scholarly journals Gene Expression and Cytokine Analyses Identify Markers of Progression from CLL-like Monoclonal B-Cell Lymphocytosis to Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3027-3027
Author(s):  
Gonzalo Blanco ◽  
Anna Puiggros ◽  
Barbara Sherry ◽  
Lara Nonell ◽  
Eulalia Puigdecanet ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Advisory Committees ◽  
Cytokine Levels

INTRODUCTION: Chronic lymphocytic leukemia (CLL)-like monoclonal B cell lymphocytosis (MBL) is considered a precursor of CLL. It is found in 5-10% of elderly healthy individuals and shows a progression rate to CLL requiring therapy of 1.1% per year. A balance between microenvironmental factors and intrinsic properties of the emerging B cell clone may be decisive for the transition from MBL to CLL, although biomarkers of progression remain unknown. The objective is to describe biological markers (B cell gene expression profiles and serum cytokine levels) that predict progression from MBL to CLL. METHODS: Gene expression profiles of clonal B cells from 14 MBL subjects (median age: 76 years, clonal B cells: 0.5-4.3 x109/L) were evaluated. With a median follow-up from analysis of 59 months (range: 10-77), 3 cases (21.4%) had progressed to CLL Binet stage A at last follow-up (clonal lymphocytosis >5x109/L, range: 6.2-7.9). Clonal B cells (CD19+CD5+) were isolated from peripheral blood by immunomagnetic methods (Miltenyi Biotec). Extracted RNA (RIN>7) was hybridized to GeneChip Human Gene 2.0 ST arrays (Affymetrix). Gene expression profiles were compared between MBL cases that progressed to CLL (P-MBL, n=3) and non-progressive MBL cases (NP-MBL, n=11). Differential gene expression was evaluated employing linear models for microarrays in R, and genes with P<0.05 and Fold Change >1.5 or <-1.5 were considered differentially expressed. To obtain insight into the functional significance of the differential genetic signatures, the Ingenuity Pathway Analysis tool (IPA, QIAGEN) was employed. On the other hand, serum levels of IL1β, IL2, IL4, IL5, IL6, IL8, IL10, IL12, IL15, IL17, IFNα, IFNγ, TNFα, GM-CSF, CCL3, CCL4, CCL19, CXCL9, CXCL10 and CXCL11 were quantified using the U-PLEX Platform (Meso Scale Discovery) and Human CXCL9/MIG Quantikine ELISA Kit (R&D Systems) in 41 MBL subjects (median age: 67 years, clonal B cells: 0.5-4.8 x109/L). With a median follow-up from analysis of 47 months (range: 0-117), 5 of them (12.2%) had progressed to CLL Binet stage A at last follow-up (clonal lymphocytosis >5x109/L, range: 6.4-17.3). Clonal B cells and cytokine levels were compared between P-MBL (n=5) and NP-MBL (N=36). For cytokine levels, the optimal cut-off values to stratify MBL cases according to their progression risk were assessed using the maxstat R package, whereas for clonal B cells a cut-off value of 3.9 x109/L was considered according to the results obtained by Kostopoulos et al (Blood Cancer J, 2017). The effect of different covariates on progression-free survival was evaluated using log-rank test. Cox proportional hazards regression models were performed to assess their independent prognostic value. P<0.05 was considered significant. RESULTS: A total of 455 genes were differentially expressed (250 upregulated and 205 downregulated in P-MBL). IPA predicted an inhibition of apoptosis as well as proteins with tumor suppressor activity (SMARCA4) in P-MBL, besides enhanced bioenergetic processes (transmembrane potential of mitochondria) and anti-inflammatory features (activation of IL13 pathway and decreased chemotaxis of phagocytes and granulocytes) (Table 1). P-MBL displayed increased clonal B cells (4.2 vs. 1.7 x109/L, P=0.003) and levels of IL10 (1.15 vs. 0.9 pg/mL, P=0.087) as well as diminished levels of IL6 (2.04 vs. 3.75 pg/mL, P=0.041). MBL cases with ≥3.9 x109/L clonal B cells, ≥1.08 pg/mL of IL10 and ≤2.04 pg/mL of IL6 had an increased risk of progression to CLL (P<0.001, P=0.006 and P=0.034, respectively) (Figure 1, Table 2). Multivariate analysis for clonal B cells and levels of IL10 maintained significance for both factors (HR=12.8, P=0.013 and HR=10.2, P=0.047, respectively) (Table 2). CONCLUSIONS: 1. P-MBL cases showed an inhibition of the apoptotic pathway and an activation of bioenergetic processes, which may account for the increased clonal B cells observed in this group. 2. P-MBL exhibited enhanced anti-inflammatory features, including augmented levels of the anti-inflammatory cytokine IL10. 3. Increased clonal B cells and IL10 levels predicted a higher risk of progression to CLL, suggesting that an augmented proliferative rate of clonal B cells together with a supporting tumor microenvironment are required for progression from MBL to CLL. ACKNOWLEDGEMENTS. PI11/01621, PI15/00437, 2017/SGR437, Fundació La Caixa, Fundación Española de Hematología y Hemoterapia (FEHH). Disclosures Gimeno: JANSSEN: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Pharmacyctics: Membership on an entity's Board of Directors or advisory committees. Abrisqueta:Roche: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, expenses, Speakers Bureau; Celgene: Consultancy, Honoraria. Bosch:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Acerta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Honoraria, Research Funding; Takeda: Honoraria, Research Funding.

