompetent hosts; and that this effect was directly linked to ICD induction, since it was abrogated in mice bearing CALRKO tumors. Second, in vitro BTZ-treated 5TGM1 cells were used as a vaccine to enhance an anti-MM immune response: injection of live tumor cells resulted in palpable tumors in non-vaccinated mice by 1 week; conversely, injection of live tumor cells in vaccinated mice did not result in detectable tumor after 30 days. In contrast, in mice similarly vaccinated with BTZ-treated CALRKO 5TGM1 cells and challenged with injection of live WT cells, only 50% of vaccinated mice were tumor-free at day 30. Next, we performed RNAseq analysis of BTZ-treated vs untreated tumors from both immunodeficient or immunocompetent mice; and also carried out an integrative analysis of RNAseq data from newly diagnosed and clinically annotated MM patients (n=360) uniformly treated with BTZ-based regimes (IFM/DFCI 2009). Importantly, increased expression of the human orthologs of the immune genes induced in mice by BTZ was strongly and positively correlated with patient clinical outcome (OS p value=0.00089). The predictive value of this signature was confirmed in an independent dataset (GSE9782) (OS p value=0.024). Moreover, by interrogating the IFM/DFCI patient dataset, we identified the gamma-aminobutyric acid receptor-associated protein (GABARAP) as a top differentially expressed gene among patients with longer survival rate (&amp;gt;5 years) as compared to those with poor survival (&amp;lt;1.5 years) after BTZ-based treatment. We found that low level of GABARAP, located on chr17p13.1, is associated with poor clinical outcome in MM patients (EFS, p= 0.0032); and that its prognostic value is still maintained even excluding HR patients with 17p deletion (EFS, p= 0.018). Interestingly, KMS11 cells that carry monoallelic deletion of GABARAP were resistant to induction of ICD by BTZ; and stable overexpression of the gene in these cells restored the functional sequelae of ICD induction upon drug exposure. Moreover, GABARAPKO in 2 ICD-sensitive cell lines abrogated the induction of ICD by BTZ; add-back experiments by pre-treatment with recombinant CALR (rCALR) or stable overexpression of GABARAP in KO clones confirmed the on-target effect GABARAPKO. Finally, mass Cytometry (CyTOF) after T cell culture with mature DCs primed by both WT and GABARAPKO AMO1 clones showed that treatment of MM cells with BTZ switched CD4+T cells towards an effector memory function; in contrast to treatment of GABARAPKO clones with BTZ, which led to T cell exhaustion. In conclusion, our studies demonstrate the clinical benefits of BTZ-induced ICD in MM; and that loss-of-function of GABARAP, particularly in HR patients with 17p deletion, abrogates induction of antitumor immunity after drug exposure. These studies provide the framework for novel combination treatments to trigger anti-MM immunity and improve patient outcome in MM. Disclosures Chauhan: Stemline Therapeutics: Consultancy; C4 Therapeutics.: Equity Ownership. Munshi:Celgene: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Abbvie: Consultancy; Amgen: Consultancy. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

A Single Nucleotide Polymorphism in SLC7A5 Was Associated With Clinical Response in Multiple Myeloma Patients.

SLC7A5 is recognized as the major mediator of melphalan uptake into multiple myeloma (MM) cells; however, its contribution to the inter-patient variability of melphalan efficacy and toxicity is yet to be well elucidated. This study aimed to investigate the impact of a single nucleotide polymorphism (SNP) rs4240803 in SLC7A5 on the gene expression, ex vivo sensitivity to melphalan, and clinical outcomes in MM patients who were undergoing autologous stem cell transplantation with high-dose melphalan. Peripheral blood mononuclear cells (PBMC) were collected from 108 MM patients prior to melphalan therapy. Clinical data were also collected from these patients following melphalan therapy. rs4240803 was associated with elevated expression of SLC7A5 mRNA, higher ex vivo sensitivity to melphalan in PBMCs, and positive 90-day response in these patients (p=0.047, 0.10, 0.049, respectively). rs4240803 impacted the expression of SLC7A5, thus contributing to the clinical response of MM patients to melphalan therapy.

Microvessel Density as a Surrogate Prognostic Marker in Patients with Multiple Myeloma: A Meta-Analysis

Background/Aims: Bone marrow (BM) angiogenesis is considered a hallmark of multiple myeloma (MM) development and progression, and can be quantified with the use of microvessel density (MVD). The purpose of this study is to provide a review and a meta-analysis of the current literature regarding the prognostic value of MVD in the overall survival (OS) of MM patients. Methods: MEDLINE was screened for studies evaluating the OS of MM patients with regard to their MVD count in BM trephine. The pooled hazard ratio (HR) and its associated 95% confidence interval (CI) among MM patients with a high and low MVD count was the primary end point. Secondary outcomes included odds ratios (OR) for 12-, 36-, and 60-month survival. Results: Ten eligible trials were identified for the analysis of the primary end point and 9 for the secondary end points. Pooled HR for OS was 1.85 (95% CI: 1.25-2.73, p = 0.002). The pooled OR of survival were 1.59 (95% CI: 1.02-2.46, p = 0.04) at 12 months, 2.90 (95% CI: 1.68-5.03, p = 0.0001) at 36 months, and 3.42 (95% CI: 2.41-4.85, p < 0.00001) at 60 months, in favor of the low MVD group. Conclusion: This meta-analysis provides persuasive evidence that MVD has significant impact on the clinical outcome of MM patients.

