Oncolytics Virus Replication Using Pelareorep (Reolysin) and Carfilzomib in Relapsed Myeloma Patients Increases PD-L1 Expression with Clinical Responses

Abstract Rationale: Immune checkpoint inhibitors, including those targeting programmed cell death protein 1 (PD-1), are tempting in immunosuppressive cancers such as multiple myeloma (MM). PD-1 inhibition alone has not been effective (Lesokhin, JCO, 2016). Evidence suggests that tumor response is determined by tumor PD-L1 expression and presence of infiltrating cytotoxic T-cell lymphocytes (CTLs). Intravenous Pelareorep (Reolysin), the infusible form of reovirus (RV), has been shown to upregulate IFN-regulated gene expression, CTL infiltration, and the PD1/PD-L1 axis in myeloma cell lines (Kelly KR et al, Leukemia, 2018) and in patients with brain tumors (Samson A et al, Sci Trans Med, 2018). Introduction:Viral oncolytic therapy with Pelareorep is supported by preclinical data indicating that its antitumor activity results from direct cytolysis and immune responses against infected MM cells. Our phase 1 single agent Reolysin trial in patients with relapsed MM confirmed the tolerability of Pelareorep and demonstrated preferential infection into myeloma cells but not microenvironmental cells in the marrow by intracellular staining for RV genome. In PART ONE of our follow-up trial, Carfilzomib (Kyprolis)-sensitive patients were accrued. Correlative studies included bone marrow aspirate pretreatment on cycle 1 day 1 and day 9 to assess RV infection of myeloma cells, replication within myeloma cells, and PD-L1 expression on myeloma cell surface. Pelareorep, Carfilzomib, and 20 mg IV Dexamethasone were all infused per the standard Carfilzomib schedule on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle. Part ONE: Carfilzomib-sensitive patients were infused at dose level one with Pelareorep 3 x 1010TCID50/day and Carfilzomib 20 mg/m2 on days 1 and 2 of cycle 1 and 27 mg/m2 thereafter, on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle. The MTD will be determined as the highest dose combination with fewer than 33% DLTs observed in cycle 1. There were 2 VGPRs, 2 PRs, 1 MR, and one patient with stable disease after cycle 1. All evaluable patients showed RV infection and replication in the post-treatment BM aspirates. In the 4 bortezomib-refractory patients in the first cohort, all have shown viral replication, and this correlated directly with activated caspase-3 in the MM cells and clinical response. Part TWO: Carfilzomib-refractory patients were treated with Carfilzomib 20 mg/m2 on days 1 and 2 of cycle 1, and 56 mg/m2 thereafter. Seven patients have been enrolled to date. In 3 patients processed to date with both pre- and post-treatment biopsies available, RV infection was detected in myeloma cells (2 patients) and endothelial cells (one patient). Replication was not seen. In these patients there was no strong evidence of increased activated caspase-3 expression in myeloma cells, nor was there a statistically significant increased CD8 cell infiltration or checkpoint protein expression after treatment. Conclusion: An increase in viral infection, viral replication, and PD-L1 expression on the surface of myeloma cells has been demonstrated in patients that had a clinical response to protocol therapy. A future study to assess the safety and tolerability of PD-1 inhibition in relapsed myeloma patients is now open to accrual (NCT03605719). Dose escalation is ongoing and the effects of the addition of Reolysin to Carfilzomib/Dexamethasone will be determined in this trial. Disclosures Hofmeister: Bristol-Myers Squibb: Research Funding; Adaptive biotechnologies: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Nuovo:Oncolytics Biotech Inc: Research Funding. Lonial:Amgen: Research Funding. Kaufman:Roche: Consultancy; Abbvie: Consultancy; Karyopharm: Other: data monitoring committee; Janssen: Consultancy; BMS: Consultancy. Nooka:GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive technologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; Spectrum 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; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen pharmaceuticals: 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. Heffner:Pharmacyclics: Research Funding; ADC Therapeutics: Research Funding; Genentech: Research Funding; Kite Pharma: Research Funding.

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Proteasome Inhibitors Sensitize Myeloma Cells to T Cell-Mediated Killing

Blood ◽

10.1182/blood.v118.21.1838.1838 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1838-1838

Author(s):

Niels W.C.J. van de Donk ◽

Henk M. Lokhorst ◽

Maarten Emmelot ◽

Tineke Aarts-Riemens ◽

Douglas W. McMillin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Fas Ligand ◽

