Methylation-Dependent Epigenetic Silencing Of Mir-152 and Mir-10b-5p Plays a Crucial Role In Modulating Tumor Progression In Multiple Myeloma

Abstract Background Epigenetic modifications including changes in DNA methylation lead to inhibited gene expressions and consequent phenotypic alterations. MicroRNAs (miRNAs) are a class of small non-coding RNAs (19∼25 nucleotides) which functions as endogenous silencers of target genes. In general, most miRNAs are downregulated in many cancers and specifically in multiple myeloma (MM). We hypothesized that the mechanism of low expression of tumor suppressor miRNAs in MM is through epigenetic silencing, specifically through CpG island hypermethylation. The aim of this study is to identify methylation-silenced miRNAs and clarify their contribution to MM development and progression. Methods MicroRNA microarray analysis was performed in a panel of six MM cell lines (MM1S, RPMI 8266, OPM2, U266, H929 and IMI9) with or without the treatment of 5 μM of 5'-aza-2'-deoxycytidine (5-Aza-CdR, DNA demethylating agent) to screen for the most commonly upregulated miRNAs in response to DNA demethylation. These results were compared to a global methylation data of patients with MM (GEO GSE21304). Of these, we further examined the role of miR-152 and miR-10b-5p in MM. Induced expression of miR-152 and miR-10b-5p with 5-Aza-CdR-treatment was validated with real-time PCR. The DNA methylation status of CpG islands of these two candidates was further evaluated by methylation specific PCR (MSP) and bisulfate sequencing PCR (BSP). The expression levels of miR-152 and miR-10b-5p in both newly diagnosed MM patients (N = 60) and normal healthy donor (N = 5) were further analyzed (GEO GSE16558). MiR-152 and miR-10b-5p mimics were transiently transfected into H929 cells, respectively and in vitro functional validation including cell proliferation, cell apoptosis and adhesion assays. Results We identified miR-152 as the most common upregulated miRNA (in all of the applied six cell lines); 7 miRNAs (miR-10b-5p, -320b, -4521, -548b-3p, -584-5p, -616-3p and -497-5p) and 77 miRNAs were upregulated by 1.5-fold or more in any four or three applied cell lines. Among them, the methylation of miR-152 and miR-10b-5p was reported in other hematologic malignancies, but little is known in MM. We first focused our attention on miR-152 and miR-10b-5p; and validated their significant upregulation in MM cell lines with 5-Aza-CdR treatment by real-time PCR. With specific methylated- or unmethylated primers for miR-152 or miR-10b-5p, MSP results indicated that miR-152 methylated PCR products had high levels in all control cells, whereas the unmethylated PCR products were significantly increased in the 5-Aza-CdR-treated cells. The BSP results of the promoter region of miR-152 showed extensive methylation throughout its promoter region (∼1000 bp upstream) in both H929 and IM9 cells, which was reversible following 5-Aza-CdR treatment. The average methylation levels of miR-152 were 77% and 84% in H929 and IM9 cells, however, after 5-Aza-CdR treatment, the methylation level decreased to 19% and 10%, respectively. Similar results were found for miR-10b-5p in both cell lines. Together, these findings confirmed that miR-152 and miR-10b-5p were suppressed through CGI methylation in MM cell lines. Compared with healthy donor, the expression of miR-152 and miR-10b-5p were significantly decreased in newly diagnosed MM patients. Moreover, overexpression experiments demonstrated that the cell proliferation of H929 cells, as detected by MTT assay, was significantly reduced after the restoration of miR-152 or miR-10b-5p (p< 0.05). Western blot results showed that cleaved PARP, caspase 9 and caspase 3 proteins were all significantly increased after miR-152 or miR-10b-5p mimics transfection. Adhesion assays showed that the adhesion capacity to stroma or fibronectin of H929 cells with miR-152- or miR-10b-5p-overexpression was significantly reduced compared with the negative control cells (p< 0.05). Conclusion As methylation-sensitive miRNAs, miR-152 and miR-10b-5p may play an important role as tumor suppressors in MM, targeting methylation of miRNAs could be a promising approach for the treatment of MM. Disclosures: Ghobrial: BMS: Advisory board Other, Research Funding; ONYX: Advisory board, Advisory board Other; NOXXON: Research Funding; Sanofi: Research Funding.

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Long Non-Coding RNAs Annotation and Their Involvement in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.4420.4420 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4420-4420 ◽

Cited By ~ 1

Author(s):

Arantxa Carrasco ◽

Teresa Ezponda ◽

Cem Meydan ◽

Marta Kulis ◽

Raquel Ordoñez ◽

...

Keyword(s):

