scholarly journals Development and Results of a Multiple Myeloma Specific Custom 77-Gene Mutation Panel for Clinical Targeted Sequencing

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 169-169 ◽  
Author(s):  
K. Martin Kortuem ◽  
A. Keith Stewart ◽  
Laura Ann Bruins ◽  
Gregory Ahmann ◽  
George Vasmatzis ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Copy Number ◽  
Research Funding ◽  
Pharmaceutical Research ◽  
Advisory Committees ◽  
Significant Information ◽  
Precision Therapy ◽  
Copy Number Changes ◽  
Salary Support

Abstract Background:A goal of “post-genomic” cancer care is development of specific, affordable, reliable and rapid next-generation sequencing-based assays determining prognosis, therapeutic selection and minimal disease monitoring, replacing older technologies in the diagnostic and monitoring workup. In this regard, we designed and deployed a custom gene panel for identification of mutations, copy-number changes and clonal frequency in multiple myeloma (MM) samples. Methods: The MM Mutation Panel Version 2.0 (M3P 2.0)1 includes 1271 amplicons from 77 genes that are either mutated in MM (genes with a published mutation incidence ≥3%), represent critical pathophysiological pathways, are targetable with small molecule-based therapies and/or are associated with drug resistance (eg. XBP-1, CRBN, IZKF, IRF4). The amplicons are spread across 21 chromosomes, allowing the identification of copy-number changes in these regions. Results: We have sequenced 38 MM cases (150 will be presented) with paired tumor-germline DNA samples, using only 20ng of DNA for library preparation. Patients (Pts) included 29 untreated and 9 MM pts with multi drug refractory disease. Coverage depth averaged 964X. Hyperdiploid MM was successfully identified in 45% of cases, del(13q) in 34%, del(1p) in 18%, del(17p) in 8% and gain of 1q in 13%. Array CGH in representative cases confirmed concordance with semiconductor sequencing. Overall, 71 exonic missense and 3 nonsense mutations were detected, and 7 frameshift indels. In 89% of the pts a mutation in at least one M3P2.0 gene was identified (range 1-8). 92% of mutations were predicted damaging (Provean, Polyphene-2 or SIFT). Most commonly mutated were KRAS (37%), NRAS (21%), BRAF (13%), TP53, CDKN1B, DIS3 (each 11%), FAM46C, STAT3, and IRF4 (each 8%). The MEK-ERK pathway was mutated in 61% of cases and in 3 cases more than one gene in this pathway (NRAS, KRAS, BRAF) was simultaneously mutated. Six mutations in BRAF were identified in 5 untreated pts, 3 of them known to be activating and targetable (Val600E). Other commonly mutated pathways were the NFKB pathway in 24% of pts and the Cyclin D pathway, mutated in 16% of cases. Genes of the cereblon pathway (CRBN, IRF4, IKZF3), essential for the action of lenalidomide (len) were mutated in 5 Pts. One newly diagnosed and len refractory Pt harbored a subclonal CRBN mutation (Asn316Lys, allelic fraction [AF] 20%) as well as 4 clonal IRF4 mutations within the DNA binding domain of the gene (Lys89Asn (AF 78%), Lys77Glu, Ser48Arg and Cys49Ser (AF 80% each). Another IRF4 mutated (Lys123Arg, AF 90%) hemodialysis dependent Pt was responsive to dose adapted treatment of len and dexamethasone (dex). Mutations in the C2H2-type 3 region of IKZF3 in a len resistant (Gly191Arg, AF 48%) pt occured as well as in a len and dex responsive (Arg242Gly, AF 52%) Pt. Furthermore, we identified a truncating XBP1 mutation (p.Leu232*, 97% AF) in a Pt refractory to bortezomib. One patient harbored a mutation in the steroid receptor NR3C1 (Pro255Leu, 22% variant reads). Finally, integrated mutation and copy-number data identified biallelic deletions of CDKN2C and FAM46C in a multi drug refractory Pt. Mutation and/or copy-number losses in both alleles were found in DIS3 in 4 Pts, in CYLD, TP53 and TRAF3 in 2 Pts and in CARD11, CDKN2A, CDKN1B, RB1and STAT3 in one Pt each. Conclusion: In summary, we report the initial results from 38 MM Pts using a custom MM sequencing panel which employs small amounts of DNA requiring very few cells and providing deep coverage in clinically meaningful timeframes. Our findings frequently include prognostically significant information, actionable targets and mutations in genes related to drug resistance. Targeted mutation profiling will likely become part of the clinical workup in MM and our M3P2.0 mutation panel is a suitable tool to provide information needed to guide precision therapy and to set the basis for individually tailored treatment decisions. 1. Kortuem KM. et al. Development Of a 47 Gene Sequencing Panel For Common Mutations Present In Multiple Myeloma: Preliminary Results In 77 p53 Deleted Patients, ASH 2013. Disclosures Stewart: Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Array BioPharma: Consultancy; Sanofi: Consultancy; Takeda Pharmaceuticals International Co.: Research Funding; Novartis: Consultancy. Bruins:OncoSpire: Salary support Other. Vasmatzis:OncoSpire: Salary Support Other. Kumar:Celgene: Consultancy, Research Funding; Millennium: The Takeda Oncology Co.: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Sanofi-Aventis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees. Fonseca:Medtronic, Otsuka, Celgene, Genzyme, BMS, Lilly, Onyx, Binding Site, Millennium, AMGEN: Consultancy, patent for the prognostication of MM based on genetic categorization of the disease. He also has sponsored research from Cylene and Onyx Other, Research Funding. Bergsagel:MundiPharma: Research Funding; OncoEthix: Research Funding; Constellation Pharmaceutical: Research Funding; Novartis: Research Funding.