Cell Clone ◽

Myeloma Cell ◽

Advisory Committees ◽

Myeloma Cells ◽

T Cell Clone

Abstract Abstract 1838 Introduction: Graft-versus-myeloma (GvM) effect following allogeneic stem cell transplantation (allo-SCT) is well established and often accompanied by graft-versus-host disease (GVHD). Unfortunately, most patients still relapse following allo-SCT, underscoring the need for new transplant strategies. Interestingly, studies evaluating the proteasome inhibitor bortezomib combined with donor lymphocyte infusions (DLI) in myeloma patients suggest enhanced efficacy of the combination. In contrast, other reports demonstrate that bortezomib has activity in preventing GVHD in various malignancies, including myeloma. Here, we investigate the effects of bortezomib on T cell-mediated myeloma cell killing in vitro. Methods: Tumor cell compartment-specific bioluminescence imaging (CS-BLI) technology was used to monitor the cytotoxic effects of T cells on myeloma cells in the presence or absence of antimyeloma agents. To this end, myeloma cell lines were stably transduced with a firefly luciferase construct. Two different T cell clones were used: 1) the alloreactive CD8+-T cell clone allo-A2, which kills all HLA-A2-positive cells, including the myeloma cell line U266; and 2) the CD4+ T-cell clone 3AB11, generated from a myeloma patient with clinical GvM/GVHD after allo-SCT. 3AB11 cells recognize a hematopoietically restricted minor antigen, also presented by the myeloma cell line UM9. Protein expression was determined by western blot analysis. Results: The CD4+-T cell clone 3AB11 and the allo-A2 specific CD8+- T cell clone displayed a time- and dose-dependent lysis of myeloma cell lines UM9 and U266, respectively. Concurrent treatment of myeloma cells with the T cell clones and bortezomib resulted in synergistic enhancement of myeloma cell death, compared to T cells alone or bortezomib alone. This synergism was observed at 24, 48, and 72 hours (but not after 4 hours) of co-treatment. Dexamethasone, which significantly inhibited myeloma cell death induced by T cells, functioned as control. When myeloma cells were treated for 12 hours with bortezomib and then extensively washed, there was still enhancement of T cell-mediated killing. However, pre-incubation of T cells with bortezomib significantly impaired their ability to induce myeloma cell lysis. These washout experiments indicate that bortezomib sensitizes myeloma cells to T cell-mediated killing, but does not enhance the intrinsic cytotoxic activity of T cells. Mechanistic studies demonstrated that antibodies blocking FAS ligand partially impaired myeloma cell lysis by 3AB11 and allo-A2 clones. More interestingly, however, blockade of FAS/FAS ligand interactions completely abrogated the synergism between bortezomib and T cells. Antibodies against TRAIL had no such effects. Furthermore, western blot analyses revealed that in UM9 and U266 cells bortezomib reduced protein levels of FLICE inhibitory protein (cFLIP), which is a potent inhibitor of FAS ligand-induced apoptosis. These results support the idea that bortezomib enhances the sensitivity of myeloma cells toward T cell-mediated lysis by restoring apoptosis signaling via the FAS/FAS ligand pathway. Conclusion: Proteasome inhibition sensitizes myeloma cells to T cell-mediated killing via a FAS/FAS ligand-dependent mechanism. Conversely, bortezomib impairs T cell function. However, the net effect is a synergistic killing of myeloma cells when T cells and bortezomib are combined. These data support further exploration of the use of bortezomib in preclinical models, with derived clinical trials in the allogeneic setting (e.g. in combination with DLI or following allo-SCT) or in the autologous setting (e.g. in combination with a tumor vaccine) potentially warranted. (CSM and TM are joint senior authors) Disclosures: van de Donk: Celgene: Research Funding. Lokhorst:Celgene: Research Funding; Jansen-Cilag: Research Funding. Hayes:Pfizer: Research Funding; Amgen: Research Funding. Munshi:Celgene: Consultancy; Millenium: Consultancy; Novartis: Consultancy; Onyx: Consultancy. Richardson:Millennium:; Celgene:; Johnson & Johnson:; Novartis:; Bristol Myers Squibb:. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: 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; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Kosan: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centocor: Consultancy, Honoraria; Amnis Therapeutics: Consultancy, Honoraria; PharmaMar: Licencing royalties; OSI Pharmaceuticals: Research Funding; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding; Genzyme: Research Funding; Johnson & Johnson: Research Funding.

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Post-Treatment Clone Size Predicts Survival Independently of IPSS-R and Response after Azacitidine Therapy for MDS.

Blood ◽

10.1182/blood-2020-139082 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-13

Author(s):

Yasuhito Nannya ◽

Magnus Tobiasson ◽

Shinya Sato ◽

Elsa Bernard ◽

Maria Creignou ◽

...

Keyword(s):

Clinical Response ◽

Board Of Directors ◽

Research Funding ◽

Post Treatment ◽

Private Company ◽

Advisory Committees ◽

Clone Size ◽

Otsuka Pharmaceutical ◽

Pre Treatment

Background DNA hypomethylating agents (HMAs), including azacytidine (AZA) have been established as key drugs for higher-risk myelodysplastic syndromes (MDS). We and others have explored the role of mutation profile before AZA administration on predicting outcomes. Actually, we have previously identified mutated-TP53 as a marker associated with higher rate of achieving complete remission (CR). In addition, mutations in TP53 and DDX41 predicted reduced and prolonged survival after treatment, respectively. However, the clinical significance of evaluating clone size changes early after treatment has not been determined. In this study, we explored the role of post-treatment clone size in predicting outcomes of AZA treatment for MDS and related diseases. Methods We enrolled 290 AZA-treated cases, including 88 from a Japanese prospective study (JALSG MDS-212 trial), 149 from Karolinska Institute, and 53 from a retrospectively collected Japanese cases. The diagnoses were MDS (n=242), MDS/MPN (n=25), and AML-MRC (n=23). For all patients, tumor samples were collected both before and after AZA administration and were analyzed for mutations in 66 genes implicated in myeloid neoplasms using targeted-capture sequencing. The median cycles of AZA treatment before sampling was 4 (range 1-7). Clone size was calculated from variant allele frequency adjusted for ploidy or allelic imbalances.Survival was calculated with a Cox regression model. Results In post-treatment samples, we identified 870 mutations in 51 genes in 255 (88%) patients with a median of 3 mutations per sample, while 943 mutations were seen in 279 (96%) patients in the pre-treatment samples. Most frequently detected mutations in post-treatment samples were seen in TET2, TP53, RUNX1, and ASXL1. Germline DDX41 mutations were excluded from clone size evaluation. Median clone sizes were 0.63 and 0.54 for pre-treatment and post-treatment samples (P=.011), respectively. The largest clone sizes (max(VAF)) in post-treatment samples had a strong negative correlation with hematological response according to IWG criteria (P < .0001). We next explored whether max(VAF) in post-treatment samples provides a more precise estimation of long-term survival than IPSS-R. Max(VAF) further stratified each IPSS-R risk group in subgroups with discrete OS (P < .0001 for IPSS-R very high and P = .0004 for high risk group). Incorporating pre-treatment mutation data (mutations in TP53 and DDX41) and max(VAF) values in addition to IPSS-R scores and clinical response, we constructed a multivariate model and found that all these factors had an independent and significant impact on OS (Figure 1A). Next, we examined whether max(VAF) combined with IPSS-R and clinical response can improve the model. For this purpose, we randomly split the cohort into 75% training and 25% validation subsets and for each split, we constructed different models using the training set, performance of which was evaluated by calculating the concordance index (c-index) using the validation set. The mean c-index in 10,000 simulation sets increased by 0.025 by adding response data to IPSS-R score (I versus IR in Fig 1B). Further improvements were obtained by adding gene mutation and max(VAF), in which the c-index increased by 0.034 (IR versus IGR in Fig 1B) and 0.010 (IGR versus IGRP in Fig 1B), respectively. For the 53 patients who received allogeneic stem cell transplantation, the median post-transplant OS was 82.6 months (range, 36.3 to not reached). Notably, max(VAF) significantly stratified OS after allo-SCT (HR, 3.3; 95%CI, 1.3 to 8.3; P = .014). Conclusions Our study revealed that post-treatment clone size significantly correlated with clinical response and the evaluation of post-treatment clone size allows for more precise prognostication after AZA treatment compared with IPSS-R and clinical response alone. Table Disclosures Naoe: NIPPON SHINYAKU CO.,LTD.: Speakers Bureau; Sysmex co.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Astellas Pharma Inc.: Speakers Bureau; Bristol-Myers Squibb Company: Speakers Bureau. Miyazaki:Celgene: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Otsuka Pharmaceutical: Honoraria; Astellas Pharma Inc.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Papaemmanuil:Kyowa Hakko Kirin: Consultancy, Honoraria; Prime Oncology: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Illumina: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; MSKCC: Patents & Royalties; Isabl: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Ogawa:Eisai Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding.