Dna Methylation ◽

Multiple Myeloma ◽

Bone Marrow ◽

B Cell ◽

Cell Lines ◽

Promoter Region ◽

Research Funding ◽

Epigenetic Mechanisms ◽

Non Coding Rnas

Abstract Increasing amount of evidence indicates that the deregulation of non-coding elements is a common feature of cancer and therefore, its investigation may uncover new molecular oncogenic mechanisms. In multiple myeloma (MM), the altered expression of a small number of long non-coding RNAs (lncRNAs) has been associated with progression and decreased survival, suggesting that these elements may play a more important role in this disease than previously expected. Nevertheless, an extensive high-throughput analysis that characterizes the deregulation of lncRNAs in MM has not yet been performed. To characterize the transcriptome, including all genomic types of lncRNAs, of MM we performed a paired end strand-specific RNA sequencing (ssRNA-seq) in 38 purified plasma cell (PC) samples from MM patients, as well as PC samples from tonsils (TPCs, n=5) and bone marrow (BMPCs, n=3) of healthy donors as controls. Principal component analysis (PCA) demonstrated that normal PC samples from tonsil and bone marrow cluster separately, suggesting that in spite of being the same cell type, their coding and non coding transcriptomes are very different. Therefore, we selected BMPCs as the normal counterparts for comparison with BM of MM samples. PCA analysis also demonstrated that the well known heterogeneity of MM patients rely not only on the coding transcriptome but also on the lncRNA expression profile. Comparison of MM to BMPCs samples showed 70 previously annotated lncRNAs that were deregulated in MM patients, with 3 lncRNAs showing higher and 67 lower expression than normal BMPCs. Moreover, we identified 40.552 novel MM-specific lncRNAs that were present in at least 3 of the 38 patients, highlighting the magnitude of the deregulation of these non coding elements in MM. To determine the functional role of altered lncRNAs in the biology of MM plasma cells we focused on the study of LINC-MSL1 (Myeloma-Specific LncRNA 1). Analysis of the expression of this lncRNA at different stages of B-cell differentiation (Naïve, Germinal Center, Memory and PC) indicated that it is not expressed at any stage, except for a modest expression in BMPCs. Interestingly, its overexpression was detected in 40% of MM specimens when compared to normal BMPCs which was validated by qPCR in an independent cohort of MM patients. To determine whether the expression of this lncRNA is regulated by epigenetic mechanisms, we studied the DNA methylation state of this gene. DNA methylation analysis in MM demonstrated that the CpGs located upstream of LINC-MSL1 were differentially methylated in comparison with normal counterpart BMPC. These CpGs showed 70% DNA methylation in control samples, about 40% in MGUS, whereas the average of MM was about 20%, showing a remarkable hypomethylation. We validated these results by pyrosequencing, which showed a significantly lower DNA methylation at the promoter region in comparison with B cell populations from tonsil, normal BMPCs and cell lines that do not overexpress LINC-MSL1. We also have observed a gain of active chromatin states analyzed by ChiP-seq in the promoter region of LINC-MSL1 in MM patient samples. These data suggest that epigenetic mechanisms, namely the progressive hypomethylation and the gain of active histone modifications, are the cause of the overexpression of LINC-MSL1 in MM. To analyze the role of the overexpression of LINC-MSL1 in MM, we engineered two MM cell lines that show high levels of LINC-MSL1, MM.1S and MM.1R, to express shRNAs against this lncRNA. Knockdown of LINC-MSL1 by two different shRNAs resulted in a reduced proliferation of the cell lines over time. This effect was not associated with a cell cycle arrest but with a marked increased in the percentage of Annexin V-positive apoptotic cells, indicating that the overexpression of LINC-MSL1 is necessary for the survival of MM cells. All together, these data demonstrate that the alteration of lncRNAs is an important an unexplored feature that contributes to MM pathogenesis. The overexpression of LINC-MSL1 is essential for MM survival and is very specific of MM BMPCs, suggesting it could be a relevant therapeutic target. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding. Melnick:Janssen: Research Funding.

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Genome Wide DNA Methylation Profiling In Patients with Multiple Myeloma.

Blood ◽

10.1182/blood.v116.21.3622.3622 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3622-3622

Author(s):

Yang Liu ◽

Shenghua Duan ◽

Xavier Leleu ◽

Yong Zhang ◽

Abdel Kareem A. Azab ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Cell Lines ◽

Promoter Region ◽

Genomic Dna ◽

Research Funding ◽

Plasma Cells ◽

Genome Wide

Abstract Abstract 3622 Introduction: Epigenetic factors such as DNA methylation have been shown to play a crucial role in the pathogenesis and progression of multiple myeloma (MM), yet studies of DNA methylation in MM are still limited. Therefore, in order to better understand the role of DNA methylation and identify specific genes that may be affected by differential methylation in MM patients, we conducted genome-wide DNA methylation profiling in cd138+ plasma cells purified from bone marrow of the patients with MM and normal donors. Methods: Genomic DNA of CD138+ Plasma cell selected from both MM patients and normal primary bone marrow was extracted using QIAGEN genome isolation kit. Following extraction, methylated DNA was isolated by Chip and hybridized to Affymetrix Human 2.0 tiling arrays. Chip assay and array hybridization was performed by Genepathway Inc. CEL files were processed and normalized using the MAT program, and methylation peaks were called from the resulting MAT scores using a custom segmentation method. Peak annotation and characterization of different genomic regions was done with custom tools and using genome annotation files from the UCSC genome database. All peaks were visualized by IGB online software. Medip-PCR was done in human MM cell lines to validate the methylation status. Methylated gene expression was determined by both Semi-quantitative PCR and real-time PCR. 5′aza was used for demethylation in human MM cell lines. Methylated gene expression with or without 5′aza treatment was determined by both Semi-quantitative PCR and real-time PCR. Results: Genomic DNA from CD138+ plasma cells from bone marrow of MM patients showed a significant increase in methylation levels compared to normal controls. We demonstrated that the hypermethylated sites were distributed across the genome in the following proportions: 3.2% in the promoter region; 45.6% in the intragenic region; 5.4 % in the 3′ end region; and 46.8 % in the intergenic region. Furthermore, around 9 % promoter CpG islands (CGIs); 11% intragenic CGIs; 15 % CGIs in 3′end region; and 14.3 % intergenic CGIs of patients genomic DNA were methylated. Moreover 2.1% promoter CGIs; 2.3 % intragenic CGIs; 2.5% CGIs in 3′end region; and 4.7% intergenic CGIs were methylated for the normal control. Medip-PCR showed that the identified methylation pattern in MM patients showed similar results in MM cell lines. Expectedly, we also observed that suppressor of cytokine signaling 1 (SOCS1) was hypermethylated at the promoter region (MAT score=19.986) as has been reported in human cell lines. Importantly, another member of SOCS family SOCS3 showed much stronger signal in the promoter region with CpG island (MAT score=31.707) in MM patients compared to normal control. Notably, the expression of two members of TNFR superfamily TNFRSF18 and TNFRSF4 which play an important role in development and programmed cell death of lymphocyte significantly have increased 283 and 141-fold after treatment with 5′aza in MM cell lines. Conclusion: These findings enhance our understanding of the role of DNA methylation in MM, as one of the epigenetic changes that may contribute to the pathogenesis of this disease. The identification and functional characterization of novel key molecules affected by DNA methylation will provide deeper insight into the molecular basis of MM disease. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Lin28B/Let-7 Axis Regulates Multiple Myeloma Proliferation By Enhancing c-Myc and Ras Survival Pathways

