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

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.

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Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v118.21.136.136 ◽

2011 ◽

Vol 118 (21) ◽

pp. 136-136

Author(s):

Ze Tian ◽

Jian-Jun Zhao ◽

Jianhong Lin ◽

Dharminder Chauhan ◽

Kenneth C. Anderson

Keyword(s):

Multiple Myeloma ◽

Tumor Suppressor ◽

Board Of Directors ◽

Expression Profiling ◽

Research Funding ◽

Advisory Committees ◽

Myeloma Cells ◽

Investigational Agent ◽

Reporter Activity

Abstract Abstract 136 Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells Ze Tian, Jianjun Zhao, Jianhong Lin, Dharminder Chauhan, Kenneth C. Anderson Medical Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, 02115 MicroRNAs (miRNAs) are 19–25 nucleotide-long noncoding RNA molecules that regulate gene expression both at the level of messenger RNA degradation and translation. Emerging evidence shows that miRNAs play a critical role in tumor pathogenesis by functioning as either oncogene or tumor suppressor genes. The role of miRNA and their regulation in response to proteasome inhibitors treatment in Multiple Myeloma (MM) is unclear. Here, we utilized MLN9708, a selective orally bio-available proteasome inhibitor to examine its effects on miRNA alterations in MM.1S MM cells. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to its biologically active form MLN2238. Our previous study using both in vitro and in vivo models showed that MLN2238 inhibits tumor growth and triggers apoptosis via activation of caspases. Moreover, MLN2238 triggered apoptosis in bortezomib-resistant MM cells, and induced synergistic anti-MM activity when combined with HDAC inhibitor SAHA, dexamethasone, and lenalidomide. In the current study, we treated MM.1S cells with MLN2238 (12 nM) for 3 hours and harvested; total RNA was subjected to miRNA profiling using TaqMan® Array Human miRNA A-Card Set v3.0 and the data was analyzed using dChip analysis. Results showed that MLN2238 modulates miRNA expression with a total of 36 miRNA changing their expression profiling (δδCT>1.5 or δδCT <-1.5; 19 were upregulated and 17 showed a downregulation). Among all miRNA, miR-33b was highly (δδCT>7) upregulated in response to MLN2238 treatment. We therefore hypothesized that miR-33b may play a role in MM pathogenesis as well as during MLN2238-induced proteasome inhibition in MM cells. We first utilized quantitative polymerase chain reaction (q-PCR) to validate the changes in miRNA expression profiling. Results confirmed that MLN2238 treatment triggers significant increase in the miR-33b expression in MM.1S cells (2.1 and 2.2 folds at 3h and 6h, respectively; P<0.001). Examination of normal PBMCs and plasma cells showed higher expression of miR-33b than patient MM cells (P<0.001). We further investigated the functional role of miR-33b in MM cells at baseline and during MLN2238 treatment. Drug sensitivity, cell viability, apoptosis, colony formation, and migration assays were performed using cell TilTer-Glo, Annexin V-FITC/PI staining, MTT staining, and Transwell assays, respectively. Signaling pathways modulated post miR-33b overexpression were evaluated by q-PCR, immunoblot, and reporter assays. Our findings show that overexpression of miR-33b significantly decreased cell viability, cell migration, colony formation, as well as increased apoptosis and sensitivity of MM cells to MLN2238 treatment. Targetscan analysis predicted pim-1 as a putative downstream target of miR-33b. Overexpression of miR-33b downregulated pim-1 mRNA and protein expression. To further corroborate these data, we co-tranfected miR-33b and Pim-1-wt or Pim-1-mt in 293T and MM.1S cell lines. In concert with our earlier findings, miR-33b decreases pim-1-wt, but not pim-1-mt reporter activity in both cell lines. Reflecting the overexpression study results, MLN2238 treatment also decreases pim-1-wt, but not pim1-mt reporter activity. Moreover, a biochemical inhibitor of pim1/2 triggered apoptosis in MM cells. Finally, overexpression of miR-33b inhibits tumor growth (P<0.001) and prolongs survival (P<0.001) in both subcutaneous and disseminated human MM xenograft models. In summary, our study suggests that miR-33b is a tumor suppressor, which plays a role during MLN2238-induced apoptotic signaling in MM cells, and provide the basis for novel therapeutic strategies targeting miR-33b in MM. Disclosures: Anderson: Millennium: 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; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership.