Pretreatment neutrophil/lymphocyte ratio but not platelet/lymphocyte ratio has a prognostic impact in multiple myeloma.

We evaluated the prognostic significance of neutrophil/lymphocyte ratio (NLR) and platelet/lymphocyte ratio (PLR) in patients with multiple myeloma (MM). In total, we retrospectively analyzed 315 newly diagnosed MM patients and calculated NLR and PLR from the complete blood count of the untreated patients. We further assessed the role of pretreatment NLR and PLR on overall survival (OS) and progression-free survival (PFS). Multiple myeloma patients with high NLR (≥2) experienced shorter OS (P=.02) and PFS (P=.01) compared with patients with low NLR (<2). Furthermore, among the patients with conventional chemotherapy, elderly patients, or patients with advanced stages, high NLR (≥2) was found to have a negative prognostic impact on OS and PFS. In the multivariate Cox analysis, we confirmed that the NLR was an independent prognostic factor for both OS and PFS (P=.000). But the differences in OS or PFS by PLR were not found in MM patients. Our study suggests that NLR not PLR can be acted as an independent prognostic factor for analyzing the clinical outcome of MM 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.

Elevated neutrophil-to-lymphocyte ratio and monocyte-to-lymphocyte ratio and decreased platelet-to-lymphocyte ratio are associated with poor prognosis in multiple myeloma.

Elevated inflammatory markers are associated with poor outcomes in various types of cancers; however, their clinical significance in multiple myeloma (MM) have seldom been explored. This study investigated the prognostic relevance of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and monocyte-to-lymphocyte ratio (MLR) in MM. Totally 559 MM patients were included in this study. NLR, PLR and MLR were calculated from whole blood counts prior to therapy. Kaplan-Meier curves and multivariate Cox proportional models were used for the evaluation of the survival. It has shown that newly diagnosed MM patients were characterized by high NLR and MLR. Elevated NLR and MLR and decreased PLR were associated with unfavorable clinicobiological features. Applying cut-offs of 4 (NLR), 100 (PLR) and 0.3 (MLR), elevated NLR, MLR and decreased PLR showed a negative impact on outcome. Importantly, elevated NLR and decreased PLR were independent prognostic factors for progression-free survival. Thus, elevated NLR and MLR, and decreased PLR predict poor clinical outcome in MM patients and may serve as the cost-effective and readily available prognostic biomarkers.

Abstract 1081: Activity of microRNA replacement reagent, MRX34, in multiple myeloma in vivo model

Abstract Background: Despite recent progress in multiple myeloma (MM) treatment with the advent of new targeted anticancer agents such as Bortezomib, Lenalidomide and, Thalidomide MM still remains incurable. To improve clinical outcome of MM patients, many research have been trying to develop novel targeted drugs. Recent studies demonstrated that microRNA Replacement reagent could be a promising therapeutic strategy in patients with hematologic malignancies. Hence, we tried to examine the efficacy of MRX34, a microRNA-34 replacement reagent, in MM animal model. Methods: We examined the anti-tumor activity of a MRX34 against 3MM cell lines (LP1, MOLP-8, and U266), and investigated the survival effects through which this novel agent shows anti-MM activity. For an animal study, we used NRG (NOD-Rag1null IL2rgnull, CB-17 SCID) mice that were injected with MM cells via tail vein. MOLP-8, LP1 and U266 cells were injected Intravenous 106 cells of NRG. When tumors were measurable, mice were assigned to 3 treatment groups receiving the vehicle alone (control), MRX34 0.3mg/kg treatment group and, MRX34 1mg/kg treatment group were given Intravenous 5 times a week. Results: Median survival of mice injected with LP1, MOLP-8 and U266 cell lines were 28days, 29 days and 34days respectively without any treatment. When mice were treated with MRX-34, median survival was 33days, 37days and 42days respectively. There was a significant survival advantage with MRX-34 treatment in MOLP-8 and U266 cell lines. For monoclonal proteins, mice treated with MRX34 group are suppressed M-protein level almost 30% less than M-protein. Summary / Conclusion: Our data indicated that MRX34 had beneficial effects in multiple myeloma animal model. Mice treated with MRX34 showed M-protein suppression and survival advantage. Our results warrant further clinical studies using this novel agent in MM. Citation Format: Woo June Jung, Youngil Koh, Sinil Kim, Hyejoo Park, Sung-Soo Yoon. Activity of microRNA replacement reagent, MRX34, in multiple myeloma in vivo model. [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 1081.