Blood ◽

10.1182/blood.v122.21.273.273 ◽

2013 ◽

Vol 122 (21) ◽

pp. 273-273

Author(s):

Salomon Manier ◽

John T Powers ◽

Antonio Sacco ◽

Michaela R Reagan ◽

Michele Moschetta ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Loss Of Function ◽

Advisory Committees ◽

Expression Levels ◽

Gain And Loss

Abstract Background MicroRNAs (miRNAs) play a pivotal role in tumorigenesis, due to their ability to target mRNAs involved in the regulation of cell proliferation, survival and differentiation. Lin28B is an RNA binding protein that regulates Let-7 miRNA maturation. Lin28B and Let-7 have been described to act as oncogenes or tumor suppressor genes, respectively, as demonstrated both in solid cancer and hematologic malignancies. However, the role of the Lin28B/Let-7 axis in Multiple Myeloma (MM) has not been studied. Method Lin28B level expression in MM patients was studied using previously published gene expression profiling (GEP) datasets. Let-7 expression levels were assessed in CD138+ primary MM cells and bone marrow stromal cells (BMSCs) by using PCR, as well as in circulating exosomes using miRNA array (Nanostring® Technology). Exosomes were collected from both normal and MM peripheral blood, using ultracentrifugation; and further studied by using electron microscopy and immunogold labeling for the detection of CD63 and CD81. The knockdown of Lin28B was performed on MM cell lines (U266, MM.1S, MOLP-8) by using a lentiviral Lin28B shRNA. Gain- and loss-of function studies for Let-7 were performed using Let-7 mimic and anti-Let-7 transfection in MM cell lines (MM1S, U266) and primary BMSCs. Cell proliferation has been evaluated by using thymidine assays. Effects of Let-7 and Lin28B on signaling cascades have been evaluated by western blot. Results Two independent GEP datasets (GSE16558; GSE2658) were analyzed for Lin28B expression, showing a significantly higher level in MM patients compared to healthy controls. In addition, high Lin28B levels correlated with a shorter overall survival (p = 0.0226). We next found that the Let-7 family members are significantly down-regulated in MM primary cells, particularly Let-7a and b (5 fold change, p < 0.05), as demonstrated by using qRT-PCR. Similarly, miRNA arrays showed a lower expression of Let-7-related miRNAs in circulating exosomes obtained from MM patients compared to healthy individuals. We further dissected the functional relevance of Lin28B in MM cells, by performing Lin28 knockdown (KD) in MM cell lines (U266, MOLP-8). This led to a significant decrease in MM cell proliferation associated with G1 phase cell cycle arrest. This was supported by up-regulation of Let-7 and down-regulation of c-Myc, Ras and Cyclin D1 in Lin28 KD MM cells. To further prove that Lin28B-dependent effects on MM cells are mediated by Let7, we next showed that let-7 gain- and loss-of-function studies regulate MM cell proliferation and Myc expression. Lin28B regulation in MM cells is dependent on Let-7, as demonstrated by an increase of both cell proliferation and c-Myc expression after anti-Let-7 transfection in the Lin28B KD cells. We therefore studied the regulation of Let-7 in MM cells through the interaction with BMSCs. Let-7 expression levels were significantly lower in BMSCs obtained from MM patients compared to healthy donors. Interestingly, the Let-7 expression level in MM cells was increased after co-culture with Let-7 over-expressing BMSCs, associated with a decrease of both cell proliferation and c-Myc expression. This suggests a potential transfer of Let-7 from BMSCs to MM cells. Conclusion This work describes a new signaling pathway involving Lin28B, Let-7, Myc and Ras in MM. Let-7 expression in MM cells is also regulated through the interaction of MM cells with BMSCs, leading to cell proliferation and Myc regulation in MM. Interference with this pathway might offer therapeutic perspectives. Disclosures: Leleu: CELGENE: Honoraria; JANSSEN: Honoraria. Daley:Johnson and Johnson: Consultancy, Membership on an entity’s Board of Directors or advisory committees; MPM Capital: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Verastem: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity’s Board of Directors or advisory committees; iPierian: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Solasia, KK: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Ghobrial:Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

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47 Genes Define Myeloma Cell Acquired Resistance to Bortezomib and Have Profound Prognostic Implications in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.499.499 ◽

2015 ◽

Vol 126 (23) ◽

pp. 499-499

Author(s):

Xenofon Papanikolaou ◽

Caleb K. Stein ◽

Ricky D Edmondson ◽

Veronica Macleod ◽

Ruslana Tytarenko ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Myeloma Cell ◽