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

Blood ◽

10.1182/blood.v126.23.722.722 ◽

2015 ◽

Vol 126 (23) ◽

pp. 722-722 ◽

Cited By ~ 1

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.

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Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Blood ◽

10.1182/blood.v128.22.311.311 ◽

2016 ◽

Vol 128 (22) ◽

pp. 311-311 ◽

Cited By ~ 4

Author(s):

Laurie Herviou ◽

Alboukadel Kassambara ◽

Stephanie Boireau ◽

Nicolas Robert ◽

Guilhem Requirand ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Newly Diagnosed ◽

Advisory Committees ◽

Myeloma Cells ◽

Ezh2 Expression ◽

Bone Marrow Plasma

Abstract Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: 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; Bristol-Myers Squibb: 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; 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; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

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Phase 1 Study of Elotuzumab in Combination with Autologous Stem Cell Transplantation and Lenalidomide Maintenance for Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.3448.3448 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3448-3448 ◽

Cited By ~ 1

Author(s):

Keren Osman ◽

Ajai Chari ◽

Samir Parekh ◽

Christine Pun ◽

Gillian Morgan ◽

...

Keyword(s):

Multiple Myeloma ◽

Stem Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Phase 1 ◽

Consolidation Therapy ◽

Open Label ◽

Advisory Committees ◽

Myeloma Cells

Abstract Introduction: Elotuzumab is a humanized monoclonal antibody directed against SLAMF7 that is approved for use in relapsed multiple myeloma patients in combination with lenalidomide and dexamethasone. This agent appears to have several modes of action, including facilitation of antibody-dependent, cell-mediated cytotoxicity (ADCC) through binding to SLAMF7 on myeloma cells and activation of natural killer (NK) cells to kill tumor cells through ligation of the target. We initiated a single-center, open label, phase 1 trial based on the hypothesis that the addition of elotuzumab and autologous peripheral blood mononuclear cell (PBMC) reconstitution to standard-of-care autologous hematopoietic stem cell transplantation (auto-SCT) and lenalidomide maintenance for consolidation therapy in myeloma patients after induction therapy will be safe and feasible. We hypothesize that early PBMC reconstitution post-auto-SCT will restore a viable NK cell population for activation by elotuzumab, which may target residual myeloma cells and promote tumor-specific humoral and cellular immune responses against myeloma cells. Subsequent maintenance therapy with elotuzumab and lenalidomide may amplify this response, resulting in long-term maintenance of the minimal residual disease state. Methods. This is a Phase 1b, open-label, trial investigating elotuzumab and autologous PBMC reconstitution with auto-SCT consolidation therapy and lenalidomide maintenance. The primary objective of this study is to assess the safety and tolerability of elotuzumab and autologous PBMC reconstitution in the setting of auto-SCT and lenalidomide maintenance in multiple myeloma patients. The secondary objectives are to assess myeloma disease status and progression-free survival (PFS) after one year of treatment. Subjects must achieve partial response or better by IMWG criteria with induction chemotherapy, be eligible for auto-SCT by institutional standards, and meet inclusion/exclusion criteria. Fifteen subjects are planned in this pilot study. The treatment plan is as follows: In addition to standard peripheral blood stem cell mobilization and harvest, subjects undergo steady-state leukopheresis for PBMC collection. Subjects receive standard melphalan conditioning (day -1) and autologous stem cell rescue (day 0). Autologous PBMC are reinfused on day +3 post-stem cell infusion and cycle 1 of elotuzumab 20 mg/kg IV is given on day +4. Subjects receive subsequent cycles of elotuzumab every 28 days up to cycle 12. Lenalidomide maintenance at 10 mg orally daily days 1-21 of every 28-day cycle begins with cycle 4 of elotuzumab, and may continue off study beyond cycle 12 at the investigator's discretion. Bone marrow aspirates and peripheral blood are collected for correlative studies at screening, cycle 2, cycle 4, and at the end of study after cycle 12. For the primary endpoint analysis, the safety population includes all subjects who received at least one dose of study treatment. The evaluable population constitutes all subjects who received at least four of the first five planned doses of elotuzumab. Results: Fourteen of the planned 15 subjects have been enrolled in the study. Demographic and staging data reflect the general transplant-eligible myeloma patient population at our institution. All 14 of these subjects are included in the safety population, having received at least 1 dose of elotuzumab. Nine of 14 subjects have completed at least 4 of the first 5 planned elotuzumab infusions and are evaluable. The majority of adverse events, including infusion reactions attributable to elotuzumab, have been grade 2 or lower. Grade 3 or higher hematologic AEs, including anemia, neutropenia, lymphopenia, thrombocytopenia, and non-hematologic AEs including nausea, vomiting, and dehydration, were attributable to the auto-SCT procedure. There were no delays in hematopoietic reconstitution observed. One episode of grade 3 hypertension was attributed to elotuzumab infusion and resolved with supportive care. No AEs were attributed to PBMC reconstitution. Conclusions: The combination of elotuzumab and PBMC reconstitution with standard auto-SCT and lenalidomide maintenance for consolidation therapy of multiple myeloma appears to be safe and feasible. One subject withdrew for personal reasons. The trial is ongoing and is expected to complete accrual and the clinical results will be updated for presentation. Disclosures Chari: Celgene: Consultancy, Research Funding; Array Biopharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pharmacyclics: Research Funding; Janssen: Consultancy, Research Funding; Amgen Inc.: Honoraria, Research Funding; Takeda: Consultancy, Research Funding. Geerlof:Bristol-Myers Squibb: Employment. Jagannath:Novartis: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Merck: Consultancy. Cho:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agenus, Inc.: Research Funding; Genentech Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Research Funding; Ludwig Institute for Cancer Research: Membership on an entity's Board of Directors or advisory committees.