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HDP101, a Novel B-Cell Maturation Antigen (BCMA)-Targeted Antibody Conjugated to α-Amanitin, Is Active Against Myeloma with Preferential Efficacy Against Pre-Clinical Models of Deletion 17p

Blood ◽

10.1182/blood-2018-99-118412 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 593-593 ◽

Cited By ~ 6

Author(s):

Ram Kumar Singh ◽

Richard J. Jones ◽

Samuel Hong ◽

Fazal Shirazi ◽

Hua Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Rna Polymerase ◽

Cell Viability ◽

Board Of Directors ◽

Stromal Cells ◽

Research Funding ◽

Myeloma Cell ◽

Advisory Committees ◽

In Vivo Models ◽

Myeloma Cells

Abstract Background: Deletion (del) of 17p involving the p53 tumor suppressor (TP53) remains an adverse prognostic factor in multiple myeloma (MM) despite the use of novel agents as well as high-dose chemotherapy with autologous stem cell rescue. Genomic TP53 deletion can cause haploinsufficiency of nearby genes, such as RNA polymerase II subunit A (POLR2A), which ia also located on 17p13.1. We therefore hypothesized that del 17p could reduce POLR2A expression and enhance sensitivity to a-Amanitin, a potent and specific inhibitor of POLR2A and RNA polymerase III. Methods: Pre-clinical studies were performed using HDP101, a monoclonal antibody-drug conjugate (ADC) targeting BCMA linked to a-Amanitin, along with unconjugated BCMA antibody, a-Amanitin, and a non-targeting control ADC in myeloma cell line models. The latter included H929, MM1.S, and MOLP-8 TP53 wild-type (WT) lines and isogenic cells in which TP53 had been knocked out (KO) using CRISPR/Cas9 genome editing techniques. To further model del 17p and POLR2A haploinsufficiency, POLR2A expression was knocked down using shRNAs. Results: Analysis of the Multiple Myeloma Research Foundation CoMMpassSM database revealed that del 17p13 by SeqFISH was associated with a significant reduction in POLR2A expression by RNASeq (26.05431 fragments per kilobase of transcript per million mapped reads (FPKM) with WT vs. 19.2983 FPKM with del 17p13; p<0.0001). Also, patients within the lower quartile of POLR2A expression, which included those with and without del 17p, had an inferior overall survival (p<0.0011) and a trend towards a worse progression-free survival, suggesting that low POLR2A levels by themselves are an adverse feature. The POLR2A inhibiting anti-BCMA/a-Amanitin conjugate HDP101 induced a time- and dose-dependent reduction in myeloma cell viability post 96-hours of drug exposure starting at concentrations as low as in the picomolar range. This reduction with HDP101 was much greater than that seen with controls, which included a-Amanitin alone, the anti-BCMA antibody without a-Amanitin, or an anti-digoxigenin antibody conjugated to a-Amanitin. When myeloma cells were co-cultured with HS-5 human marrow stromal cells, only a-Amanitin and, to a much greater extent, HDP101 induced a loss of cell viability in myeloma cells, while the stromal cells were spared by HDP101. Loss of cell viability due to HDP101 was associated with induction of apoptosis as judged by the appearance of an increased population of cells that had a sub-G0/G1 DNA content, and that stained positively with Annexin V. Moreover, HDP101 treatment caused the appearance of cleaved fragments of Caspase 9 and 3, and loss of the mitochondrial trans-membrane potential. H929, MM1.S, and MOLP-8 TP53 KO cells were more sensitive to both HDP101 and, less potently to a-Amanitin than were their isogenic TP53 WT parental cells. As H929 cells expressed high levels of BCMA regardless of TP53 or POLR2A status, these were further examined for their sensitivity to HDP101. Notably, the preferential impact upon TP53 KO cells was associated with increased expression of Activating transcription factors -4 and -6, suggesting enhanced induction of endoplasmic reticulum stress, and at least two arms of the unfolded protein response. Interestingly, shRNA-mediated knockdown (KD) of POLR2A expression alone was sufficient to increase baseline levels of apoptosis in both H929 TP53 WT and KO cells, with a greater impact in the latter, supporting the promise of this target. Importantly, KD of POLR2A expression to further model del 17p was also associated with enhanced sensitivity to HDP101 in both the TP53 WT and KO cells compared to the control treatments. Evaluation of HDP101 in primary samples and with in vivo models is underway, and will be presented at the Annual Meeting. These studies were supported by a Leukemia & Lymphoma Society Specialized Center of Research (SCOR-12206-17). Conclusions: Our preliminary data support the possibility that del 17p myeloma may have a therapeutic vulnerability to the POLR2A inhibitor a-Amanitin through loss of TP53, and that this sensitivity is further enhanced by decreased POLR2A expression, which is common among del 17p patients. Moreover, they suggest that HDP101 is a novel potent and specific therapeutic that could show enhanced activity in the clinic especially against high-risk multiple myeloma, where effective therapies are still needed to improve patient outcomes. Disclosures Pahl: Heidelberg Pharma AG: Employment. Orlowski:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Genentech: Consultancy; Poseida: Research Funding; BioTheryX, Inc: 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, Research Funding; Millenium Pharmaceuticals: Consultancy, Research Funding.