Advisory Board ◽

University Of Arkansas ◽

Newly Diagnosed ◽

Medical Sciences ◽

Gene Probes ◽

The Common

Abstract The proteasome inhibitor Bortezomib (Bz), the first agent of a new class of drugs in Multiple Myeloma (MM), has shown remarkable activity and forms an integral part of modern MM treatment. Nevertheless, resistance to Bz eventually develops in a significant proportion of patients, with adverse effects on survival. Numerous publications have addressed this issue through in vitro developed models of acquired Bz resistance (BzR). However the results were quite different in each publication, none of the produced Bz myeloma cell lines was provably stable, no common mechanism of resistance could be demonstrated, and hence were of minimal relevance to the clinical setting. In order to address these issues an effort was made for the development of an in vitro model of acquired BzR that would resemble the clinical reality in the most accurate way. Two myeloma cell lines were used, one resembling a multisensitive (JJN3) and the other a multiresistant (U266) drug behavior, that were both sensitive to Bz. An at least 20 fold increase in the 48h Bz IC50 was noted for both cell lines. The increase in the IC50 was able to be verified a year after culturing the cell lines in normal medium thus ensuring a stable resistance phenotype. To delineate the molecular mechanisms that underlie the development of BzR a combined genetic/Gene Expression Profile (GEP) and functional/Proteomics approach was used with emphasis in the common elements of both cell lines. The hypothesis was that if certain pathways are activated in the cells that actually produce the phenotype of BzR they must fulfil two important criteria: 1) They must be present in all the levels of the BzR, 2) The gene changes have to be verified in the level of the gene encoded proteins thus securing their functional importance. GEP of the naïve cell lines along with the GEP of the Bz resistant cells at different levels of BzR (5-fold, 10-fold, 20-fold) were used. The statistical analysis revealed 100 gene probes common in both cell lines that achieved their highest change as soon as BzR was established and remained stable at that level for all later versions (P<0.1, q<0.1) and 115 gene probes common in both cell lines that their change was proportional to the level of BzR (P<0.001, q <0.005). The proteomics analysis of the Bz resistant cell lines at their latest level of resistance (20-fold) revealed 262 proteins common in both cell lines that were up-regulated and 263 common in both cell lines that were down-regulated (change >10% to be considered significant). The intersection of the list of the common genes with the list of the common proteins revealed 47 gene-proteins all but one novel in MM. They can be grouped in distinct biological categories with the most prominent ones being the ROS/Mitochondrial Factor category comprising of 10 gene-proteins, the E3 Ubiquitin Pathway 6 genes-proteins and Translation Regulation 5 genes-proteins. Even more importantly 30 of them have profound survival implications in MM -all of them novel in MM- both for Overall Survival (OS) and Progression Free Survival (PFS) in both Bz (TT3) and non Bz (TT2) containing protocols implying that myeloma cells apply both Bz specific and non-specific mechanisms to acquire BzR. Based on these 30 genes-proteins a GEP risk score (GEP-30) was constructed that was able to achieve remarkable statistical power in both Bz containing and non containing trials of both newly diagnosed (TT2 with and without thalidomide i.e. TT2+ and TT2-, TT3a, TT3b, HOVON, MRC IX, Figure 1A,B,C) and relapsed MM (TT6 , OS: NR vs 1.52 yr P<0.00001, PFS: NR vs 1.13 yr P<0.00001 for low and high risk) Figure 1. KM plots for OS and PFS of GEP-30 for newly diagnosed MM Figure 1. KM plots for OS and PFS of GEP-30 for newly diagnosed MM Figure 1B. Figure 1B. Figure 1C. Figure 1C. Disclosures Stein: University of Arkansas for Medical Sciences: Employment. Barlogie:University of Arkansas for Medical Sciences: Employment. Epstein:University of Arkansas for Medical Sciences: Employment. Heuck:Janssen: Other: Advisory Board; Celgene: Consultancy; Millenium: Other: Advisory Board; Foundation Medicine: Honoraria; University of Arkansas for Medical Sciences: Employment.

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CXCR4 Is a Regulator Of Disease Involvement Of Extramedullary Myeloma Confirmed By a Novel Mouse Model For Extramedullary Disease

Blood ◽

10.1182/blood.v122.21.5320.5320 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5320-5320 ◽

Cited By ~ 1

Author(s):

Yuji Mishima ◽

Michele Moschetta ◽

Michaela R Reagan ◽

Yong Zhang ◽

Ilyas Sahin ◽

...

Keyword(s):

Multiple Myeloma ◽

Protein Expression ◽

Cell Lines ◽

Chemokine Receptors ◽

Research Funding ◽

Advisory Board ◽

Confocal Imaging ◽

Extramedullary Disease ◽

Gene And Protein Expression

Abstract Background Extramedullary disease (EMD) in patients with multiple myeloma occurs mostly in advanced disease or relapse. EMD seems to have a different pathogenesis from medullary myeloma and is often characterized by a more aggressive clinical course. To date molecular mechanisms of development of EMD have not been fully understood. Methods: Human MM cell lines, IM-9 and MM1S were serially selected in immune-deficient mice. IM-9 and MM1S cells were inoculated intravenously and harvested from the tumors developed in the bone marrow (BM) and liver (site of extramedullary disease), aiming to establish BM-prone and liver-prone clones. Tumor progression was periodically checked by bioluminescence (BLI) and in vivo live confocal imaging. After three rounds of in vivo selections, the cells of both BM- and liver-prone were characterized by gene and protein expression and cellular functional assays. Results: We obtained three liver-prone sub-clones in both IM-9 and MM1S after serial in vivo selections. These cells had equal proliferation rates in vitro compared to the original or BM-prone cells, but exhibited more aggressive phenotype in vivo. Liver-prone clones had significantly higher migration ability than BM-prone clones (11.6% vs 6.1% migration, respectively p=0.018). Gene and protein expression analysis revealed that each liver-prone clone had a higher expression of sets of chemokine receptors specifically CXCR4. Using thein vivo metastatic model CXCR4-over expressing myeloma cells exhibited higher metastatic property to the extramedullary organs whereas CXCR4-knockdown cells has less tumor metastasis to the liver. Discussion: We established EMD-prone human multiple myeloma cell lines that reproducibly developed liverinvolvement consistent with human extramedullary disease. These cells exhibited higher migratory ability and increased expression of several chemokine receptors, specifically CXCR4. We validated the effect of CXCR4 on developing extramedullary myeloma using our established in vivo mode. Further studies to determine the role of CXCR4 for therapeutic targeting of extramedullary disease in MM are ongoing. Disclosures: Ghobrial: Sanofi: Research Funding; Noxxon: Research Funding; BMS: Advisory board, Advisory board Other, Research Funding; Onyx: Advisoryboard Other.

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Treatments and Outcomes of Newly Diagnosed Multiple Myeloma Patients > 75 Years Old: A Retrospective Analysis

Blood ◽

10.1182/blood-2020-142985 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-15

Author(s):

Abdullah S. Al Saleh ◽

Alissa Visram ◽

Harsh Parmar ◽

Angela Dispenzieri ◽

Eli Muchtar ◽

...