Identification of Initiating Trunk Mutations and Distinct Molecular Subtypes: An Interim Analysis of the Mmrf Commpass Study

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 722-722 ◽  
Author(s):  
Jonathan J Keats ◽  
Gil Speyer ◽  
Legendre Christophe ◽  
Christofferson Austin ◽  
Kristi Stephenson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Copy Number ◽  
Interim Analysis ◽  
Research Funding ◽  
Molecular Subtypes ◽  
Bayesian Clustering ◽  
Clustering Method ◽  
Advisory Committees

Abstract The Multiple Myeloma Research Foundation (MMRF) CoMMpass trial (NCT145429) is a longitudinal study of 1000 patients with newly-diagnosed multiple myeloma from clinical sites in the United States, Canada, Spain, and Italy. Each patient receives a treatment regimen containing a proteasome inhibitor, immunumodulatory agent, or both. Clinical parameters are collected at study enrollment and every three months through the five-year observation period. To identify molecular determinants of clinical outcome each baseline and progression tumor specimen is characterized using Whole Genome Sequencing, Exome Sequencing, and RNA sequencing. This will be the first public presentation of the interim analysis seven cohort with 760 enrolled patients of whom 565 are molecularly characterized. This cohort of patients includes 14 patients with baseline and secondary samples along with 7 patients with characterized tumor samples from the bone marrow and peripheral blood. Although the median follow-up time for the cohort is only 260 days the patients on proteasome and IMiD based combinations are currently showing a PFS and OS benefit compared to those receiving combinations with each agent alone. From the raw mutational analysis we identified 24 significant genes that are recurrently mutated and the mutated allele is detectably expressed in all but one, DNAH5. Suggesting these mutations are likely contributing to myelomagenesis through an unconventional mechanism. Interestingly, DIS3 mutations are independent of KRAS, NRAS, and BRAF indicating a potential mechanistic link while PRKD2 mutations are associated with t(4;14). To identify events driving the initiation of myeloma we performed a detailed clonality analysis using a bayesian clustering method that corrects for copy number abnormalities and tumor purity to assign mutations into distinct clonal branches versus the initiating trunk mutations. On average 63.8% of mutations are trunk mutations and in 86.7% of patients at least one trunk mutation is associated with somatic hypermutation of an immunoglobulin gene as expected in a late stage B-cell malignancy. This identified many expressed trunk mutations that did not come out in the classic significance analysis like ATM, EGR1, and CCND1. To identify molecular subtypes we performed unsupervised clustering using a consensus clustering approach on independent discovery and validation cohorts, which identified 12 distinct subtypes, using a combination of silhouette score and cumulative distribution of consensus scores. This analysis identified two distinct groups associated with t(4;14) with mutations in FGFR3 and DIS3 being exclusive to one subgroup. In addition, this analysis separates patients with cyclin D translocations into three different groups, with one group having the second lowest PFS proportion. Three patients without CCND1 or CCND3 translocations were found to have IgH translocations targeting CCND2. The MAF subgroup was associated with the lowest OS and PFS proportion, and the three MAF/MAFB translocation negative patients in the subgroup all had MAFA translocations. The remaining 6 subgroups are associated with hyperdiploid copy number profiles and harbor the majority of the IgH-MYC translocation events. Two of the hyperdiploid groups are associated with a low level of NFKB activation compared to the remaining four, one of these is defined by the highest proliferation index but paradoxically the other has the second worst OS proportion. Another group is enriched with FAM46C and NRAS mutations. The genomic profiles of the paired tumors isolated from the peripheral blood and bone marrow are highly similar indicating these are not genetically distinct tumor compartments, at least in this subset of seven patients. Applying our bayesian clustering method to the serial samples resolved additional clonal clusters as mutations with similar cancer cell fractions at diagnosis clearly diverged at later timepoints. These analyses have identified tumor initiating mutations and new subtypes of myeloma, which are associated with distinct molecular events and clinical outcomes. Disclosures Jagannath: Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Merck: Honoraria; Janssen: Honoraria. Siegel:Celgene Corporation: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Merck: Speakers Bureau. Vij:Takeda, Onyx: Research Funding; Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy. Zimmerman:Amgen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Onyx: Honoraria. Niesvizky:Celgene: Consultancy, Speakers Bureau. Rifkin:Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Consultancy, Membership on an entity's Board of Directors or advisory committees. Lonial:Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding.

Combined Inhibition of HDAC and AKT As a Strategy to Overcome Multi-Drug Resistance in Patients with Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4325-4325
Author(s):  
Mitsuhito Hirano ◽  
Yoichi Imai ◽  
Takahiko Murayama ◽  
Kota Sato ◽  
Junichi Yamamoto ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Monoclonal Antibodies ◽  
Mrna Expression ◽  
Cell Lines ◽  
Research Funding ◽  
Cytotoxic Effect ◽  
Hdac Inhibitors ◽  
Pharmaceutical Research ◽  
Adcc Activity