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Regulation Of DKK-1 Expression By p38 MAPK Signaling Via CREB Myeloma Cells

Blood ◽

10.1182/blood.v122.21.1917.1917 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1917-1917

Author(s):

Jing Yang ◽

Yuping Zhong ◽

Jin He ◽

Zhiqiang Liu ◽

Huan Liu ◽

...

Keyword(s):

Board Of Directors ◽

P38 Mapk ◽

Binding Sites ◽

Research Funding ◽

Transcriptional Activity ◽

Bone Destruction ◽

Myeloma Cell ◽

Chip Assay ◽

Advisory Committees ◽

Myeloma Cells

Abstract Bone destruction is a hallmark of multiple myeloma and severely compromises a patient’s quality of life. Recently, we have demonstrated that p38 mitogen-activated protein kinase (MAPK), which is constitutively activated in myeloma cells, is a master regulator of myeloma-mediated bone destruction. We have observed that myeloma cell p38 MAPK upregulated the production of dickkopf-1 (DKK-1), of which inhibits osteoblast differentiation and bone formation by inhibiting Wnt signaling and enhances osteoclast differentiation and bone resorption by upregulating RANKL production. Our results have shown that treatment of SB203580 (SB20), a p38 MAPK inhibitor, downregulated, while treatment of anisomycin, a p38 MAPK activator, upregulated DKK-1 expression. However, the mechanism underlying the regulation of DKK-1 expression by p38 MAPK in myeloma cells remains poorly elucidated. Previous studies have suggested that CREB is a p38 MAPK-targeted transcriptional factor. We therefore hypothesized that myeloma cell p38 MAPK may upregulate the transcriptional levels of DKK-1 through CREB. To verify whether p38 MAPK regulates CREB phosphorylation in myeloma cells, ARP-1 and U266 cells were treated with SB20 or anisomycin. Western blotting results showed that SB20 treatment significantly reduced, while anisomycin treatment enhanced phosphorylation of CREB in both cell lines. Database analysis of DKK-1 promoter regions predicted a couple of potential CREB-binding sites localized around 1.3 kb upstream from the starting codons. CHIP assay further indicated that CREB specifically bound to the one of the putative binding sites (-1,354 bp). To validate the CHIP assay results, we designed three constructs: construct 1 containing the whole promoter (-2,542 bp to -220 bp), which has previously been shown to have strong transcriptional activity; construct 2, as a truncated portion of the promoter containing the putative CREB-binding sites (-1,392 bp to -220 bp); and construct 3, as a truncated portion of the promoter containing neither CREB-binding sites (-1073 bp to -220 bp). These constructs were amplified by PCR and cloned into a luciferase reporter gene vector (pGL2) respectively. To examine the effect of CREB on the transcription activities of DKK1 in myeloma cells, reporter constructs were transfected into ARP-1 and U266 cells. In line with our findings in CHIP assay, we observed that in both myeloma cells, the construct 2 had similar strong transcriptional activity as the construct 1 did, the construct 3 had little activity. These results demonstrated that there was a CREB-binding site in DKK-1 promoter, and is required for p38 MAPK-upregulated DKK-1 expression in myeloma cells. In conclusion, our results uncover a mechanism of myeloma cell p38 MAPK in osteoblast inhibition and osteoclast activation by which p38 MAPK transcriptionally upregulates DKK-1 expression via CREB. Disclosures: Orlowski: Bristol-Myers Squibb: 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; Millennium: The Takeda Oncology Company: Honoraria, 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, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

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A Novel Evolutionary Pattern Revealed Using Deep Sequencing of Immunoglobulin Loci at Diagnosis and over the Course of Treatment in Multiple Myeloma Patients

Blood ◽

10.1182/blood.v128.22.238.238 ◽

2016 ◽

Vol 128 (22) ◽

pp. 238-238 ◽

Cited By ~ 2

Author(s):