Distinct Gene Expression Signatures in Patients with Richter's Syndrome and Chronic Lymphocytic Leukemia with Prior Exposure to Ibrutinib

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Hanyin Wang ◽  
Shulan Tian ◽  
Qing Zhao ◽  
Wendy Blumenschein ◽  
Jennifer H. Yearley ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Activation ◽  
Expression Profiles ◽  
Lymphocytic Leukemia ◽  
B Cell Activation ◽  
Advisory Committees ◽  
Upregulated Genes

Introduction: Richter's syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive lymphoma with dismal prognosis. Transcriptomic alterations have been described in CLL but most studies focused on peripheral blood samples with minimal data on RS-involved tissue. Moreover, transcriptomic features of RS have not been well defined in the era of CLL novel therapies. In this study we investigated transcriptomic profiles of CLL/RS-involved nodal tissue using samples from a clinical trial cohort of refractory CLL and RS patients treated with Pembrolizumab (NCT02332980). Methods: Nodal samples from 9 RS and 4 CLL patients in MC1485 trial cohort were reviewed and classified as previously published (Ding et al, Blood 2017). All samples were collected prior to Pembrolizumab treatment. Targeted gene expression profiling of 789 immune-related genes were performed on FFPE nodal samples using Nanostring nCounter® Analysis System (NanoString Technologies, Seattle, WA). Differential expression analysis was performed using NanoStringDiff. Genes with 2 fold-change in expression with a false-discovery rate less than 5% were considered differentially expressed. Results: The details for the therapy history of this cohort were illustrated in Figure 1a. All patients exposed to prior ibrutinib before the tissue biopsy had developed clinical progression while receiving ibrutinib. Unsupervised hierarchical clustering using the 300 most variable genes in expression revealed two clusters: C1 and C2 (Figure 1b). C1 included 4 RS and 3 CLL treated with prior chemotherapy without prior ibrutinib, and 1 RS treated with prior ibrutinib. C2 included 1 CLL and 3 RS received prior ibrutinib, and 1 RS treated with chemotherapy. The segregation of gene expression profiles in samples was largely driven by recent exposure to ibrutinib. In C1 cluster (majority had no prior ibrutinb), RS and CLL samples were clearly separated into two subgroups (Figure 1b). In C2 cluster, CLL 8 treated with ibrutinib showed more similarity in gene expression to RS, than to other CLL samples treated with chemotherapy. In comparison of C2 to C1, we identified 71 differentially expressed genes, of which 34 genes were downregulated and 37 were upregulated in C2. Among the upregulated genes in C2 (majority had prior ibrutinib) are known immune modulating genes including LILRA6, FCGR3A, IL-10, CD163, CD14, IL-2RB (figure 1c). Downregulated genes in C2 are involved in B cell activation including CD40LG, CD22, CD79A, MS4A1 (CD20), and LTB, reflecting the expected biological effect of ibrutinib in reducing B cell activation. Among the 9 RS samples, we compared gene profiles between the two groups of RS with or without prior ibrutinib therapy. 38 downregulated genes and 10 upregulated genes were found in the 4 RS treated with ibrutinib in comparison with 5 RS treated with chemotherapy. The top upregulated genes in the ibrutinib-exposed group included PTHLH, S100A8, IGSF3, TERT, and PRKCB, while the downregulated genes in these samples included MS4A1, LTB and CD38 (figure 1d). In order to delineate the differences of RS vs CLL, we compared gene expression profiles between 5 RS samples and 3 CLL samples that were treated with only chemotherapy. RS samples showed significant upregulation of 129 genes and downregulation of 7 genes. Among the most significantly upregulated genes are multiple genes involved in monocyte and myeloid lineage regulation including TNFSF13, S100A9, FCN1, LGALS2, CD14, FCGR2A, SERPINA1, and LILRB3. Conclusion: Our study indicates that ibrutinib-resistant, RS-involved tissues are characterized by downregulation of genes in B cell activation, but with PRKCB and TERT upregulation. Furthermore, RS-involved nodal tissues display the increased expression of genes involved in myeloid/monocytic regulation in comparison with CLL-involved nodal tissues. These findings implicate that differential therapies for RS and CLL patients need to be adopted based on their prior therapy and gene expression signatures. Studies using large sample size will be needed to verify this hypothesis. Figure Disclosures Zhao: Merck: Current Employment. Blumenschein:Merck: Current Employment. Yearley:Merck: Current Employment. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Parikh:Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kenderian:Sunesis: Research Funding; MorphoSys: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Gilead: Research Funding; BMS: Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Juno: Research Funding; Mettaforge: Patents & Royalties; Torque: Consultancy; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Mutation Burden in Multiple Myeloma Is Captured By Gene Expression Profiles