Nowadays, patients with multiple myeloma (MM) have multiple choices of therapy including monoclonal antibodies, proteasome inhibitors, and immunomodulatory drugs (IMiDs), whereas some patients still develop resistance to these drugs and require novel therapeutic modalities. Here, we focused on inhibition of HDAC and AKT to overcome drug resistance. Lenalidomide (Len) selectively binds to cereblon (CRBN), which mediates recruitment of specific substrates like IKZF1 to E3 ubiquitin ligase and subsequent degradation, resulting in downregulation of IRF-4 and c-Myc. Then, we developed Len-resistant myeloma cells by RNAi-mediated downregulation of CRBN. Treatment of these cells with HDAC inhibitors reduced IKZF1 mRNA, suggesting potential efficacy of HDAC inhibitors against CRBN-low expressing or mutated MM. According to the integrated database for expression profile and disease prognosis (GenomicScape, http://www.genomicscape.com), higher expression of MICA was significantly associated with better overall survival in MM. MICA is an NK cell-activating ligand and plays an important role in ADCC. We observed that ADCC activity of both daratumumab and elotuzumab against MM cells was enhanced in the presence of HDAC inhibitors, which was compatible with our previous data that HDAC inhibitors upregulated MICA mRNA expression via inhibition of IKZF1 (ASH2018 abstract #4435). We also observed that HDAC inhibitors upregulated MICA mRNA in CRBN-deficient cells, suggesting promise of the combination of HDAC inhibitors and monoclonal antibodies against Len-resistant MM. Len-resistance is also affected by phosphorylation status of GSK-3. PI3K/AKT pathway is frequently activated in MM cells, and AKT inactivates GSK-3 by direct phosphorylation, resulting in c-Myc stabilization. Enhanced phosphorylation of GSK-3 was observed in CRBN-deficient H929 cells after long-term culture with Len, and such a phosphorylation status of GSK-3 was correlated with less CRBN amount and higher Len concentration (Figure 1). Afuresertib, an AKT inhibitor, suppressed GSK-3 phosphorylation (p-GSK-3) with or without ACY-1215, an HDAC inhibitor, leading to a substantial decrease of c-Myc (Figure 2). On the other hand, CHIR 99021, a GSK-3 inhibitor, partially counteracted to cytotoxic effect of afuresertib on H929 cells (Figure 3). These results suggest that increased p-GSK-3 is involved in acquired Len-resistance, and that combined inhibition of HDAC and AKT can overcome Len-resistance through decreased p-GSK-3. Furthermore, we examined the efficacy of CUDC-907, a dual HDAC and PI3K inhibitor. CUDC-907 had a cytotoxic effect on the MM cell lines including those had low CRBN expression. Bortezomib, doxorubicin, and dexamethasone resistant MM cell lines were also sensitive to CUDC-907. CUDC-907 upregulated MICA mRNA expression, but downregulated IKZF1 mRNA expression. Treatment of RPMI-8226 cells with CUDC-907 enhanced the ADCC activity of daratumumab (Figure 4). Furthermore, CUDC-907 was effective on primary MM cells which were resistant to bortezomib and Len (Figure 5). Thus, dual inhibition of HDAC and AKT with or without monoclonal antibodies is a promising therapeutic approach to multi-drug resistant MM. Disclosures Imai: Celgene: Honoraria, Research Funding; Janssen Parmaceutical K.K.: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Futami:Torii Pharmaceutical: Research Funding. Ri:Janssen Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Sanofi: Honoraria, Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; MSD: Research Funding; Novartis Pharma: Research Funding; Gilead Sciences: Research Funding; Astellas Pharma: Research Funding; Teijin Pharma: Research Funding. Yasui:TokioTHERA Holdings, Inc.: Equity Ownership. Iida:Teijin Pharma: Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Astellas: Research Funding; Gilead: Research Funding; Sanofi: Research Funding; MSD: Research Funding; Abbvie: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Daichi Sankyo: Honoraria, Research Funding. Tojo:Torii Pharmaceutical: Research Funding; AMED: Research Funding.

Disruption of DEPTOR/mTORC1/mTORC2 Signaling Cascade Using a Novel Selective mTOR Kinase Inhibitor AZD8055 Results In Growth Arrest and Apoptosis In Multiple Myeloma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 791-791 ◽  
Author(s):  
Diana Cirstea ◽  
Teru Hideshima ◽  
Loredana Santo ◽  
Samantha Pozzi ◽  
Sonia Vallet ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Growth ◽  
Cell Survival ◽  
Board Of Directors ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Advisory Committees ◽  
Mtor Kinase

Abstract Abstract 791 Targeting PI3K/Akt/mTOR signaling is among one of the promising therapeutic strategies in multiple myeloma (MM), since it facilitates MM cell survival and development of drug resistance in the context of the bone marrow microenvironment. Specifically, regulation of PI3K activity, which mediates MM cell growth and drug resistance, by mTOR complex 1 (mTORC1) provides the rationale for use of rapamycin analogs for MM treatment. However, rapamycin alone fails to overcome bone marrow-induced proliferation of MM cells, at least in part, because of the mTORC1-dependent feedback loops which activate PI3K/Akt. More recently, extensive studies of the mTOR network have identified mTORC2 as a “rapamycin-insensitive” complex. Sharing mTOR kinase as a common catalytic subunit, mTORC1 and mTORC2 mediate two distinct pathways: mTORC1 controls cell growth by phosphorylating key regulators of protein synthesis S6 kinase 1 (P70S6K) and the eIF-4E-binding protein 1 (4E-BP1); mTORC2 modulates cell survival and drug resistance by phosphorylating target proteins including Akt and serum/glucocorticoid regulated kinase 1(SGK1)/N-myc downstream regulated 1 (NDRG1). Moreover, studies have also revealed overexpression of a novel mTOR-interacting protein DEP domain containing 6 (DEPTOR), which can modulate mTOR activity and promote PI3K/mTORC2 signaling in primary MM tumor cells and in MM cell lines while mTORC1 remains silenced. We therefore hypothesized that targeting mTOR may disrupt DEPTOR/mTOR interaction and silence mTORC1/mTORC2 signaling, thereby overcoming mTOR resistance in MM cells. To confirm this idea, we used AZD8055, an orally bioavailable selective ATP-competitive mTOR kinase inhibitor, in our MM preclinical models. AZD8055- treatment of MM.1S inhibited phosphorylation of both mTORC1 and mTORC2 substrates: P70S6K; 4E-BP1 including the rapamycin-resistant T37/46 – downstream targets of mTORC1; as well as Akt and NDRG1 – effectors of mTORC2 refractory to rapamycin. Interestingly, AZD8055-mediated mTORC1/mTORC2 downregulation was associated with DEPTOR upregulation, which is consistent with the finding that DEPTOR expression is negatively regulated by mTORC1 and mTORC2. Moreover, inhibition of mTORC1 alone by rapamycin resulted in reduction of DEPTOR, associated with Akt activation. Furthermore, we observed that DEPTOR expression was decreased in MM.1S cells cultured with IL-6, IGF-1 or bone marrow stromal cells (BMSCs), which stimulate PI3K/Akt/mTOR signaling, evidenced by enhanced P70S6K and Akt phosphorylation. Unlike rapamycin, AZD8055 reversed those effects and inhibited MM.1S proliferation, even in the presence of these cytokines or BMSCs. AZD8055-induced growth inhibition was associated with apoptosis, evidenced by caspase-9, -3 and PARP cleavage in a time-dependent fashion (80% apoptotic cells at 72 hour culture as detected by Annexin V/PI staining). Moreover, AZD8055 induced cytotoxicity even in rapamycin resistant MM cell lines and primary patient MM cells. Finally, AZD8055 demonstrated significant anti-MM activity in an in vivo human MM cell xenograft SCID mouse model. Taken together, our data show that disruption of DEPTOR/mTORC1/mTORC2 cascade in MM cells results in significant anti-tumor effects, providing the framework for future clinical trials of AZD8055 to improve patient outcome in MM. Disclosures: Guichard: AstraZeneca: Employment, Shareholder AstraZeneca. Anderson:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Onyx: Consultancy; Merck: Consultancy; BMS: Consultancy; Acetylon: Membership on an entity's Board of Directors or advisory committees, Ownership interest (inc stock options) in a Start up company. Raje:AstraZeneca: Research Funding; Acetylon: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Presence of Extrachromosomal DNA (ecDNA) Impacts Both Progression Free and Overall Survival and Is an Independent Poor Prognostic Marker in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 461-461
Author(s):  
Parth Shah ◽  
Anil Aktas-Samur ◽  
Mariateresa Fulciniti ◽  
Raphael Szalat ◽  
Masood A. Shammas ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Overall Survival ◽  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
P Value ◽  
Newly Diagnosed ◽  
Extrachromosomal Dna ◽  
Publicly Traded ◽  
Advisory Committees