Nikhil C. Munshi ◽

Joaquin Martinez-Lopez ◽

Victoria Carlton ◽

Stephanie Minvielle ◽

Yu-Tzu Tai ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Post Treatment ◽

Large Cohort ◽

Differential Sensitivity ◽

Advisory Committees ◽

Myeloma Cells ◽

Equity Ownership

Abstract Introduction: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. As multiple myeloma is considered a clonal disease originating from the transformation of a single plasma cell, myeloma cells are traditionally thought to have one clonal Ig gene sequence that remains stable throughout the course of the disease. We previously observed that multiple Ig sequences related by somatic hypermutation (SHM) may be present in some MM patients at diagnosis. Here we provide an expanded observation in a very large cohort of the patients, and perform mutational analysis of the oligoclonal myeloma clonotypes observed at diagnosis and post-treatment, revealing changes in the relative frequency of the MM clonotypes and emergence of new Ig clones. Methods : 620 MM patientsenrolled in IFM/DFCI and Hospital 12 de Octubre trials were included in this analysis. The next-generation sequencing (NGS)-based immunosequencing platform was used to detect evidence of oligoclonality at the Ig heavy chain loci. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from DNA and/or RNA isolated from purified CD138+ MM cells collected at the time of diagnosis. MM-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic (dx) sample. The highest frequency MM clonotype in a dx sample is termed the "index clonotype." DNA and/or RNA isolated from dx AND post-treatment bone marrow samples were assessed for evidence of evolved MM clonotypes. Results: We identified Ig clones in 367 RNA samples and 430 DNA samples from the cohort. We first looked for cases with evidence that myeloma cells have two unrelated origins. We found 11/620 (1.8%) cases at diagnosis, which had evidence of unrelated clones as evident by having three IgH or two functional sequences. In 8 of the 11 cases (72.6%), we had multiple samples to analyze, including two samples at diagnosis or diagnosis/post-treatment pairs. In 4 of the 8 samples, we saw dramatically different relative frequencies of unrelated clones in these samples suggesting that these unrelated clones are likely to be present in two distinct cells. We then considered cases where we found two IgH sequences that are related to each other by SHM at diagnosis. Overall 79 (12.7%) of 620 samples had more than one evolved clone; of these 63/367 (17.2%) of RNA dx samples showed evidence of evolved clones via SHM, while 22/430 patients (5.1%) showed evidence of evolved clones related to the index clone via SHM in DNA samples from diagnosis. Mutant clonotypes had an average of 3.9 to 4.5 mutations in the CDR3 region. We also noted mixed isotypes in 13 clones from 13 patients at diagnosis. The majority of related clones observed in the RNA samples are present at very low frequencies (<10-4), as the greater sequencing depth in RNA allows for identification of low frequency clones. 304 post-treatment samples from 206 patients were MRD positive and were assessed for the presence of clonal evolution. In 27/304 follow-up samples (8.8%) and 7/206 patients (3.4%), an evolved clone related to the index clone was observed even though the period between diagnosis and post-treatment samples was only 6 months. In 6 patients, a substantial change in the relative index and unrelated clone frequencies was observed from the dx to post-treatment time points suggesting a differential sensitivity to treatment. Conclusions: We confirm presence of multiple evolved clonotypes in a substantial percentage of diagnostic MM samples in a large cohort of patients. The evolution of multiple clones related by SHM indicates that SHM remains active after myeloma development and may also impact other non-Ig sites. These findings shed light on the biology and pathogenesis of MM and may provide prognostic information. The very high depth of our sequencing also indicates that the emergence of new IgH clones may be newly acquired mutations in the Ig gene, driven by some ongoing genomic mutation process. Thus, these evolved myeloma clonotypes may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy. Disclosures Munshi: Takeda: Consultancy; Pfizer: Consultancy; Merck: Consultancy; Celgene Corporation: Consultancy; Oncopep: Consultancy, Equity Ownership. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Attal:amgen: Consultancy, Research Funding; celgene: Consultancy, Research Funding; janssen: Consultancy, Research Funding; sanofi: Consultancy. Moreau:Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Other: Scientific Founder; Oncopep: Other: Scientific Founder. Faham:Adaptive Biotechnologies Corp: Employment. Avet-Loiseau:janssen: Consultancy; sanofi: Consultancy; celgene: Consultancy; amgen: Consultancy.

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Targeting PI3K-AKT-mTOR Signaling in Multiple Myeloma Mesenchymal Stem Cells Mediates Antiproliferative Effect on Myeloma Cells

Blood ◽

10.1182/blood-2021-150413 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1600-1600

Author(s):

Luca Heinmann ◽

Helal Mohammed Mohammed Ahmed Noman ◽

Klara Möllers ◽

Subbaiah Chary Nimmagadda ◽

Kaiyan Sun ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Therapeutic Strategies ◽

Advisory Committees ◽

Myeloma Cells ◽

Bm Niche ◽

Novel Therapeutic Strategies ◽

Novel Therapeutic

Abstract Introduction: Multiple myeloma (MM) is a B-cell malignancy characterized by an abnormal proliferation and infiltration of malignant plasma cells in the bone marrow (BM). Mesenchymal stromal cells (MSCs) represent a crucial component of the BM niche and mediate essential signalling via cytokines and cell-cell interactions. The interplay of MM cells and BM-MSC is complex and relies on multiple signaling pathways leading to MM progression and therapeutic resistance. Objectives: MM remains an incurable disease so far. Distinctive for this disease is a long-lasting polarization of the BM niche influencing MM progression and prognosis. We, therefore, focussed on MSCs to identify enrichment for different hallmark gene sets and their aberrant signaling contributing to the pathogenesis of the disease, therapy response and to further identify novel therapeutic strategies. Methods: BM-MSCs were isolated from patients with MM at diagnosis (MM-D-MSC) and in remission (MM-R-MSC) as well as from donors with other malignant diseases (CTR-MSC). RNA sequencing and Western Blot were used for examination of enriched pathways. Various functional assays for proliferation, apoptosis and cell cycle were performed either using a mono-culture or co-culture protocol of MSC and the MM-cell lines MM.1S and SKMM2 treating the cells with the pan-PI3K-inhibitor GDC-0941. Results: MM-D-MSCs supported the growth of myeloma cell lines better (3 fold, p<0.01) than MM-R- and CTR-MSCs. Our results demonstrate that MM-D-MSCs have a distinct gene expression profile compared to CTR-MSC indicating potential nodes of crosstalk and therapeutic importance. Amongst others, the PI3K-AKT-mTOR hallmark gene set was significantly enriched in MM-D-MSCs as compared to CTR-MSCs (p<0.001). We confirmed these findings on a proteomic level. We found evidence for the upregulation of PI3Kα, AKT, pAKT and mTOR in MM-D-MSC comparing to the other MM-R- and CTR-MSCs (p<0.05). We treated these MSC and the MM-cell lines MM.1S and SKMM2 with the PI3-Kinase inhibitor GDC-0941. The treatment reduced the signaling PI3Kα, AKT and mTOR in both, MSC and MM-cells. As stated MM-D-MSC supported the growth of myeloma cells better than other MSC types. However, upon GDC-0941 treatment, the proliferation of MM-D-MSCs was significantly reduced compared to the other MSC-types. In addition, the inhibition of proliferation of myeloma cell lines MM1S and SKMM2 was much more pronounced when they were cocultured with MM-D-MSC (32 and 34 %, p=0.04) compared to the growth of myeloma cells in coculture with MSC types, either in remission or other malignancies. Conclusion: We here identified functionally distinct differences in MM-D-MSCs compared to MM-R-MSCs or CTR-MSCs. Our data further provides a deeper insight into the molecular signature of MM-MSCs, a predictive of patient prognosis and treatment outcome. Targeting MSCs as a crucial part of the MM-BM niche by using PI3K-inhibitors could contribute to novel therapeutic strategies to effectively block MM-MSC interaction improving overall patient survival. Disclosures Raab: Roche: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: 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. Khandanpour: BMS/Celgene: Honoraria; Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; GSK: Honoraria.

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Longitudinal T Cell Immunoprofiling of Patients with Relapsed and/or Refractory Myeloma Who Receive Daratumumab Monotherapy: A Subanalysis of a Phase 2 Study (the REBUILD Study)

Blood ◽

10.1182/blood-2019-124655 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3167-3167 ◽

Cited By ~ 1

Author(s):

Elisavet Vlachonikola ◽

Anna Vardi ◽

Eftathios Kastritis ◽

Evdoxia Hatjiharissi ◽

Eirini Katodritou ◽

...