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A Multicenter Phase II Single Arm Trial of Isatuximab in Patients with High Risk Smoldering Multiple Myeloma (HRSMM)

Blood ◽

10.1182/blood-2019-123205 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3116-3116 ◽

Cited By ~ 5

Author(s):

Elisabet E. Manasanch ◽

Sundar Jagannath ◽

Hans C. Lee ◽

Krina K. Patel ◽

Connor Graham ◽

...

Keyword(s):

Multiple Myeloma ◽

High Risk ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Infusion Reaction ◽

Advisory Committees ◽

Myeloma Cells ◽

History Of ◽

Smoldering Myeloma

Background High risk smoldering multiple myeloma (HRSMM), defined as having immunoparesis and at least 95% abnormal plasma cells/all plasma cells by advanced flow cytometry, has a risk of progression to multiple myeloma of about 75% after 5 years of diagnosis. These patient have no symptoms and current standard is to follow them without treatment. Isatuximab is an IgG1 monoclonal antibody that binds to CD38 highly expressed in myeloma cells. Isatuximab has activity as monotherapy (overall response rate (ORR) 35%), with lenalidomide/dexamethasone (ORR 56%) and pomalidomide/dexamethasone (ORR 62%) in relapsed MM. We designed a phase II study to test the efficacy of isatuximab in high risk smoldering myeloma. Our study is registered in clinicaltrials.gov as NCT02960555. Methods The primary endpoint of the study is the ORR of isatuximab 20 mg/kg IV days 1, 8, 15, 22 cycle 1; days 1, 15 cycles 2-6 and day 1 cycles 7-30 in high risk smoldering myeloma. 24 patients were accrued in the first stage (of maximum 61 patients). Secondary endpoints are PFS, OS, clinical benefit rate (CBR). Exploratory endpoints are quality of life analysis (QoL), MRD, molecular/immune characterization using DNA/RNA sequencing of myeloma cells and the microenvironment before and after treatment. Results 24 patients with HRSMM were accrued from 02/08/2017 until 12/21/2018 (Table 1). All patients are evaluable for response. Best responses: ORR (≥PR) 15(62.5%), CR MRD- flow at 10-5 1 (5%), VGPR 4 (17%), PR 10 (42%), minor response (MR) 4 (18%), stable disease 5 (21%); CBR (≥MR) 79%. Median number of cycles received were 11.5 (range 6-30). Five patients have stopped treatment (one has completed the study, one with heavy history of smoking was diagnosed with squamous cell cancer of the tongue, one could no longer travel to treatments due to relocation, two progressed to active multiple myeloma after 16 and 6 cycles of treatment, respectively). There have been no deaths. DNA/RNA seq is ongoing for biomarkers of response. There were 5 grade 3 severe treatment-related adverse events (RAE) which resolved to baseline: dyspnea -related to infusion reaction (n=2), headache (n=1), ANC decrease (n=1), urinary tract infection (n=1). Most common grade 1-2 related adverse events (n): nausea (7), vomit (5), WBC decrease (3), diarrhea (3), fatigue (6), headache (4), mucositis (4), myalgia (4) and infusion reaction (3). In patients with available QoL functional