Abstract Background Focal amplifications and rearrangements drive tumor growth and evolution in cancer. Focally amplified regions often involve the juxtaposition of rearranged segments of DNA from distinct chromosomal loci into a single amplified region and nearly half of these regions can be explained by circular, extrachromosomal DNA (ecDNA) formation. Cancer-associated ecDNA shows a unique circular placing ecDNA at the interface of cancer genomics and epigenetics. As formation of ecDNA represents a manifestation of genomic instability, we have investigated presence and prognostic impact of ecDNA in multiple myeloma (MM). Methods Whole genome (WGS) and transcriptome (RNAseq) sequencing data from CD138 purified MM cells from 191 uniformly-treated newly diagnosed MM patients were used for this analysis. Copy number variants (CNV), single nucleotide variants (SNV) and structural variants (SV) were identified on all WGS samples using Facets, Mutect2 and Manta. Seed data from these CNV results was passed to the AmpliconArchitect tool to determine presence of focally amplified and rearranged segments of DNA. Seed CNV thresholds were set for a minimum CNV size of 100kb and a copy number of equal or greater to 5. Extrachromosomal calls were then annotated using the Amplicon Classifier to determine the presence of ecDNA. Multivariate survival analysis was performed after segregating samples into the conventional myeloma risk classifications including translocations, copy number alterations, ISS, age and mutations associated with risk. Differential expression analysis was performed on transcriptomic data using DEseq2. Results We identified 6.8% of the newly diagnosed patients with ecDNA, 12.5% with complex non-cyclic DNA amplifications and 10.1% with linear amplifications. ecDNA and complex events were targeting MM dependent genes, including MYC/PVT1, IRF4 as well as known driver genes such as CDYL and TRAF2. We further evaluated association between ecDNA, complex rearrangements, linear amplification and patients with none of these amplification types and found that patients with ecDNA had significantly poor PFS (median PFS 22 months vs. 41 months) and OS (median OS 41 months vs. 105 months). Patients having ecDNA in their MM cells did not show any significant enrichment for known translocations, double hit or TP53 mutations. In a multivariate model including ecDNA and all other known MM risk features, ecDNA was found to be an independent predictor of progression free survival.(HR 2.6, CI: 1.26 -5.6, p=0.0082) and overall survival (HR 7.94 CI:3.5-17.9 p < 0.0001). Patients with ecDNA have higher mutational load probability(8798 vs 6982, effect size = 0.64 , probability is 91.1). However, this was not reflected in heterogeneity by using MATH score. We found that patients with ecDNA are likely to have BRAF mutations (OR= 25.07 [2.57 - 330 95% CI], p value = 0.002), however overall RAS/RAF pathway mutations were similar to other patients. Patients with ecDNA showed fragile DNA with more breaks (median segments 197 vs. 125.5, p value = 0.001). Although ecDNA is defined as copy number gain with fragments having 5 or more copies, overall genomic gain between ecDNA and other patients were similar. However, overall genomic loss in patients with ecDNA were higher than others (7% vs. 4.2%, p = 0.06). By differential gene expression analysis we noted 98 differentially expressed genes in MM cells with ecDNA. The downregulated geneset involved pathways responsible for cell death as well as the RAS pathway. Interestingly, CD38 was upregulated in the ecDNA dataset suggesting greater potential for CD38 targeting therapies in these patients. Conclusions ecDNA, as an unique marker of perturbed genomic integrity, is observed in a subset of patients and is an independent prognostic marker in newly diagnosed MM patients. As patients with ecDNA are not fully captured by other risk features its incorporation in an expanded definition of a high risk group of multiple myeloma should be investigated. Future studies will endeavor to explore the biological mechanism through which ecDNA are formed and influences outcomes in myeloma. Figure 1 Figure 1. Disclosures Richardson: Sanofi: Consultancy; GlaxoSmithKline: Consultancy; Karyopharm: Consultancy, Research Funding; AstraZeneca: Consultancy; AbbVie: Consultancy; Oncopeptides: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy; Protocol Intelligence: Consultancy; Celgene/BMS: Consultancy, Research Funding; Secura Bio: Consultancy; Regeneron: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding. Perrot: Abbvie: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Moreau: Abbvie: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Sanofi: Honoraria; Celgene BMS: Honoraria; Oncopeptides: Honoraria. Thakurta: Oxford University: Other: Visiting Professor; BMS: Current Employment, Current equity holder in publicly-traded company. Anderson: Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Munshi: Legend: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Adaptive Biotechnology: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Celgene: Consultancy; Pfizer: Consultancy.

Prospective Investigation of Genetic Alterations in Osteolytic Lesions Compared to Paired Random Aspirates

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4419-4419
Author(s):  
Sandra Sauer ◽  
Jens Hillengass ◽  
Barbara Wagner ◽  
Daniel Spira ◽  
Marc Andre Weber ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Iliac Crest ◽  
Genetic Alterations ◽  
Advisory Committees ◽  
Single Nucleotide ◽  
Osteolytic Lesions ◽  
Myeloma Cells