Keyword(s):

Flow Cytometry ◽

T Cell ◽

Clinical Response ◽

Board Of Directors ◽

Research Funding ◽

Post Treatment ◽

Secondary Endpoint ◽

Median Frequency ◽

Advisory Committees ◽

Pre Treatment

Recent evidence suggests immunomodulatory effects of daratumumab in heavily pre-treated Multiple Myeloma (MM) patients (pts); however, the precise effects remain under-characterized, particularly at the molecular level. REBUILD is an ongoing prospective, multicenter, non-comparative, open-label, phase II study that evaluates the effects of daratumumab monotherapy on bone metabolism of pts with relapsed and/or refractory MM (RRMM) who have had ≥2 prior lines of therapy, including lenalidomide and a proteasome inhibitor. Secondary endpoint of the study included the evaluation of T cell dynamics by comprehensive analysis of the T cell receptor (TR) repertoire employing next generation sequencing (NGS) and multi-color flow cytometry. Herein we report the results of this secondary endpoint for the first 14 pts who completed 3 cycles of daratumumab monotherapy. In total, we analyzed 28 paired samples collected at screening (n=14) and on Day 1 of Cycle 4 (C4D1, n=14) of treatment in order to assess potential changes in relation to treatment and clinical response. Patients were grouped based on best responses into responders (i.e. patients with partial response [PR, n=1] and very good PR [VGPR, n=6]), and non-responders (i.e. patients with minimal response [MR, n=2], stable disease [SD, n=4], or progressive disease [PD, n=1]). Starting material was peripheral blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw NGS reads (n=6,715,406 | median 221,145/sample) were processed through a previously published, purpose-built bioinformatics pipeline. Only productive TRBV-TRBD-TRBJ rearrangements were taken into consideration (n=3,097,565 | median 101,670/sample) for the computation of clonotypes (i.e. TRB rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence). Overall, 151,153 distinct clonotypes (median 5,084 clonotypes/sample) were assessed. Both groups (responders/non-responders) displayed clonal T cell expansions both pre- and post-treatment. Clonality was found to be increased after treatment for both responders and non-responders, with statistical significance in the former (median cumulative frequency of the 10 most expanded T cell clonotypes/sample: 31% pre-treatment versus 40% post-treatment, respectively | p=0.04). In both groups, the clonotype repertoire appeared to be renewed with only a small fraction of pre-treatment clonotypes remaining after treatment (1% for non-responders; 0.6% for responders). Interestingly, in the responders' group we noticed a significant shift in the major clonotype repertoire at screening vs C4D1. In particular, in the responders' group the 10 most expanded clonotypes/sample at C4D1 represented expansions of clonotypes present at very low frequency at screening, whereas the most expanded clonotypes at screening decreased or even diminished post-treatment, suggesting that daratumumab treatment led to the emergence of anti-myeloma T cell clones which contributed to clinical response. On the contrary, the 10 most expanded pre-treatment clonotypes in the non-responders' group tended to dominate also the post-treatment repertoire. Of note, 13 shared clonotypes were identified amongst the post-treatment repertoires of different patients (responders/non-responders); shared clonotypes were not found in other entities in public databases, raising the possibility that they may be "disease-specific" and selected by common tumor-associated antigens. With a single exception, shared clonotypes were detected in cases with relevant HLA restrictions, which is noteworthy given the random HLA background of our cohort. Finally, flow cytometry analysis revealed a significant increase post treatment in the percentage of CD3+ T cells (median frequency at screening 60% versus 83% at C4D1 | p=0.003), driven mostly by the expansion of the CD8+ T cell compartment (median frequency at screening 30.8% versus 48.9% at C4D1 | p=0.03) in both groups. In conclusion, TR clonality increases post-treatment through a renewal mechanism; however, pre-treatment clones significantly expanded post-treatment in responders, alluding to the existence of clonotypes with anti-MM properties that may be activated after treatment with daratumumab, arguably contributing to clinical response. Disclosures Kastritis: Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria; Amgen: Honoraria, Research Funding. Hatjiharissi:Janssen: Honoraria. Katodritou:Genesis: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Gavriatopoulou:Genesis: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses; Takeda: Honoraria, Other: Travel expenses; Amgen: Honoraria. Delimpasi:Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria; Genesis: Honoraria, Other: Travel grant. Symeonidis:Sanofi: Research Funding; MSD: 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; Pfizer: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tekeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Janssen: Honoraria, Other: Travel expenses, Research Funding; Medison: Honoraria; Genesis: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Celgene: Honoraria; Takeda: Honoraria, Other: Travel expenses, Research Funding. Chatzidimitriou:Janssen: Honoraria.

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p53 Functional Assessment and Correlation with 17p Deletion and/or TP53 Mutation Status: Final Report of the ICLL001 Bomp Trial

Blood ◽

10.1182/blood-2018-99-116722 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3127-3127

Author(s):

Magali Le Garff-Tavernier ◽

Claire Quiney ◽

Lauren Veronese ◽

Florence Nguyen-Khac ◽

Pauline Robbe ◽

...

Keyword(s):