scores (n=9 at baseline and n=7 after 6 months of therapy), isatuximab was effective in reducing their anxiety and worry of progression to multiple myeloma. Isatuximab also improved general QoL scores by the end of cycle 6 of treatment which were now comparable to those in the general population (Figure 1). Conclusion Isatuximab is very well tolerated, results in high response rates in HRSMM and has the potential to change the natural history of this disease. In ongoing QoL analysis, initial data shows improvement in QoL and decreased cancer worry after isatuximab treatment. Immune-genomic analysis is ongoing and may identify patients that benefit the most from treatment. Disclosures Manasanch: celgene: Honoraria; merck: Research Funding; quest diagnostics: Research Funding; sanofi: Research Funding; BMS: Honoraria; Sanofi: Honoraria. Jagannath:Multiple Myeloma Research Foundation: Speakers Bureau; BMS: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Medicom: Speakers Bureau; Merck: Consultancy. Lee:Daiichi Sankyo: Research Funding; Celgene: Consultancy, Research Funding; GlaxoSmithKline plc: Research Funding; Sanofi: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Patel:Poseida Therapeutics, Cellectis, Abbvie: Research Funding; Oncopeptides, Nektar, Precision Biosciences, BMS: Consultancy; Takeda, Celgene, Janssen: Consultancy, Research Funding. Kaufman:Janssen: Other: travel/lodging, Research Funding. Thomas:Xencor: Research Funding; BMS: Research Funding; Celgene: Research Funding; Amgen: Research Funding. Mailankody:Takeda Oncology: Research Funding; Juno: Research Funding; Celgene: Research Funding; Janssen: Research Funding; CME activity by Physician Education Resource: Honoraria. Lendvai:Janssen: Employment. Neelapu:Acerta: Research Funding; Celgene: Consultancy, Research Funding; BMS: Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Incyte: Consultancy; Merck: Consultancy, Research Funding; Allogene: Consultancy; Cellectis: Research Funding; Poseida: Research Funding; Karus: Research Funding; Pfizer: Consultancy; Unum Therapeutics: Consultancy, Research Funding; Novartis: Consultancy; Precision Biosciences: Consultancy; Cell Medica: Consultancy. Orlowski:Poseida Therapeutics, Inc.: Research Funding. Landgren:Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Theradex: Other: IDMC; Abbvie: Membership on an entity's Board of Directors or advisory committees; Merck: Other: IDMC. OffLabel Disclosure: Isatuximab for the treatment of smoldering myeloma

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Identification of Novel Regulatory Pathway for Immunoglobulin Production Provides Rational Treatment for Bortezomib-Resistant Multiple Myeloma Patients

Blood ◽

10.1182/blood-2020-140673 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 40-42

Author(s):

Alexander Vdovin ◽

Michal Durech ◽

Tomas Jelinek ◽

Tereza Sevcikova ◽

Juli R. Bago ◽

...

Keyword(s):

Mass Spectrometry ◽

Multiple Myeloma ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Patient Survival ◽