Keyword(s):

Multiple Myeloma ◽

Clinical Trials ◽

Elderly Patients ◽

Research Funding ◽

Staging System ◽

Complete Response ◽

The Elderly ◽

Advisory Board ◽

Newly Diagnosed ◽

Elderly Age

Introduction: In general, the use of an immunomodulator (IMiD), proteasome inhibitors (PI) and dexamethasone (dex) for the treatment of MM is associated with better outcomes. The management of elderly patients with multiple myeloma (MM) is challenging due to difficulty in managing their co-morbidities and inability to tolerate treatment side effects. We evaluated therapies and outcomes of elderly patients with newly diagnosed MM. Methods: This is a retrospective study of patients with MM who were >75 years old treated at the Mayo Clinic, Rochester from January 2004 to January 2018. We included patients who were treated on clinical trials as well as off-trials. Patients were classified as receiving treatment with IMiD+PI+dex, alkylator+PI+steroid, IMiD+dex, PI+dex, alkylator+IMid+steroid, and other (alkylator with steroid or steroid only). Treatment response was documented as well as the progression-free (PFS), defined as the time from therapy initiation to disease relapse or death from any cause and overall survival (OS), defined as the time from start of treatment to death from any cause. A multivariate analysis for factors affecting OS was done including the following variables: being on a triplet combination (alkylator+PI+steroid, IMid+PI+dex, or alkylator +IMiD+steroid), revised international staging system (R-ISS)(stage 3 vs. 1-2), bone marrow plasma cell percentage (BMPC%)(>60% vs. ≤60%), and receiving treatment during or after 2010 vs. before 2010. Analysis was done for patients treated off-trials, as well as, including trial patients. Results: We identified 394 patients with MM who were >75 years old and 246 (62%) were male. For non-trial patients (n=350), IMiD+dex (32%) was the most commonly used regimen followed by alkylator with steroid or steroid only (20%), alkylator+PI+steroid (18%), and IMid+PI+dex (13%). The remaining patients were treated with PI+dex (12%) and alkylator +IMiD+steroid (5%). Forty-four patients (11%) were treated in clinical trials with alkylator+IMid+steroid (47%), IMiD+dex (25%), IMiD+PI+dex (14%), and alkylator+PI+steroid (14%). The median follow up was 45.9 months with an interquartile range of 28.2 to 75.6 months. Overall, achieving very good partial response or complete response was more likely in patients who were treated with an IMid+PI+dex (58%) or alkylator+PI+steroid (47%), compared to in other therapies (5-30%)(P<0.0001). The PFS and OS for non-trial patients are displayed in Figure 1 (A,B) and for all, including trial patients in (C,D). Overall, the median OS was significantly longer in patients who were treated with a triplet in non-trial as well as all patients. In a multivariate for OS including non-trial patients, predictors for better OS included receiving a triplet (HR: 0.63, P=0.02) and not having an R-ISS stage 3 (HR: 0.39, P=0.001). This was also found when including trial patients (using a triplet, HR: 0.65, P=0.01 and not having an R-ISS stage 3, HR: 0.35, P=0.0002). Conclusion: In MM patients >75 years old, being able to receive triplet therapy is associated with better survival. This study provides better understanding of the natural history of MM outside of trials in the elderly age group. Disclosures Dispenzieri: Celgene: Research Funding; Alnylam: Research Funding; Pfizer: Research Funding; Intellia: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Dingli:Bristol Myers Squibb: Research Funding; Alexion: Consultancy; Millenium: Consultancy; Rigel: Consultancy; Sanofi-Genzyme: Consultancy; Apellis: Consultancy; Janssen: Consultancy; Karyopharm Therapeutics: Research Funding. Kapoor:GlaxoSmithKline: Research Funding; Amgen: Research Funding; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; Janssen: Research Funding; Cellectar: Consultancy; Celgene: Honoraria. Gertz:Spectrum: Other: personal fee, Research Funding; Janssen: Other: personal fee; Prothena: Other: personal fee; Alnylam: Other: personal fee; Ionis/Akcea: Other: personal fee; Springer Publishing: Patents & Royalties; Proclara: Other; DAVA oncology: Speakers Bureau; Johnson and Johnson: Speakers Bureau; Teva: Speakers Bureau; Sanofi: Other; Research to Practice: Other; Celgene: Other; Abbvie: Other; Aurora Bio: Other; Physicians Education Resource: Other: personal fee; Medscape: Other: personal fee, Speakers Bureau; Amgen: Other: personal fee; Appellis: Other: personal fee; Annexon: Other: personal fee. Kumar:Carsgen: Other, Research Funding; Tenebio: Other, Research Funding; BMS: Consultancy, Research Funding; Karyopharm: Consultancy; MedImmune: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Kite Pharma: Consultancy, Research Funding; Oncopeptides: Consultancy, Other: Independent Review Committee; IRC member; Genentech/Roche: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Celgene/BMS: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Adaptive Biotechnologies: Consultancy; Merck: Consultancy, Research Funding; Amgen: Consultancy, Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments, Research Funding; Janssen Oncology: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Takeda: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; AbbVie: Other: Research funding for clinical trials to the institution, Consulting/Advisory Board participation with no personal payments; Dr. Reddy's Laboratories: Honoraria; Cellectar: Other; Genecentrix: Consultancy.

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Advanced Imaging and Targeted Myeloma Lesion Biopsies to Enhance Global Response Assessment and Evaluate Spatial Heterogeneity in Multiple Myeloma

Blood ◽

10.1182/blood-2020-136478 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 20-22

Author(s):