Functional Status ◽

Clinical Response ◽

Board Of Directors ◽

Research Funding ◽

Gene Disruption ◽

Tp53 Mutation ◽

Tp53 Gene ◽

Functional Assay ◽

Type B ◽

Advisory Committees

Abstract Introduction: The 17p deletion (del(17p)) resulting in loss of the TP53 gene is associated with impaired response to genotoxic agents and has an impact on PFS following BTK inhibitor and possibly also venetoclax. The del(17p) usually coincides with TP53 mutation, leading to the impairment of the p53-associated pathway. Sole TP53 mutations appear also associated with poor outcome in prospective trials. The iwCLL guidelines recommend to look for del(17p) and TP53 mutation before each line of treatment. An original approach is the functional assay, which highlights the functional abnormalities of p53 whether it is a TP53 gene disruption (del(17p) and/or TP53 mutation) or a defect of another actor in the p53 pathway. We aim to validate this functional assay on a prospective trial and to study the impact of p53 status on the clinical response regardless of the biological method. Methods: Clinical and biological data were collected from 74 CLL patients (pts) enrolled in the BOMP phase II trial of the French Innovative Leukemia Organization (FILO) (NCT01612988) evaluating 6 monthly courses of BOMP including bendamustine, ofatumumab and high dose methylprednisolone in fit pts with relapsing CLL. In addition to conventional screening, we focused on p53 evaluation at time of inclusion. FISH analysis for del(17p) was done with a 5% cut-off for positive result. TP53 gene mutation screening was performed by Sanger sequencing of the coding region (exons 2-11). A targeted NGS screening (19 genes including TP53, Illumina MiSeq) was also performed. The p53 functional status was determined by a flow cytometry assay based on induction of p53 and p21 protein expression after etoposide and nutlin-3 exposition, as previously described (Le Garff-Tavernier M., 2011), which allows the detection of 3 types of p53 dysfunction (A, B and C), irrespective of an ATM default. Clinical response was evaluated by PFS, OS and TTNT Kaplan-Meier analyses (MedCalc stat). Results: Data from the whole cohort are available. Median age was 64 yrs. Pts had a median of 1 (1-3) lines of treatment previous to this trial, including FCR in >90%. Concerning p53 evaluation, a del(17p) was found in 30% of cases by FISH (22/73 pts with a median of 68% positive cells, range 10-98). The percentage of p53 abnormalities increased to 41% when TP53 mutations were screened (30/73 pts with 1 to 8 mutations, median VAF 10 %, range 1.6-90). Results from the p53 functional assay were available for 69 pts showing the highest level of p53 abnormalities. Indeed, p53 dysfunction was observed in 48% of pts (33/69) including type A (n=11), type B (n=17) and type C (n=5) dysfunction. Thus, the sensitivity and specificity of the p53 functional assay to detect pts with del(17p) and/or TP53 mutation were of 87% and 84% respectively (n=68 pts for which the 3 tests were available). Interestingly, discordant results were observed in 10 pts: 4 pts with a functional p53 despite a TP53 gene disruption (3 with TP53 mutation only and 1 with del(17p) only) and conversely 6 pts with a p53 dysfunction (all with type B dysfunction) but without any TP53 gene disruption, suggesting alternative alterations of the p53 pathway. The only similarity for those latter pts is the occurrence of at least one ATM abnormality (del(11q) and/or ATM mutation). The combination of the 3 assays defines 3 groups: (1) "intact p53" (no TP53 disruption and functional p53, n=32), (2) "altered p53" (TP53 disruption and p53 dysfunction, n=26) and (3) "discordant p53" (n=10). PFS and TTNT were higher in pts without (n=38) compared to those with TP53 gene disruption (n=30) (p=0.04 for both). The OS, even though not significant, presented a similar trend. When considering the functional status, a similar profile is observed but with a better discrimination between pts with normal p53 function (n=36) and pts with p53 dysfunction (n=32) (p=0.002 and 0.003, respectively). Combining the 3 assays, PFS and TTNT of the group 3 "discordant p53" profiles' appeared intermediate (Figure 1). Conclusion: This study shows that a p53 functional analysis can predict with an acceptable sensitivity the presence of a TP53 gene disruption. Interestingly, this functional assay coupled with cytogenetic and mutational screening could reveal a sub-group of pts with discordant results for which PFS and TTNT appeared intermediate. Evaluation of other discordant cases is mandatory to confirm these results and could lead to a wider use of this global functional approach. Figure 1. Figure 1. Disclosures Feugier: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sylvain:Gilead: Other: scientific advisor board. Schuh:Giles, Roche, Janssen, AbbVie: Honoraria. Guieze:abbvie: Honoraria; janssen: Honoraria; gilead: Honoraria. Leblond:Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Sandoz: Honoraria; Amgen: Honoraria.

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Chromosomal Translocation t(11;14) Induced By the Cre-Loxp System in Normal B Cell-Derived Ips Cells for the Study of Myeloma-Initiating Cells

Blood ◽

10.1182/blood-2020-134572 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 18-19

Author(s):

Misaki Sugai ◽

Naohiro Tsuyama ◽

Yu Abe ◽

Yusuke Azami ◽

Kenichi Kudo ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

B Lymphocytes ◽

Research Funding ◽

Chromosomal Translocation ◽

Stem Cell Differentiation ◽

Advisory Committees ◽

Myeloma Cells ◽

Company Limited

The cellular origin of multiple myeloma (MM) has not yet been identified. Based on immunoglobulin heavy chain (IgH) gene analysis, myeloma cells are derived from mature B cells. Chromosomal aberrations such as trisomy and chromosomal translocation (cTr) play a critical role in the early tumorigenesis of MM. We hypothesized that the abnormal cells from which myeloma cells originate might be mature B lymphocytes with chromosomal or genetic changes in the reprogrammed state that enable them to acquire the potential to become tumors in the process of redifferentiation into B lymphocytes. We established induced pluripotent stem cells (iPSs) from normal B lymphocytes (BiPSCs: BiPSC13 & MIB2-6); these BiPSCs have the same VDJ rearrangement of IgH as the original B lymphocytes and differentiate into CD34+/CD38- hematopoietic progenitor cells co-culture with stromal cells, AGM-S3 (Sci Rep, 2017). We then established a method to induce reciprocal cTr t(11;14), which is a reciprocal cTr between IgH and CCND1 and the most frequent cTr in MM, using the CRISPR/Cas9 system; cTr was induced by infection of IgH-CCND1 lentiCRISPRv2 lentivirus, which targets the human IgH Eµ region and 13kb upstream of the CCND1 coding sequence, to BiPSCs (Oncol Lett, 2019). Subsequently, we established cell lines carrying reciprocal cTr t(11;14) between CCND1 and either an allele in which VDJ rearrangement of IgH had been completed or an allele in which VDJ rearrangement had not been completed (stopped at DJ joining) in BiPSC13 t(11;14) (AZ & AX) and MIB2-6 t(11;14) (BC & BG), respectively. These BiPSCs differentiated into CD34+/CD38-/CD45+/-/CD43+/- hematopoietic progenitors cells in co-culture with AGM-S3 or in stem cell differentiation medium; this was subsequently confirmed by the differentiation into granulocytes, macrophages, and erythroblasts in a colony-formation assay. We are now trying to produce BiPSCs in which cTr t(11;14) is induced when they differentiate into mature B cells expressing CD27. First, we used the Cre-loxP recombination system to induce cTr t(11;14) in BiPSCs. BiPSCs were transfected with IgH loxP-Neo-loxP knock-in vector and IgH lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying loxP-Neo-loxP in IgH were transfected with iCre-EGFP. After removing the loxP-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP were transfected with CCND1 loxP-FRT3-Neo-FRT3 knock-in vector and CCND1 lentiCRISPRv2 vector. Subsequently, G418-resistant BiPSCs carrying IgH-loxP in IgH and loxP-FRT3-Neo-FRT3 in CCND1 were transfected with Flpo-EGFP. After removing the FRT3-Neo site from EGFP-positive cells, BiPSCs carrying IgH-loxP in IgH and CCND1-loxP-FRT3 in CCND1 were transfected with iCre-HygR. Hygromycin B-resistant cells were picked, the reciprocal cTr t(11;14) was confirmed by polymerase chain reaction, and we established BiPSCs with der(11)t(11;14) and BiPSCs with der(14)t(11;14). We also developed a system in which Cre is expressed along with CD27 expression in the B cell lymphoma cell line Raji. These BiPSCs could be useful for the study of myeloma-initiating cells, but whether they would be able to be redifferentiated into B lymphocyte is important. Disclosures Hanamura: Mundipharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SHIONOGI Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis Pharma K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; DAIICHI SANKYO COMPANY, LIMITED: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Eisai Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; NIPPON SHINYAKU CO.,LTD.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Japan Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ono Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