Abstract Background: Bone disease is the most frequent clinical manifestation of multiple myeloma. In this prospective study we ask whether osteolytic lesions (OL) are driven by myeloma cells showing a different background of genetic alterations in terms of chromosomal aberrations and expressed single nucleotide variants (SNVs) compared to random aspirates (RA) from diffuse myeloma cell infiltration at the iliac crest (spatial genetic heterogeneity). Material and Methods: Consecutive sample-pairs (n=41) were prospectively obtained by CT-guided biopsies of OLs as well as simultaneous random bone marrow aspirates of the iliac crest, the latter undergoing CD138-purification of myeloma cells, in transplant eligible patients with previously untreated symptomatic multiple myeloma, after written informed consent. Peripheral blood mononuclear cells were used as germline control. Plasma cell infiltration in biopsies was quantified histologically. Samples pairs (n=8) were subjected to RNA-sequencing (Illumina HiSeq2000), gene expression profiling using DNA-microarrays (Affymetrix U133 2.0), whole exome sequencing (Illumina NextSeq 500), and arrayCGH (Affymetrix cytoscan array). Results and Discussion: Expressed single nucleotide variants.The spectrum of mutated genes in our samples comprises two of the most frequently mutated in symptomatic myeloma, i.e. KRAS and FAM46C, alongside those implicated in myeloma pathophysiology, e.g. mutations in IRF4, FGFR3, and CD200. In total, 1-10 clonal expressed non-synonymous SNVs were exclusively found in OL compared to RA, comprising e.g. WHSC1, FAM46C, and ROCK1P1. In 2/8 patients (25%), no expressed clonal differences between RA and OL were present. Single nucleotide variants.In investigated samples, 77-1569 non-synonymous SNVs appear with an allele frequency of ≥10% in OL and RA, clustering in 4-5 groups. The clonal constitution can vary, but subclones are detectable in both. Subclonal complexity is maintained (subclones remain present) in OL compared to RA, and the vast majority of subclonal changes is present in both, especially for expressed non-synonymous SNVs, incompatible with an "osteolytic clonal variant" driving OL in the majority of patients. Copy number alterations and loss of heterozygosity.Subtle differences in copy number between OL and RA are present. However, only 1/8 patients (12.5%) showed further "gained" aberrations in OL compared to RA, i.e. deletions on chromosome 7p, 8p, and 11p as well as 19p gain. Loss of heterozygosity was observed in 3/8 patients (37.5%) with a shared pattern between OL and RA in all of them. Conclusions: In our prospective study, the majority of alterations is shared between RA and OL. Spatial heterogeneity is present, but nature and frequency of alterations detectable exclusively in OL make them unlikely candidates in most myeloma patients for being causative for generation of OL. Disclosures Hillengass: Novartis: Research Funding; Sanofi: Research Funding; BMS: Honoraria; Celgene: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Goldschmidt:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Durie:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

scholarly journals Mutations and Copy Number Changes Predict Progression from Smoldering Myeloma to Symptomatic Myeloma in the Era of Novel IMWG Criteria

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4456-4456
Author(s):  
Eileen M Boyle ◽  
Cody Ashby ◽  
Ruslana G. Tytarenko ◽  
Yan Wang ◽  
Michael A Bauer ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Copy Number ◽  
Research Funding ◽  
Targeted Sequencing ◽  
Advisory Committees ◽  
Kras Mutations ◽  
Free Survival ◽  
Bonferroni Adjustment ◽  
Smoldering Myeloma ◽  
Copy Number Changes

Abstract Introduction: Despite novel International Myeloma Working Group (IMWG) criteria, Smoldering Myeloma (SMM) remains a heterogeneous disease for which correctly identifying patients that will eventually progress to myeloma (MM) is essential. The genetic and molecular factors that underlie disease progression are not well elucidated, therefore, we examined samples from SMM patients in order to identify molecular determinants of progression. Methods: CD138-sorted and control samples from 77 non-treated SMM patients according to IMWG 2014 underwent targeted sequencing and gene expression profiling (GEP). The median follow-up was 4.81 years (95% CI: 4.19-6.16). Targeted sequencing consisted of 140 genes and additional regions of interest for copy number, as well as tiling of the immunoglobulin and MYC loci for detection of translocations and was performed on a NextSeq500 using 75 bp paired end sequencing. Results were aligned to the hg19 genome and mutations, translocations and copy number were determined. Nonnegative matrix factorization (NMF) (NMF package in R) was used to identify mutation signatures. The median mean coverage was 365 (88-696) and 783 (161-1559) for translocations (Tx) and mutations respectively. We compared these samples to 199 newly diagnosed MM samples. Results: Significant differences in the frequencies of mutated genes were seen, including fewer NRAS, KRAS, FAM46C, LRRK2 and TP53 mutations and more PCLO and MAFB mutations than expected in comparison to MM (p<0.05). Regarding structural changes, there was no difference in the incidence of Tx (including those involving MAF and MAFB) but significantly fewer del(1p), del(12p) and del(14q) cases in SMM (p<0.05). There was no difference in the incidence of MYC translocations (19% of cases) and MYC rearrangements (23%). The 4-year progression rate was 25 percent. The presence of KRAS mutations (n=9) and del(6q) (n=11) were statistically associated with shorter progression free survival (PFS) [median 49m (26-∞) vs 147m (67.6-∞) and 26m (9.6-∞) vs 147m (80-∞) for KRAS and del(6q) respectively] and treatment free survival (TFS) [median 6m (9.6-∞) vs 19m (9.6-∞) and 9 m (13.4-∞) vs 16m (13.4-∞) for KRAS and del(6q) respectively]. MYC alterations and NF-κB alterations (BIRC2 and BIRC3 loss, TRAF2 and TRAF3 mutation or gain, CYLD loss, MAP3K14 mutations) did not influence progression. There were no double-hit patients in this cohort defined by bi-allelic-TP53 or ISS III with amp(1q). Ten percent of patients were identified as high-risk according to GEP4 risk-score. Pearson correlation was performed between patients that progressed (n=24) against those who did not (n=53) for genetic events with n≥6. Del(6q) [χ2=0.32, p=0.004], LRP1B [χ2=0.27, p=0.015] and KRAS mutations [χ2=0.28, p=0.01] were positively correlated to progression, but only del(6q) remained significant after Bonferroni adjustment. Of particular interest, we did not identify the APOBEC mutational signature in the t(14;16) SMM samples, which is heavily associated with a poor prognosis in t(14;16) MM (4/11 in MM and 0/5 in SMM). Discussion: As previously reported, copy number changes, Tx and mutations predate MM. The lower frequencies of copy number changes and mutations suggest an ongoing process whereby cells acquire successive events eventually leading to MM. KRAS and del(6q) were significant predictors of both PFS and TFS with hazard ratios of 2.8 and 3.71, respectively. We comprehensively analyzed both the NF-κB pathway mutations and copy number changes, that did not bear, unlike previous reports, any clear relationship to PFS. Although we are limited by the power of this analysis, this supports the idea that the NF-κB dependency preexists symptomatic myeloma and is present throughout disease stages. Further analysis of the NF-κB 11-gene signature expression are ongoing. This is the first broad analysis of both MYC rearrangements and Tx in SMM. Previous studies have focused on FISH analysis of IGH-MYC Tx that underestimate the extent of MYC rearrangements present. Finally, our data also shows that absence of an APOBEC signature in SMM may account for the rather indolent phenotype of MAF and MAFB Tx in comparison to MM. Conclusion: KRAS mutations as well as del(6q) were associated with shorter PFS and TFS in this dataset. The absence of APOBEC signature may explain part of the indolent phenotype of the MAF and MAFB translocation SMM patients. Disclosures Boyle: Gilead: Honoraria, Other: travel grants; Amgen: Honoraria, Other: travel grants; Celgene: Honoraria, Other: travel grants; Abbvie: Honoraria; Takeda: Consultancy, Honoraria; La Fondation de Frace: Research Funding; Janssen: Honoraria, Other: travel grants. Facon:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Dumontet:Janssen: Honoraria; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding; Sanofi: Honoraria. Morgan:Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding. Davies:Abbvie: Consultancy; MMRF: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; TRM Oncology: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; ASH: Honoraria; Janssen: Consultancy, Honoraria.