Advisory Committees ◽

Myeloma Cells ◽

Ubiquitinated Proteins ◽

Ig Synthesis

Introduction Monoclonal immunoglobulin (Ig) is a valuable diagnostic marker in patients with multiple myeloma (MM). An inevitable consequence of extensive Ig synthesis is overload of misfolded proteins that saturate proteasome capacity making the myeloma cells highly sensitive to proteasome inhibitors (PI). Even though PI are regularly used in the clinic, resistance often emerges leaving clinicians with limited treatment options. Therefore, there is a need for a robust marker selecting MM patients for precise PI-based combination therapy. Methods We performed a multiple database search for genes associated with Ig production and MM patients' survival. Additionally, we compared gene expression profiles (RNAseq) of primary MM cells with low and high Ig levels. Next, we validated the identified hits by shRNA knockdown and overexpression studies using myeloma cell lines, primary MM samples, and mouse models. We also applied mass spectrometry-based proteomic analysis, advanced biochemical approaches, and genetic models to reveal the Ig production pathway components and function. Finally, we performed a limited rational drug screening to select suitable compounds for combination treatment. Results RNAseq and database mining revealed a strong association between the expression of plasma cell-specific deubiquitinase OTUD1, Ig production, and MM patient survival. Suppression of OTUD1 with shRNAs in RPMI8226 and MM1.S cell lines reduced Ig levels, increased proliferation, and induced bortezomib resistance. Conversely, inducible OTUD1 overexpression enhanced Ig production, slowed down proliferation, and increased bortezomib sensitivity. In the xenografts mouse models cells with high OTUD1 levels synthesized more Ig and developed smaller tumors. Intriguingly, the transcription of Ig genes was not influenced by OTUD1 expression suggesting that OTUD1 functions as a posttranslational regulator of Ig assembly. To gain mechanistic insight into the Ig pathway regulation by OTUD1, we utilized the biotin proximity labeling method (Turbo-ID) combined with mass spectrometry analysis. We found several novel OTUD1 interaction partners including the E3 ubiquitin ligase KEAP1 and endoplasmic reticulum (ER) redox protein PRDX4. We demonstrated that KEAP1 acts upstream of OTUD1 by regulating OTUD1 ubiquitination and stability. Consistently, survival analysis revealed that MM patients with high KEAP1 expression (low OTUD1) had a worse prognosis than patients with low levels of KEAP1 (high OTUD1). PRDX4 regulates disulfite bonds formation during protein folding and is uniquely expressed in fully differentiated plasma cells. Here, we revealed that OTUD1 specifically deubiquitinates and thus stabilizes PRDX4 inside the ER. Additionally, we performed rescue genetic experiments and found a direct link between the OTUD1-PRDX4 axis and Ig production. The increase in OTUD1 expression (high Ig) led to a dramatic increase in the total pool of ubiquitinated proteins formed mainly by misfolded Ig, while OTUD1 knockdown (low Ig) had an opposite effect. We showed that changes in the level of ubiquitinated proteins correlated with PI sensitivity. Of note, OTUD1 did not affect the expression of proteasome subunits, either their enzymatic activity. Our mechanistic findings prompted us to propose a novel therapeutic opportunity in PI resistant MM patients. We hypothesize that the resensitization of Ig low MM cells to PI could be achieved by enhancing ER stress leading to an increase in misfolded proteins that would ultimately saturate proteasomes. Indeed, from clinically relevant drugs tested so far, the HSP-90 inhibitor (17-AAG) reverted the PI resistance in OTUD1 low (Ig low) myeloma cells. An in vivo validation of the combination treatment and testing of Ig involvement in PI sensitivity and proliferation of MM cells is ongoing. Conclusion Here we present the discovery of a novel regulatory mechanism for Ig production in plasma cells. Based on our results and previously published studies, we conclude that Ig synthesis is a clinically significant factor related to PI response and MM patient survival. Our findings suggest that the intracellular Ig level is an important biomarker to identify patients benefiting the most from PI-based therapies. Finally, we provide a rational solution for selective, combination therapy to overcome PI resistance in MM patients with a decreased capacity to synthesize Ig. Figure Disclosures Hajek: Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; PharmaMar: Consultancy, Honoraria; Oncopeptides: Consultancy.

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

Blood ◽

10.1182/blood-2021-151647 ◽

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.

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Impact on Response and Survival of DNA Repair Single Nucleotide Polymorphisms In Relapsed or Refractory Multiple Myeloma Patients Treated with Thalidomide

Blood ◽

10.1182/blood.v116.21.4041.4041 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4041-4041

Author(s):

Carlos Fernández de Larrea ◽

María Teresa Cibeira ◽

Alfons Navarro ◽

Tania Díaz ◽

Dolors Fuster ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Repair ◽

Single Nucleotide Polymorphisms ◽

Board Of Directors ◽

Research Funding ◽

Response Rate ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Advisory Committees ◽