Sabrina L. Browning ◽

Terri L. Parker ◽

Noffar Bar ◽

Tara Anderson ◽

Madhav V. Dhodapkar ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

High Risk ◽

Board Of Directors ◽

Research Funding ◽

Response Assessment ◽

Advisory Board ◽

Newly Diagnosed ◽

Advanced Imaging ◽

Advisory Committees

Background: Multiple myeloma (MM) is a heterogeneous plasma cell neoplasm with significant genetic and biologic complexity. Limitations remain in our standard assessment of response to therapy, as random bone marrow biopsy may misrepresent the varied histologic and molecular features of this multifocal disease. Advanced imaging is crucial in evaluating bone and extramedullary (EM) lesions. We aim to refine global response assessment in MM, with incorporation of advanced imaging-guided lesion biopsies, to improve knowledge of residual tumor burden critical to patient outcomes. Methods: Patients ≥18 years of age with standard or high risk newly diagnosed clinical MM were eligible to participate in this study. Advanced imaging with positron emission tomography/computed tomography (PET/CT) or whole body magnetic resonance imaging (WB-MRI) based on access, standard bone marrow biopsy and aspiration, and targeted lesion biopsy occurred at enrollment and after 4 cycles of carfilzomib, lenalidomide, and dexamethasone (CRd). Carfilzomib was administered intravenously at a dose of 36 mg/m2 twice weekly, lenalidomide orally 25 mg daily days 1-21, and dexamethasone orally 40 mg weekly, with dose modifications as needed. Conventional clinical response, using IMWG Response Criteria (Kumar S et al, 2016), was assessed after each cycle of treatment. Results: An interim analysis was completed on 17 patients enrolled between June 2018 and March 2020, with 14 evaluable for global treatment response. Median age was 61 years (range, 43-76 years) and 82.4% of patients were male. 76.5% had Revised International Staging System (R-ISS) stage II or III disease and 58.8% had EM disease arising from bone (EM-B, 41.2%) or independently in soft tissue (EM-S, 17.6%). 70.6% of patients had at least one high risk feature at the time of diagnosis (Table 1). Of the 16 patients with conventional skeletal survey (CSS) at study entry, 68.8% had at least 1 myeloma-defining lesion on advanced imaging that was missed on CSS. Four patients had adequate sample from initial lesion biopsy for cytogenetics and fluorescence in situ hybridization (FISH), 3 of whom demonstrated discordant FISH results when compared to standard bone marrow samples. Clinical response rates after 4 cycles of CRd were notable with 85.7% of patients achieving ≥ very good partial response (VGPR) and 3 patients with stringent complete response (sCR) and minimal residual disease (MRD) negativity by flow cytometry with a sensitivity of 10-5. However, of the 12 patients with ≥ VGPR by conventional response assessment, 9 had residual disease on advanced imaging with PET/CT (2 patients), WB-MRI (6 patients), or total spine MRI (1 patient) (Figure 1). Repeat myeloma lesion biopsy was limited to 6 patients with targetable lesions after induction therapy, with diagnostic yield impacted by the presence of sclerotic tissue and insufficient marrow elements in some of the lesions sampled (Table 2). 85.7% of patients continued CRd or proceeded to high dose therapy and autologous stem cell rescue, with no patients transitioning directly to maintenance treatment after 4 cycles of CRd. At a median follow-up of 8 months, 14.3% (2/14) of patients have had progression of disease. Both individuals had residual lesions on imaging at end of treatment, despite one with flow MRD-negative sCR and normal FISH by standard assessment. There were no grade 4 serious adverse events or deaths. Conclusions: In our cohort of high risk newly diagnosed MM, CRd induction was potent and well-tolerated. While deep clinical responses were observed by conventional clinical assessment, two thirds of patients had persistent abnormalities on advanced imaging with concern that these sites could give rise to progressive MM. Our patients demonstrated spatial heterogeneity, highlighting the limitations of standard bone marrow evaluation. Use of advanced imaging and targeted lesion biopsies in response assessment enhances our understanding of tumor growth pattern in MM and consideration could be given to integrating these into clinical care when available. Current limitations of this study include a small number of patients with lesions amendable to repeat biopsy and their incomplete diagnostic yield. Ongoing investigation includes whole exome sequencing of paired bone marrow and focal lesion biopsies and application of a WB-MRI lesion scoring system to further augment this novel response assessment method. Disclosures Anderson: Celgene: Speakers Bureau; Janssen: Speakers Bureau; Takeda: Speakers Bureau; Amgen: Speakers Bureau. Dhodapkar:Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Lava Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Kite: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board. Prebet:Jazz Pharmaceuticals: Consultancy, Research Funding. Xu:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Haims:Pfizer: Consultancy. Neparidze:Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Advisory board; Eidos Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Diagnostic committee member ; GlaxoSmithKline: Research Funding; Janssen: Research Funding. OffLabel Disclosure: Carfilzomib has been shown to have significant anti-myeloma activity in relapsed myeloma. Phase I/II studies as well as one phase III study also showed favorable outcomes with carfilzomib-based regimens in newly diagnosed multiple myeloma, including in patients with high risk disease. We utilized an induction regimen with carfilzomib, lenalidomide, and dexamethasone given that patients enrolled in this study were required to have bone or soft tissue disease on advanced imaging, indicating a likely high risk feature with potentially aggressive disease biology. It has been shown that the combination of carfilzomib, lenalidomide, and dexamethasone is a safe regimen for patients with multiple myeloma. This combination is approved in the relapsed/refractory setting and included in NCCN guidelines for newly diagnosed multiple myeloma.

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Prolonged Follow-up Confirmed a Role for Upfront Tandem Auto-Allo Transplant in Multiple Myeloma Also in the Era of New Drugs

Blood ◽

10.1182/blood.v128.22.3469.3469 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3469-3469

Author(s):

Luisa Giaccone ◽

Andrea Evangelista ◽

Francesca Patriarca ◽

Roberto Sorasio ◽

Massimo Pini ◽

...

Keyword(s):