scholarly journals COVID-19 in Patients with Hematologic Malignancies: A Single-Center Experience

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 36-37
Author(s):  
Lindsay Wilde ◽  
Adam F. Binder ◽  
Matthew Carabasi ◽  
Joanne Filicko-O'Hara ◽  
John L. Wagner ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Research Funding ◽  
Pharmaceutical Research ◽  
B Cell Lymphoma ◽  
Hematologic Malignancies ◽  
Cell Transplant ◽  
Advisory Committees ◽  
Presenting Symptoms ◽  
Oncology Research

Introduction :The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and subsequent coronavirus-19 (COVID-19) pandemic has impacted hematologic malignancies (HM) care worldwide. Reported risk factors for severe COVID-19 presentation include older age, medical comorbidities, and cardiac disease - many of which apply to patients with HM (Guan et al., 2020; Zhou et al., 2020). Additionally, patients with HM may be at even higher risk of infections with or complications from SARS-CoV-2 due to immune dysfunction from their underlying disease or treatment (He et al., 2020). However, data regarding rates of infection and outcomes in this population are limited. Here we describe the demographic characteristics, coexisting conditions, presenting symptoms, treatment, and outcomes of a cohort of patients with HM and COVID-19 infection at network sites across the Sidney Kimmel Cancer Center- Jefferson Health. Methods : We created an HM-specific COVID-19 database within our health system. Patients were identified for inclusion in the database by physician referral and query of the electronic medical record. Epidemiological, clinical, and laboratory data, therapy details, and outcomes on patients were obtained by accessing electronic medical records. A retrospective study of patients with a diagnosis of a HM- within 5 years of COVID-19 diagnosis-and a confirmed diagnosis of COVID-19 were was conducted using this database. A confirmed diagnosis of COVID-19 was defined as a positive result on a real-time RT-PCR assay of a specimen collected by nasopharyngeal swab. Results: More than 3,000 telehealth or in-person patient visits were conducted for patients with HM in the Jefferson Health Network between March 9, 2020 and July 15, 2020. During that period, 21 patients with HM had a confirmed diagnosis of COVID-19. Median age was 67 years (range 21-89). The majority of patients (86%) had at least 1 comorbid medical condition, and 76% had a history of tobacco use. The most common HM was multiple myeloma (7/21, 33%), followed by diffuse large B-cell lymphoma (3/21, 14%). 12/21 (52%) patients were on active cancer treatment at the time of COVID-19 diagnosis, and patients had received a median of 2 lines of cancer therapy (range 0-6). All 12 patients who were on active therapy at the time of COVID-19 diagnosis experienced a treatment interruption. Two patients had undergone prior autologous stem cell transplant (SCT) and 1 had undergone prior allogeneic SCT. Details on HM diagnosis and treatment are presented in Table 1. Twenty patients required hospital admission at the time of COVID-19 diagnosis, 7/21 were admitted to the ICU, and 6/21 required intubation. The most common presenting symptoms were fever (48%), cough (43%), and shortness of breath (43%), and lymphopenia (absolute lymphocyte count (ALC) &lt;1,000 B/L) was common at presentation (56%). More than half (13/21, 62%) of patients received some COVID-19 directed therapy, while 8 were treated with supportive care alone. As of July 15, 2020 18/21 (86%) patients were alive. Characteristics of the 3 patients who died are described in Table 2. Conclusion : In contrast to published reports, we found that the number of confirmed COVID-19 in patients with HM at our center was surprisingly low, with only 21 cases in 4 months. Furthermore, the mortality rate of 14% was lower than expected when compared to published cohorts of similar patients, which have shown mortality rates as high as 40% (He et al., 2020; Malard et al., 2020; Martín-Moro et al., 2020). Postulated reasons for the low number of infections include the early adoption of universal masking and robust utilization of telehealth to promote social distancing. In our small cohort, multiple myeloma was the most frequent HM diagnosis associated with COVID-19 infection, but this may be related to the prevalence of MM in our geographic area. The vast majority of HM patients with symptomatic COVID-19 were former smokers. Disclosures Binder: Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy. Alpdogan:Seattle Genetics: Consultancy; Kiowa Kirin: Consultancy. Kasner:Otsuka Pharmaceutical: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Outschoorn:Otsuka Pharmaceutical: Research Funding. Palmisiano:Genentech: Research Funding; AbbVie: Research Funding. Flomenberg:Tevogen: Consultancy, Honoraria. Porcu:Cell Medica: Research Funding; Daiichi: Consultancy, Honoraria; Galderma: Research Funding; Innate Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kiowa Kirin: Research Funding; Kura Oncology: Research Funding; Miragen: Research Funding; Verastem: Consultancy; Viracta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding.