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4450-4450
Author(s):  
John D Shaughnessy ◽  
Samir Parekh ◽  
Hearn Jay Cho ◽  
Alessandro Lagana ◽  
Ajai Chari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Board Of Directors ◽  
Research Funding ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Training Set ◽  
Advisory Committees ◽  
Test Set ◽  
Kegg Pathways ◽  
Mutation Burden

Abstract In the current study we sought to determine whether mutation burden (MB) is reflected in gene expression patterns. We generated 389 gene expression profiles and 311 mutation profiles from 182 cases, split into a training set of 97 and test set of 84. Copy number variants (CNV), rearrangements (TX) and short variant mutations (SV) were identified with FoundationOne Heme (F1) and U133Plus2.0 (u133) gene expression data, GEP70 and GEP80 risk scores, subgroup and CNV calls provided by Signal Genetics. 145 Kegg pathways, transcription factor binding sites (TFbs) for 195 TF mapped to u133 genes and 1161 F1 features and u133 GEP signatures/scores were evaluated for enrichment of defined genes. u133 data for normal plasma cells (n=22), MGUS (n=44), smoldering MM (n=12), relapsed MM (n=76), and MM cell lines (n=42) were derived from GSE31162. Newly diagnosed MM samples came from GSE31162 (n=584), GSE19784 (n=321), GSE15695 (n=247) and E-MTAB-317 (n=233). Of 593 genes assayed by F1, 293 were mutated at least twice. There were a total of 3454 mutations (average = 11, minimum = 3, and maximum = 37). KRAS was mutated in 40 tumors, while TP53 was mutated 45 times in 31 tumors. TP53 and 62 other genes had 2 or more unique mutations in a single tumor. A linear curve of MB exhibited a sharp upward inflection at 19 mutations. We sought to determine if GEP could identify characteristic features of MM flanking the inflection point. A training set of 97 (86 <= 15 MB and 11 >= 21 MB) and a test set of 84 (49 <= 15 MB, 28 >15 but < 21 MB and 7 >= 21MB) was produced. A mean ratio identified 576 genes exhibiting 2-fold higher expression in MM with high MB (hiMB) and 1617 genes from low MB (loMB). Notably, forty-four of the 293 mutated genes were in this list of genes. A geometric mean ratio of the two gene sets was then calculated for all samples. The mean of the resulting score (MB.2) was higher in MM with hiMB (1.66) than MM with loMB (-0.235) in the training set. MM with hiMB (0.329) had a higher MB.2 score than the group with intermediate MB.2 (0.158) and both higher than MM with loMB (-0.122). MB.2 was lowest in normal PC (-0.518) and progressively increased with disease progression: MGUS (-0.341), SMM (-0.308), MM (0.199), relapsed MM (0.334), MM cell lines (1.168).[SP1] 57% of the 182 cases harbored only SV mutations, 32% had SV and CNV, 32% SV and TX and 7% had SV, CNV and TX mutations. SV only mutations were present in 76% of MB.2 quartile 1 (MBq1) and 30% of MBq4. SV, CNV and TX mutations were present in 4% of MBq1 and 17% of MBq4. MB.2 was positively correlated with GEP70, GEP80, proliferation index, and TP53 target genes in MM and genes modulated by thalidomide and dexamethasone in PGx studies, in at least 6 of the 7 cohorts studied. The CD2 subtype, a myeloid classification and GEP70 low risk were significantly overrepresented in both MBq1 and GEP70q1 in all 7 cohorts. Conversely, the MF, MS, and PR subtypes, GEP70 and GEP80 high risk, as well as +1q, amp1q21, and del13q were significantly overrepresented in MBq4 and GEP70q4 in all 7 cohorts. MB.2 [SP2] genes derived from MM with loMB where enriched in 45 of 148 Kegg pathways. Notable were Hedgehog, Prostaglandin, Tx factors in cancer, HOX, MYB signaling, ephrin-B reverse signaling and embryonic stem cells. Five pathways related to B-cell biology were enriched. Mitotic cell cycle, integrin signaling, chromatin acetylation, ubiquitin ligation, and G1 to G1/S were underrepresented. MB.2 genes from MM with hiMB were enriched in TP53, lipid lysis, complement cascade, adherens junctions, Wnt regulation of CYR61, cyclins, prostaglandins, cell cycle, and MYC target pathways. Interferon signaling, TNF-NF-kB, EGFR, NOD, endoplasmic reticulum, ubiquitin ligation, Wnt-Hedgehog-NOTCH and BMP-SMAD modules were underrepresented. An enrichment of Rel and NF-kB TFbs was observed for genes negatively correlated with MB.2. and genes positively correlated with MB.2 and GEP70 were enriched for E2F and TP53 binding site. In conclusion, we show that MB can be captured by GEP in MM, that MB increases with disease progression, and pathways enriched by hiMB and loMB are different and may imply differences in pathogenesis as well as treatment. [SP1]This is an important finding - suggest emphasizing more [SP2]Starting with this para suggest referring to groups are low vs high mutation burden for improved readability. Disclosures Shaughnessy: Signal Genetics: Consultancy, Patents & Royalties. Cho:Genentech Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agenus, Inc.: Research Funding; Janssen: Consultancy, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ludwig Institute for Cancer Research: Membership on an entity's Board of Directors or advisory committees. Chari:Novartis: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Array Biopharma: Consultancy, Research Funding; Amgen Inc.: Honoraria, Research Funding; Pharmacyclics: Research Funding. van Laar:Signal Genetics, Inc.: Employment. Jagannath:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myer Squibb: 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. Barlogie:Signal Genetics: Patents & Royalties.