Single Nucleotide

Abstract Abstract 4041 Background: Thalidomide was the first of the so-called new drugs incorporated in the treatment of multiple myeloma (MM). In this era of emerging novel agents, there is a real need for increased knowledge of the pre-treatment genetic profile of patients who will potentially benefit from each drug. The analysis of polymorphisms in drug metabolism pathways and in immune system genes can help to identify patients with possible different treatment response and outcome. Single nucleotide polymorphisms (SNPs) are the most frequent type of genomic polymorphisms and are involved in chemotherapy response in different tumors, including MM. We examined SNPs in 12 genes and correlated our findings with response, toxicity and overall survival (OS) to thalidomide in patients with relapsed MM. Methods: Twenty-eight patients (13M/15F; median age 59 years, range 40 to 82 years) with relapsed or refractory MM from November 1999 to December 2003 were treated with single agent thalidomide at a single institution. The median duration of thalidomide treatment was 4 months and the median dose was 400 mg/day. Median follow-up for alive patients was 103 months (range 86 to 112). Genomic DNA was isolated from bone marrow slides using a commercial assay (Qiagen). SNPs were analyzed by TaqMan assay in an ABI Prism 7500 Sequence Detection system (Applied Biosystems). The genes explored were those related to multidrug resistance (ABCB1 [rs3842, rs1045642]), drug metabolic pathways (NR1I2 [rs1523130, rs1523127], GSTT1 [rs4630], SULT1C2 [rs1402467]), DNA repair systems (XPA [rs1800975], ERCC1 [rs735482], ERCC2 [rs13181], ERCC5 [rs17655, rs1047768], XRCC1 [rs25487], XRCC5 [rs1051677, rs1051685], TOP2A [rs13695]) and cytokines (VEGFA [rs10434, rs2010963]). Results: Partial response (PR) was attained in 17.9% (5/28) and minimal response (MR) in 28.6% (8/28), while 3 (10.7%) and 12 (42.9%) patients showed no response (NR) or progressive disease (PD), respectively. The response rate (PR+MR vs. NR+PD) to thalidomide was higher (66.7%) in patients with hetero- (AC) or homozygous (CC) SNPs in ERCC1 (rs735482) than in those with wild type (AA) (33.3%) (p=0.006). Patients with the ERCC5 heterozygous SNP rs17655 (CG) had a higher response rate (77.8%) than those with the homozygous SNP (GG) or wild type (CC) (31.6%) (p=0.04). Patients with heterozygous XRCC5 (AG) polymorphism rs1051685 showed a higher response rate (100%) than those with wild type (AA) (34.8%) (p=0.013). Longer OS was associated with the homo- and the heterozygous SNP in ERCC1 (AC + CC vs. AA; p=0.005) and with the heterozygous SNP in XRCC5 (rs1051685) (AG vs. AA; p=0.02) (Figures 1 and 2). A trend to longer OS was also observed in patients with polymorphisms in XRCC1 (p=0.06). The heterozygous polymorphism in GSTT1 (CT vs. TT) was associated with a lower frequency of thalidomide-induced peripheral neuropathy (p=0.04). Conclusion: SNPs in ERCC1 and XRCC5 were strongly associated with higher response rate and longer OS to thalidomide in patients with relapsed/refractory MM. SNPs in ERCC5 were also associated with greater response rate. Since the polymorphism in XRCC1 was not related to a better response to thalidomide, it can be speculated that the trend to prolonged OS could be related to other molecular mechanisms. Our findings could be useful to identify patients with MM who are more likely to benefit from thalidomide-based therapies. Disclosures: Cibeira: Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosiñol:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Blade:Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Myeloma-Induced Alterations of Glutamine Metabolism Impair Bone Microenvironment Niche in Multiple Myeloma Patients

Blood ◽

10.1182/blood-2018-99-111337 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4481-4481

Author(s):

Denise Toscani ◽

Martina Chiu ◽

Giuseppe Taurino ◽

Emanuela Vicario ◽

Valentina Marchica ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Bone Disease ◽