scholarly journals Hexokinase-2 Regulates Hypoxia-Inducible Autophagy, Leading to Enhance Anti-Apoptotic Capability of Refractory Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1787-1787
Author(s):  
Sho Ikeda ◽  
Fumito Abe ◽  
Matsuda Yuka ◽  
Akihiro Kitadate ◽  
Takahiro Kobayashi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Lines ◽  
Research Funding ◽  
Pharmaceutical Research ◽  
Proteasome Inhibitors ◽  
Myeloma Cell ◽  
Hypoxic Stress ◽  
Myeloma Cells ◽  
Viable Cells

(background) The drug resistance of multiple myeloma (MM) cells is thought to be induced by various factors of the bone marrow microenvironment. Of these factors, hypoxic stress may be associated with drug resistance in various hematologic malignancies, including MM. Hypoxic stress lead MM cells to induce distinct gene expressions. It has been reported that oncogenic transcription factors such as IRF4 and Myc are suppressed under hypoxia. Instead, accumulation of another transcription factor, HIF-1α upregulates anti-apoptotic proteins, increases glycolysis, and enhances neovascularization leading MM cells to represent anti-apoptotic phenotype. Autophagy is an intracellular process that encapsulates cytoplasmic components, which are directed to the lysosome for degradation. Autophagy and proteasomal degradation prevent apoptosis caused by endoplasmic reticulum (ER) stress. Although proteasome inhibitor such as bortezomib, is a key drug for MM, it may induce treatment resistance. This might be because autophagy is induced in hypoxic microenvironment. Autophagy associated molecules might be therapeutic target in MM cells adapted to hypoxia. (Aim and methods) To clarify the association of hypoxia inducible genes and autophagy, and to obtain rational basis for a new therapeutic strategy against MM, we performed following experiments in vitro using myeloma cell lines (MM.1S, KMS-12-PE, KMS-11, and H929) and primary samples (n=6) that were subjected to hypoxia (1% O2). (Results) First, we examined volcano plot analysis on our cDNA microarray data (GSE80545) of patient samples incubated in normoxia or hypoxia for 48 hours. 546 probes were significantly elevated in hypoxia (fold change > 2.0, p < 0.05). Gene ontology analysis revealed that "Glycolytic Process" contained 13 genes such as PFKFB4, ENO2, ALDOC, PFKFB3, HK2, PFKP, GPI, PGK1, LDHA, ALDOA, ENO1, PKM, and GAPDH. We focused on hexokinase-2 (HK2) because it has been reported that HK2 activates autophagy under stress conditions. Western blot analysis for patient samples revealed that HK2 expression was remarkably upregulated under hypoxia. Apoptosis assay showed that viable cells of HK2 knockdowned cell lines were significantly lower than that of control cells under hypoxia, but not under normoxia. Also, in hypoxia, we found that number of 3-bromopyruvate (3-BrPA, a HK2 inhibitor) subjected viable cells were significantly lower than that of normoxia. This suggested that HK2 contributes to anti-apoptotic phenotype of MM cells under hypoxia. Next, we examined the role of HK2 in autophagy under hypoxia. Because degradation of p62 and increase of LC3-II/LC3-I ratio is considered to be useful for autophagy detection, we examined these factors by Western blot analysis. We found that hypoxic stress decreased expression of p62 and increased the ratio of LC3-II/LC3-I in myeloma cell lines, indicating that hypoxia activates autophagy. However, under hypoxia, these changes were canceled by HK2 knockdown. We confirmed that the number of autophagosome were significantly decreased in HK2-knockdowned myeloma cells by electron microscopy analysis. These data suggested that HK2 is required for hypoxia-inducible autophagy in MM. Finally, we examined the effect of combined inhibition of HK2 and proteasome. In hypoxia, apoptosis by bortezomib was significantly increased in HK2-knockdowned myeloma cells when compared with control. Moreover, we found that the combination of 3-BrPA and bortezomib increased apoptotic cells in both normoxia and hypoxia. These results suggested that HK2-inhibition can induce apoptosis against MM cells with enhancement of sensitivity to proteasome inhibitors. (Conclusion) These results suggest that hypoxia induced HK2 promotes autophagy and inhibits apoptosis. Thus, the combination of proteasome inhibitors and HK2 inhibition may bring about a deep response against treatment resistant MM. Disclosures Ikeda: Nippon Shinyaku Research Grant: Research Funding. Takahashi:Bristol-Myers Squibb: Speakers Bureau; Eisai Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Chug Pharmaceuticals: Research Funding; Ono Pharmaceutical: Research Funding; Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; Asahi Kasei Pharma: Research Funding.

scholarly journals Inhibition of Sialylation Impairs Adhesion on Madcam-1 and E-Selectin and Sensitize Multiple Myeloma Cells to Bortezomib in a Xenograft Mouse Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3204-3204
Author(s):  
Alessandro Natoni ◽  
Mariah Farrell ◽  
Heather Fairfield ◽  
Lucy Kirkham-McCarthy ◽  
Matt Macauley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Cell ◽  
Sialyl Lewis X ◽  
Advisory Committees ◽  
Selectin Ligands