Multiple Myeloma ◽

Cumulative Incidence ◽

Research Funding ◽

Advisory Board ◽

New Drugs ◽

Main Concern ◽

Newly Diagnosed ◽

Post Transplant ◽

The Impact

Abstract Introduction: Before the introduction of new drugs, we designed a trial where treatment of newly diagnosed multiple myeloma (MM) patients with double autografts or autograft followed by nonmyeloablative allograft was based on the presence or absence of HLA identical siblings (Bruno B et al. N Engl J Med 2007) We reported an update with special focus on long term outcomes. Methods: From September 1998 to July 2004, 162 consecutive patients with newly diagnosed MM up to the age of 65 years and at least one sibling were enrolled at 5 Italian centers, and divided into 2 groups: donor (N=80) vs no donor (N=82). First-line treatments consisted of a cytoreductive autograft followed by a HLA identical sibling nonmyeloablative allograft or a second melphalan-based autograft (N=58 and N=46, respectively, completed the protocol). Results: Median follow-up was 12.3 years (range, 7.7-15.3) from allograft, and 12.1 years (range, 10.5-15.4) from second autograft. The 5-year cumulative incidence of non-relapse mortality was 17.2% (95%CI: 7.4 to 27.1) in the allograft arm and 4.3% (95%CI:0 to 10.3) in the autograft arm. One of the main concern post allograft is the impact of chronic graft-versus-host disease (cGVHD): in our setting its 2-year cumulative incidence was 67.2% (95%CI: 54.9 to 79.5). We also evaluated the cumulative incidence of immunesuppression discontinuation in patients with cGVHD, considering both death and relapse as competing events: 26.8% of cGVHD patients (95%CI: 13 to 40.6) at 24 months and 39% (95%CI:23.6 to 54.4) at 60 months were alive and without therapy. Median overall survival (OS) and progression-free survival (PFS) from second transplant were 137 and 43 months in the allograft arm and 46 and 18 months in the autograft one (p=0.006 and p=0.001, respectively). In the allograft arm, 33 out of 58 patients relapsed at least once, and first salvage treatments included donor lymphocyte infusion (DLI, N=13), thalidomide (N=10) and bortezomib (N=8). Of note, 2 patients lost complete remission status but did not require further therapy. Nineteen out of 33 patients required a second post-transplant salvage treatment: 1 received chemotherapy, 1 DLI, 1 received debulking treatment with bortezomib followed by a second allograft, and 16 patients were treated with new drugs containing regimens. In the autograft arm, 36/46 patients relapsed and received salvage treatments consisting of: allograft (N=1), 3rd autograft prepared with a new-drugs containing regimen (N=6), thalidomide (N=17), bortezomib (N=7), lenalidomide (N=1), chemotherapy alone (N=4). Among these, 19 required a third-line treatment: 1 received an allograft, and 18 a regimen containing new drugs. Median OS from 1st relapse was 89.8 months (95%CI: 33.3 to n.r.) in the allograft arm vs 23.5 months (95%CI: 12.5 to 50.5) in the autograft (p=0.009). Conclusions: Our update showed that more then a third of patients developing cGVHD were relapse-free and cGVHD-free at 5-years post-transplant and that the advantage in OS in the allograft arm is maintained also after relapse, suggesting a synergism between graft-vs-myeloma effect and new agents. Upfront allograft in MM remains a matter of debate, and it should be performed only within clinical trials. The main limit of the present study was the lack of novel agents as part of the pre-transplant approach, nevertheless our results suggested that allograft may have a role, and it might be considered in young patients with high-risk features such as del [13], t(4;14), del(17p), and t(14;16), who remain at poor prognosis even in the era of new drugs. Disclosures Bringhen: Mundipharma: Other: ADVISORY BOARD; Amgen: Other: ADVISORY BOARD; Janssen-Cilag: Honoraria; Celgene: Honoraria; BMS: Honoraria; Karyopharm: Other: ADVISORY BOARD. Massaia:Janssen: Other: advisory board; Roche: Other: advisory board, research support; Gilead: Other: advisory board. Palumbo:Janssen Cilag: Honoraria; Takeda: Employment, Honoraria. Boccadoro:CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; SANOFI: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Mundipharma: Research Funding; Abbivie: Honoraria; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding.

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The Synergistic Effect of Melphalan and XPO1 Inhibition in Pre-Clinical Models of Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.5662.5662 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5662-5662 ◽

Cited By ~ 2

Author(s):

Yan Cui ◽

Joel G Turner ◽

Jana L Dawson ◽

Juan A Gomez ◽

Kenneth H. Shain ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Damage ◽

Western Blot ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Single Agent ◽

Newly Diagnosed ◽

Advisory Committees ◽

Genetics Research

Abstract Introduction:Multiple myeloma (MM) is an incurable cancer of plasma cells. It accounts for approximately 10% of all hematologic malignancies. In the US, it is estimated that there will be approximately 30,330 new cases and 12,650 deaths in 2016. In the past decade, responses/survivals have been significantly increased by newer therapies. However, almost all of the patients will eventually die from multi-drug resistant disease. Materials and Methods:Weused XPO1 inhibitors (XPO1i) selinexor (300nM) or KPT-8602 (300nM) +/- melphalan (15 μM) to treat human MM parental RPMI8226 and U266 cells, and melphalan resistant LR5 and LR6 cell lines for 20 hours and then assayed for apoptosis and viability by flow cytometry. DNA damage was assayed by the comet assay and phospho-H2AX protein expression in H929 human myeloma cells. p53, NFkB, IKKα, FANCF, and FANCL were assayed by Western blot in H929 MM cells. We also treated cells from patients with newly diagnosed or relapsed/refractory MM with the XPO1i (300nM)/ melphalan (10μM) combination and assayed for apoptosis. In addition, selinexor/melphalan treated NOD/SCID-gamma mice with U226 MM tumors were assayed for tumor growth, survival, and toxicity. Results:Cell viability of all tested MM cell lines was decreased synergistically and apoptosis increased by XPO1i/melphalan treatment (selinexor/melphalan, P = 2.2x10E-6 to 0.0032, KPT-8602/ melphalan, P = 1.2X10E-7 to 0.0031). Comet assays showed that the XPO1i/ melphalan drug combination increased DNA damage more than single agent melphalan or XPO1i alone. Phospho-H2AX expression also was increased (selinexor/ melphalan, P = 0.005 and KPT-8602/melphalan, P = 0.001). Western blot analysis showed that XPO1i treatment can increase p53 and decrease NFkB, IKKα, FANCF, and FANCL in MM cells. Apoptosis assays showed that both melphalan-resistant and parental MM cell lines were sensitized to melphalan by XPO1i. In addition, CD138+/light chain+ MM cells from newly diagnosed and relapsed/refractory MM patients were sensitized (20-fold and 5 to10-fold respectively) by XPO1i to melphalan. XPO1i/melphalan combination treatment demonstrated a strong synergistic anti-tumor effect when compared to single-agent melphalan (selinexor, P = 0.0024 and KPT-8602, P = 0.0030) in NOD/SCID-gamma mice challenged with U266 MM tumors. XPO1i/ melphalan treated mice had increased survival and no significant toxicity. Conclusions:XPO1i's can improve the response of human MM cell lines and patient MM cells to melphalan both in vitro and ex vivo. The mechanism of this synergy reversing melphalan resistance may be due to increased nuclear p53, in combination with decreased NFkB and IKKα, and decreased DNA repair proteins FANCL and FANCF of the Fanconi Anemia/BRCA pathway. Our preliminary data suggest that the synergistic cell kill may be because XPO1i's increase melphalan-induced DNA damage and block the repair of the DNA damage. Thus using combination therapies of XPO1i, especially the clinical compounds selinexor and KPT-8602 +/- melphalan may have potential to improve the treatment outcomes of MM. Based on these promising pre-clinical data, we designed a phase 1/2 clinical trial evaluating the combination of selinexor and high-dose melphalan as a conditioning regimen for autologous hematopoietic cell transplantation in patients with multiple myeloma (NCT02780609). Disclosures Shain: Novartis: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Signal Genetics: Research Funding; Takeda/Millennium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen/Onyx: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baloglu:Karyopharm Therapeutics Inc: Employment, Other: stockholder. Nishihori:Signal Genetics: Research Funding; Novartis: Research Funding.