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Comparison of Steady-State Imatinib (IM) Trough Levels, Clinical Response, and Safety Between Caucasian and Asian Patients with Chronic Lyeloid Leukemia in Chronic Phase (CML-CP) Treated with 400mg and 800mg Daily Doses of IM in the Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) Study.

Blood ◽

10.1182/blood.v114.22.1127.1127 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1127-1127

Author(s):

Dong-Wook Kim ◽

Camille Granvil ◽

Eren Demirhan ◽

John Reynolds ◽

Yu Jin ◽

...

Keyword(s):

Adverse Event ◽

Steady State ◽

Clinical Response ◽

Board Of Directors ◽

Research Funding ◽

Average Dose ◽

Advisory Committees ◽

Equity Ownership ◽

Grade 3 ◽

Trough Levels

Abstract Abstract 1127 Poster Board I-149 Background In the TOPS study, IM trough levels (Cmin) were collected from different race groups, mainly Caucasian and Asian, but also Black and others. Inter-ethnic differences in the PK of a drug are known to be important factors accounting for inter-individual variation in drug responsiveness. This analysis reports the comparison between Caucasian and Asian CML patients (pts) treated at doses of 400 mg QD and 400 mg bid (800 mg daily) of IM Cmin on Day 29 of initial treatment, clinical response, safety and tolerability. Methods Steady state IM Cmin on Day 29 and clinical response and safety data obtained during the first 12 months (mos) of treatment were obtained from pts randomized 2:1 to 800 mg or 400 mg daily IM. The steady-state Cmin was defined as predose concentration collected approximately within ±3 hours of the scheduled dosing time on Day 29. The associations of race with the rates of major molecular response (MMR) and complete cytogenetic response (CCyR) were evaluated. Correlation of IM exposure with clinical response (MMR and CCyR) was assessed by grouping pts into quartiles based on their measured IM Cmin levels at Day 29. The safety endpoint for each pt was the presence or absence of an adverse event (AE) of grade 3 or higher in the first 12 mos from the first dose. Results IM Cmin levels were available from 229 pts in TOPS including 54 Caucasians, 18 Asians, and 14 Black and others at 400 mg (total 86) and 103 Caucasians, 29 Asians, and 11 Black and others at 800 mg (total 143). For the first 12 mos, the means of the average dose intensities for Asian (mean [range], 362 [267-400] in 400 mg and 666 [226-800] in 800 mg) were not significantly different from Caucasian (386 [204-597] in 400 mg and 666 [289-800] in 800 mg) (P=0.070 and P=0.995 for the 400 mg and 800 mg arms, respectively). Mean (± SD) of IM Cmin levels (ng/mL) with respect to race are shown in Table 1. IM Cmin was slightly over-proportional to dose and showed large interpatient variability (CV=42-60%) for both dose groups regardless of the race group. In the lower quartile Cmin group (Cmin<1290 ng/mL), the differences in CCyR and MMR rates between Asian and Caucasian pts were significant (P=0.031 and P=0.022 respectively), which was probably due to a higher rate of dose interruptions in the 1st 12 mos in Asian pts. A definitive conclusion cannot be drawn due to the small number of Asian pts. Occurrences of at least one grade 3 or 4 adverse event were found to be significantly higher in Asian pts (69% and 75% in the 1st 6 and 12 mos respectively) compared to Caucasian pts (53% and 57% in the 1st 6 and 12 mos respectively) (P=0.028 and P=0.008 respectively). Conclusion The results of this analysis from TOPS show that IM Cmin levels were similar between Caucasian and Asian CML pts in each treatment arm. There were no major differences in efficacy, as measured by MMR and CCyR rates by 12 mos, between Asian and Caucasian pts. There were no unexpected differences in patterns of AEs between Caucasian and Asian pts; however, occurrences of one or more grade 3 AEs were higher in Asian. Further analysis on a larger group of CML pts will be needed to evaluate the impact of AE rate differences between Caucasian and Asian pts. Disclosures Kim: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Wyeth: Research Funding. Granvil:Novartis: Employment. Demirhan:Novartis: Employment. Reynolds:Novartis: Employment. Jin:Novartis: Employment. Wang:Novartis: Employment, Equity Ownership. Baccarani:Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau. Cortes-Franco:Novartis: Honoraria, Research Funding, Speakers Bureau; Wyeth: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding. Hughes:Bristol-Myers Squibb: Advisor, Honoraria, Research Funding; Novartis: Advisor, Honoraria, Research Funding. Guilhot:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria.

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