Research Funding ◽

Bone Cells ◽

Bone Microenvironment ◽

Advisory Committees ◽

Osteolytic Lesions ◽

Osteolytic Bone Disease ◽

The Relationship

Abstract Multiple myeloma (MM) cells are characterized by tight dependence on the bone marrow (BM) microenvironment that exerts a permissive role on cell growth and survival. In turn, MM cells markedly modify their microenvironment leading, in particular, to the development of osteolytic bone lesions. Recently, we demonstrated that metabolic alterations is a major feature of MM cells showing that BM plasma of MM patients is characterized by lower levels of Glutamine (Gln) and higher levels of Glutamate (Glu) and ammonium when compared with patients with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). In the majority of MM patients MM cells are Gln-addicted since they strictly depend on extracellular Gln, do not express Glutamine Synthetase (GS), the enzyme that synthetizes Gln from Glu and ammonium, and are endowed with high levels of the Gln transporter ASCT2. Based on this evidence, we have hypothesized that the peculiar Gln metabolism of MM cells may have a significant impact on the relationship with the bone microenvironment and contribute to the development of osteolytic lesions. We firstly characterized a panel of human MM cell lines (HMCLs) for their GS expression and response to decreasing levels of Gln. The majority of HMCLs, which did not express GS, consumed large amounts of extracellular Gln but secreted nearly half of the amino acid as Glu. Two HMCLs, MM1.S and U266, with a sizable GS expression, were less sensitive to Gln deprivation and secreted less Glu in the extracellular space compared with GS-negative HMCLs. Consistently, the activity of the Glu exchanger x-CT (the product of SLC7A11 gene) was lower in GS-positive than in GS-negative cells. The response to Gln starvation was then studied in mesenchymal stromal cell line (MSC), as well as in osteoblastic (HOBIT) and pre-osteocytic cells (HOB-01). HOBIT and HOB-01 were more sensitive to Gln depletion than MSC. Indeed, while MSC showed a low EC50 for Gln (0.064mM), which is 10-times lower than the physiological blood Gln concentration (around 0.6 mM), the EC50 values of HOBIT and HOB-01 cells were 0.250 mM and 0.297mM, respectively. Furthermore, L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, emphasized the effects of Gln deprivation on all the cell lines tested. Indeed, Gln deprivation enhanced the expression of GS, suggesting that both stromal and osteoblastic cells exploit the enzyme to counteract Gln deprivation. On the basis of these data, we assessed the effects of Gln and Glu on osteogenic differentiation by incubating MSC, either immortalized or primary, with an osteogenic medium containing different concentrations of Gln and Glu. After 2 weeks, compared with cells differentiated in high Gln/high Glu conditions, MSC incubated in the presence of decreased Gln and increased Glu showed lower osteogenic ability, as assessed by real time PCR and ALP staining. Lastly, MSC co-cultured for 72 hours with GS-negative, but not with GS-positive HMCLs, showed reduced viability and increased GS expression. Lastly, to put in a translational perspective these in vitro observations, we analyzed the BM plasma levels of Gln and Glu in a cohort of 41 patients with newly diagnosed MM, including 9 smoldering MM (SMM) and 32 active MM patients (20 of them with osteolytic bone disease, 12 of them without bone disease). All 20 osteolytic MM patients had more than three osteolytic lesions. We found that MM patients had lower Gln levels and higher Glu levels than SMM patients. Moreover, when compared with MM patients without bone disease, MM patients with bone disease showed lower levels of Gln and higher levels of Glu. The results of these analyses are being continuously updated increasing the number of samples tested. Overall, these results indicate that MM cells are able to create a low-Gln/high-Glu bone marrow microenvironment that sustains GS expression in bone cells and impairs their differentiation and viability. Thus, the peculiar metabolic milieu in the MM bone microenvironment affects the relationship between neoplastic and bone cells and may contribute to the development of osteolytic bone disease in MM patients. Disclosures Aversa: Astellas: Honoraria; Merck: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Basilea: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Giuliani:Takeda Pharmaceutical Co: Research Funding; Celgene Italy: Other: Avisory Board, Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding.

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Molecular Characterization Using Oncoscan Chromosome Microarray in an International Cohort of 51 Patients with Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN)

Blood ◽

10.1182/blood-2021-152922 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3497-3497

Author(s):

Eli Williams ◽

Stefano A Pileri ◽

Maria Rosaria Sapienza ◽

Carlos Barrionuevo ◽

Carlos Bacchi ◽

...