Abstract Introduction Multiple myeloma (MM) is a cancer of clonal plasma cells that hijack the bone marrow (BM) niche to create a drug resistant, incurable malignancy. Aberrant sialylation has been linked to immune cell evasion, drug resistance, and metastasis in cancer; indeed sialyltransferases, including ST3GAL1, ST3GAL4 and ST3GAL6, are aberrantly expressed in many cancers (Glavey et al., 2014). We have previously shown that targeting ST3GAL6 in MM cells inhibits their ability to extravasate and colonize the BM in mouse models (Glavey et al., 2014). Moreover, we also showed that a subpopulation of MM cells expresses functional E-Selectin ligands which, upon expansion, gives rise to a more aggressive disease and resistance to bortezomib in mice (Natoni et al., 2017). Based off these findings, we herein investigated whether inhibiting sialylation in E-selectin-enriched MM cells with 3Fax-Neu5Ac, a small molecule sialyltransferase inhibitor, could alter the ability of these cells to home in the BM and restore bortezomib sensitivity in vivo. We hypothesized that inhibiting homing of MM cells to the BM will improve survival and that co-treatment with bortezomib and 3Fax-Neu5Ac will have a synergistic effect. Methods E-selectin ligands enriched MM1S cells (either positive or negative for GFP/Luciferase) were derived from parental cells by cell sorting using the HECA-452 antibody, which recognize sialofucosylated E-selectin ligands. We then determined the 3Fax-Neu5Ac dose and exposure times needed to decrease sialylation on these MM cells without causing toxicity. HECA-452-enriched MM1S cells were pretreated with 3Fax-Neu5Ac or vehicle for 7 days before being injected into SCID-beige mice and then treated with vehicle or bortezomib (0.3 mg/kg twice a week). Mice were analyzed via bioluminescence imaging (BLI) to monitor tumor progression and weighed twice a week. Mice were euthanized when they began to show paralysis under our IACUC protocol. 3Fax-Neu5Ac pretreated HECA-452 MM1S cells were also tested in vitro for their ability to adhere and roll on VCAM-1, MAdCAM-1 and E-Selectin under shear stress and to respond to bortezomib in co-culture with HS5 cells. Results Treatment of HECA-452 MM1S cells with 3Fax-Neu5Ac, at 300 μM for 7 days significantly reduced sialylation on these cells. Importantly, reducing sialylation with 3Fax-Neu5AC reduced tumor burden and increased survival, although this did not reach significance for survival (Figure 1A). Both vehicle- and 3Fax-Neu5Ac-treated cells significantly responded to bortezomib in the first 5 weeks of the in vivo study (Figure 1B). However, the HECA-452 MM1S cells did not show increased survival when treated with bortezomib suggesting an acquired mechanism of resistance in vivo. Importantly, pretreatment of the HECA-452 MM1S with 3Fax-Neu5Ac could improve survival of these mice preventing bortezomib resistance. In vitro, the HS5 stromal cells protected the HECA-452 MM1S cells from bortezomib and pretreatment with 3Fax-Neu5Ac partially reverted this protection. Moreover, the HECA-452 MM1S cells pretreated with 3Fax-Neu5Ac displayed reduced adhesion on MAdCAM-1 and E-selectin. Conclusions Sialylation plays an instrumental role in bone homing, BM colonization, and drug resistance of MM cells. Pretreatment of HECA-452 MM1S cells with 3Fax-Neu5Ac decreased their sialylation, restored sensitivity to bortezomib in vivo and prolonged survival in mice. This is likely because 3Fax-Neu5Ac pretreatment has multiple effects on MM cells including reducing cell adhesion mediated-drug resistance and adhesion to key molecules involved in BM homing such as MAdCAM-1 and E-selectin. The reduced adhesion on E-selectin is most likely due to the disruption of E-selectin ligands on the surface of MM cells as they require Sialyl Lewis X to function. Notably, we also found that de-sialylation impairs adhesion on MAdCAM-1 (3Fax-Neu5Ac vs DMSO P=0.038) which, together with E-selectin, is another critical BM homing receptor. This data suggests for the first time that sialylation may controls the affinity of integrin α4β7 and its counter-receptor MAdCAM-1. In turn, this would reduce BM homing and increase MM cells in the circulation were they are more prone to the cytotoxic effects of bortezomib. This study supports the importance of targeting sialylation in MM and provides a strong rationale for further clinical translation of this novel approach. Disclosures O'Dwyer: Glycomimetics: Research Funding; Celgene: Research Funding; BMS: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Evolution of Genomic Changes and Their Significance in Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 605-605
Author(s):  
Masood A Shammas ◽  
Herve Loiseau ◽  
Cheng Li ◽  
Leutz Buon ◽  
Samir B Amin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Clinical Outcome ◽  
Genomic Instability ◽  
Copy Number ◽  
Research Funding ◽  
Chromosome 1 ◽  
Poor Clinical Outcome ◽  
Genomic Changes ◽  
Aggressive Phenotype ◽  
Copy Number Changes

Abstract Abstract 605 Despite therapeutic advances and high response rates, most if not all patients with multiple myeloma (MM) develop drug resistance and relapse and curative outcomes remain elusive. A prominent feature of MM is striking genomic instability that evolves with the progression of disease. This genomic instability is considered responsible for development of aggressive phenotype associated with relapsed disease and for development of drug resistance. The molecular basis for the generation of this genetic diversity in cancer cells thus has important implication in understanding cancer progression and therapy. However the genomic evolution in MM patient samples has not been documented. Here, we have utilized single nucleotide polymorphism (SNP) arrays to monitor genome-wide changes in heterozygosity and copy number, in two CD138+ MM cell samples collected 6 months apart from 14 MM patients. Genomic changes acquired in the late tumor samples were identified using early samples as baseline. We defined an event as detectable change in heterozygosity/copy number in three or more consecutive SNPs. All 14 MM patients acquired new genomic change in the later sample at a frequency ranging from 0.021 - 2.674 % (i.e. per 100 informative loci investigated). Although the rate of mutation varied, 12 out of 14 patients had acquired >100 mutational events. Chromosomes 1, 13, and × showed large areas of copy number change in several patients. We also evaluated if genomic changes correlate with changes in expression of corresponding genes. Selecting larger areas of genome, we observed that copy number changes correlate well with the change in expression of genes in these areas. As expected, we also observed a correlation among changes in copy number, heterozygosity, and gene expression at several chromosomal loci. In a number of instances frequently recurrent changes were observed. For example, recurrent copy number changes in areas spanning 1q42.13-1q44 and 1p12-1p12 of chromosome 1 were seen in majority (12 out of 14 and 13 out of 14) patients, whereas copy number changes in the p arm of chromosome × were present in all patients. Similarly the region of chromosome × spanning xq42.13-xq44 showed change in heterozygosity in majority of patients. We also observed that some of the newly acquired changes in late samples correlated with genomic markers of poor clinical outcome. We evaluated prognostic significance of these changes in 192 uniformly treated patients with MM with genomic gains and losses data from SNP array and survival information. Changes in chromosomal regions 1p12 and xp22.1-xp22.33 frequently observed in late samples were significantly (p = 0.017 and 0.037) associated with poor survival in these patients. These data suggest that MM cells acquire changes associated with aggressive phenotype and shorter survival. In conclusion we observe that MM patients acquire genomic changes at a very high rate; and certain chromosomal regions are more vulnerable predicting poor clinical outcome. These data also suggest a need to target mechanisms mediating genomic instability for therapeutic application. Disclosures: Richardson: Keryx Biopharmaceuticals: Honoraria. Anderson:Millenium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

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