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Trial in Progress: A Prospective Phase I/II Trial to Jointly Optimize the Administration Schedule(s) and Dose(s) of Melphalan for Injection (Evomela) As a Preparative Regimen for Autologous Hematopoietic Stem Cell Transplantation in Newly Diagnosed Multiple Myeloma

Blood ◽

10.1182/blood-2020-139853 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 37-38

Author(s):

Qaiser Bashir ◽

Peter F Thall ◽

Dawen Sui ◽

Cristina Knape ◽

Jitesh Kawedia ◽

...

Keyword(s):

Multiple Myeloma ◽

Phase I ◽

Propylene Glycol ◽

Cell Transplantation ◽

Research Funding ◽

Room Temperature ◽

Advisory Board ◽

High Dose ◽

Newly Diagnosed ◽

Preparative Regimen

Background: High-dose chemotherapy followed by autologous hematopoietic cell transplantation (auto HCT) is considered to be the standard of care treatment for transplant-eligible patients with multiple myeloma (MM). The most commonly used conditioning regimen in this setting is high-dose melphalan by intravenous administration. Conventional melphalan formulations, when administered at high doses, can put patients at risk of potential propylene glycol-associated toxicities. Melphalan for injection (Evomela) is propylene glycol free (PGF), can be dissolved directly using saline, and as a PGF reformulation of Alkeran, incorporating Captisol brand of beta-cyclodextrin sulfobutyl ethers sodium salts, overcomes previous formulation limitations. In 2016 Evomela was the first product approved by the US FDA for high-dose conditioning treatment prior to HCT in MM patients and it is also indicated for the palliative treatment of patients with MM for whom oral therapy is not appropriate. Reconstituted Evomela solution can be stored in the vial for up to 1 hour at room temperature or up to 24 hours at 2-8 °C with no significant degradation. After storage in the vial, it remains stable for an additional 3 to 29 hours after, preparation of admixture solution in infusion bags at concentrations of 0.25 to 5.0 mg/mL, respectively. As well, Evomela solution in saline, at concentration of 5.0 mg/mL melphalan, was bacteriostatic through 72 hours when stored at 2-8 °C. This stability allows for less frequent handling by pharmacy and nursing staff, resulting in a concomitant decrease in exposure risks, increased convenience and administration flexibility, suggestive of an improved ease of handling and administration, when compared to Alkeran. Further, Evomela may actually be less toxic due to the absence of propylene glycol. Although increased melphalan doses have previously demonstrated signals of improved response, the most commonly used dose of melphalan is 200 mg/m2, primarily due to concerns of toxicity. Emerging data regarding higher stability and potentially less toxicity of PGF melphalan (Evomela) supports dose escalation evaluation in order to improve the outcomes. Previous trial data have shown that continuous infusion or frequent fractionated-dose delivery increases the antitumour activity of several drugs. Due to the instability of currently available Alkeran at the room temperature, infusional studies have not been feasible. This limitation is overcome by Evomela, being that the compound is stable for several hours at the room temperature, thus allowing evaluation of infusional schedules in addition to the traditional 30-60 minute bolus doses. Here, we describe a trial designed to assess whether the above noted characteristics of Evomela allow for the escalation of dose and prolongation of infusion time, in order to increase the efficacy of melphalan in patients undergoing auto-HCT. Study Design/Methods: This is a two-stage phase I-II trial to optimize the dose and schedule of Evomela given as a single agent preparative regimen for auto-HCT. Up to 60 participants may be included if they are 18-70 years of age, with non-relapsed MM, have a Karnofsky performance score ≥70%, who have received at least two cycles of initial systemic therapy, and are within 2 to 12 months of the first dose. Patients will be randomized 1:1 to two different infusion schedules (30-60 minute infusion or 8-9 hour infusion) using Evomela (2mg/ml) at two dose cohorts (200mg/ml2 or 225mg/m2). Because 2 mg/mL Evomela is stable for 10 hours, patients receiving the 8-9 hour infusion will receive the total dose in one single infusion bag. The primary objectives are to determine the optimal dose and schedule of Evomela before auto-HCT for MM and collect pharmacokinetic data and compare the exposure-response evaluations between the two infusion schedules. Secondary outcomes will include incidence of treatment-related mortality, rate of minimal residual disease negative complete response at 90 days post auto-HCT, progression-free survival and overall survival after auto-HCT in newly diagnosed myeloma patients treated on different schedules and doses of Evomela. The active study follow-up period will be up to one-year post auto-HCT. Figure Disclosures Bashir: Acrotech: Research Funding; StemLine: Research Funding; Takeda: Other: Advisory Board, Research Funding; Celgene: Research Funding; KITE: Other: Advisory Board; Amgen: Other: Advisory Board; Purdue: Other: Advisory Board. Qazilbash:Amgen: Research Funding; Bioclinica: Consultancy; Angiocrine: Research Funding; Bioline: Research Funding; Janssen: Research Funding.

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