Keyword(s):

Board Of Directors ◽

Copy Number ◽

Research Funding ◽

Plasmacytoid Dendritic Cell ◽

Chromosome 9 ◽

Single Nucleotide Variants ◽

Advisory Committees ◽

Single Nucleotide ◽

Cell Neoplasm ◽

Chromosome Microarray

Abstract Introduction Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematological malignancy with multi-organ and frequent skin involvement, and poor clinical outcomes. Based on the limited available data, the estimated incidence is 0.44% of all hematologic malignancies, representing less than 1% of acute leukemias, and 0.7% of cutaneous lymphomas. Due to the rarity of this entity, there have been relatively few studies characterizing the molecular profile of BPDCN. We examined a cohort of 51 patients with BPDCN using OncoScan chromosome microarray, which provides genome-wide copy number abnormality (CNA) analysis. Methods An international cohort of BPDCN cases were collected from centers in Brazil (Laboratorio de Patologia, Botucatu), Swtizerland (University of Zurich), France (Hospital St. Louis, Paris), Peru (Instituto Nacional de Enfermedades Neoplasicas, Lima), Canada (Department of Pathology, University of Montreal), Italy (Derpartment of Pathology, University of Bologna), and US (Department of Pathology - The Ohio State University, Department of Hematopathology - MD Anderson Cancer Center; and Department of Pathology - University of Virginia). A total of 58 tissue blocks from 51 patient samples were retrieved. The diagnosis of BPDCN was done and confirmed by at least three independent hematopathologists or dermatopathologists in accordance with the WHO classification (Lyon 2017). For the purpose of the molecular analysis substratification, cases were classified as 'BPDCN' if they were positive for TCF4, and 'BPDCN-like' if they were negative for TCF4. Immunohistochemistry for CD123, CD4, and CD56 was performed in all cases. Exclusion criteria included expression of MPO, lysozyme, CD3, CD19, CD20, CD22, and/or EBV. DNA was extracted from FFPE samples via standard techniques and processed on OncoScan CNV Plus microarray (ThermoFisher Scientific) according to manufacturer's recommended protocol. Copy number abnormalities and select single nucleotide variants and insertions/deletions (74 mutations in 9 genes) were analyzed on Chromosome Analysis Suite software (ChAS v4.1; ThermoFisher Scientific). Additional analysis was performed using Nexus Copy Number (BioDiscovery, version 10.0). Results To date, we have successfully analyzed 45 cases of BPDCN with Oncoscan, revealing widespread CNA in the vast majority of cases (44/45; 98%). Alterations of chromosome 9 were common in this cohort, particularly CNAs involving CDKN2A/B at 9p21.3. Twenty-five cases (56%) demonstrated CNA including CDKN2A/B, with ten of these cases demonstrating a homozygous loss of CDKN2A/B (22%). Alterations of chromosome 13 were also frequently detected with loss of RB1 (located at 13q14.2) detected in 24 cases (53%). The RUNX1 gene (21q22.12) was a common target of CNAs in this cohort, seen in nine cases (20%). Eight of these cases showed a copy number gain of RUNX1, which is a recurrent finding in a variety of hematological malignancies, particularly myeloid neoplasms. The remaining case with RUNX1 CNA showed a focal, homozygous loss of the gene, demonstrating that dysregulation of RUNX1 through CNA is a common event in BPDCN. We observed frequent deletions of ETV6 (53%), IKZF1 (33%), and TP53(16%) in our cohort. The ARHGAP26 gene (5q31.3), which is associated primarily with juvenile myelomonocytic leukemia, was included in CNA in 13 cases (29%), with both gains and losses observed in this cohort. Oncoscan can detect a limited number of single nucleotide variants in nine genes that are frequently mutated in cancers (BRAF, EGFR, IDH1, IDH2, KRAS, NRAS, PIK3CA, PTEN, and TP53). Mutations were detected in ten cases (22%), with NRAS and TP53 variants detected in three cases each and KRAS and IDH2 variants detected in two cases each. Conclusions Our preliminary data demonstrates complex genomic alterations in BPDCN, with the RB1 locus on chromosome 13, the CDKN2A/B locus on chromosome 9, and the ETV6 locus on chromosome 12 most commonly detected. However, widespread genomic alterations were detected involving a variety of cancer-associated genes further characterizing CNA in BPDCN. Analysis of additional BPDCN cases is progress. Disclosures Khoury: Kiromic: Research Funding; Angle: Research Funding; Stemline Therapeutics: Research Funding. Porcu: Viracta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Daiichi: Honoraria, Research Funding; Kiowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Spectrum: Consultancy; DrenBio: Consultancy. Gru: StemLine: Honoraria, Research Funding, Speakers Bureau; CRISPT Therapeutics: Research Funding; Innate Pharma: Research Funding.

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