scholarly journals FcmR Shapes BCR Signaling in IgM-Positive Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2620-2620
Author(s):  
Alexandra da Palma Guerreiro ◽  
Cornelia Dorweiler ◽  
Ismini Halmer ◽  
Olaf Merkel ◽  
Elena Maria Hartmann ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Research Funding ◽  
Functional Role ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Malignant Cells ◽  
Support Research

Abstract Background: The Fc receptor for IgM (FcmR/ TOSO) is significantly overexpressed on chronic lymphocytic leukemia (CLL) cells from peripheral blood, but becomes down-regulated in the tumor microenvironment by e.g. CD40:CD40L interaction. Since the functional role of FcmR on lymphomagenesis is still not understood, we developed a conditional knockout mouse with B cell-specific FcmR-depletion. These mice were crossbred with the Eµ-TCL1 murine model, which develops a CLL-like phenotype. Results: The depletion of FcmR/TOSO in TCL1 mice (Eµ-Tcl1tg/wt FcmRfl/fl CD19cre/wt; further on called TCT) revealed a significantly shorter overall survival (296 days; n=40) compared to the TOSO expressing control mice (Eµ-Tcl1tg/wt FcmRwt/wt CD19cre/wt; TC; 344 days; n=106; Log-rank p<0.0001). In addition, these mice show a significantly higher blood leukocyte count and lower platelet and erythrocyte count. Leukocytes could be identified as CLL-characteristic leukemic CD19+/CD5+ B cells. Altogether TCT exhibited a faster progress of disease. Spleen immunohistochemistry revealed the transformation of most TCT (14/17 transformed) into an even more aggressive phenotype with increased splenomegaly and change in tissue and cell morphology compared to TC (9/9 not transformed). While characterizing these cells by flow cytometry, we identified a significantly higher expression of IgM on malignant B cells from TCT in comparison to TC mice. This finding indicates that the BCR itself might have a different contribution to lymphomagenesis in FcmR knock-out settings. Therefore, to validate the functional role of FcmR in the process of lymphomagenesis, we performed transcriptome profiling by RNA-Seq using splenic leukemic cells (CD19+ CD5+) from 36-week old TC (n=4) and TCT (n=4) mice. 2089 genes were found to be significantly modulated in the malignant cells of TCT mice, from which 1221 were downregulated and 868 showed an upregulation (significant change in mean expression; p<0.05). To investigate the role of IgM on TCT mice, purified malignant B cells were incubated for two hours with F(ab')2 goat anti-mouse IgM. Strikingly, TCT mice showed 3941 genes (2054 downregulated, 1887 upregulated) with significant difference in expression compared to TC (p<0.05). The gene expression profiles of the anti-IgM treated mice revealed a stronger regulation of BCR signalling in TCT mice, suggesting that FcmR represents an important factor in these processes. We examined the gene expression profiles, using Ingenuity Pathway Analysis Software. Analysis revealed that the most deregulated functions include interferon-signalling, recruitment of leukocytes, infection of cells and cellular movement. Conclusion: Here we present functional evidence that loss of FcmR results in increased IgM/BCR on the surface of non-switched leukemia. Moreover, malignant cells with loss of FcmR are more susceptible to BCR stimulation and show a signature of signalling pathways, which contribute to inflammation in B cell malignancies. Disclosures Fingerle-Rowson: MorphoSys: Employment. Pallasch:Gilead: Research Funding. Wendtner:Abbvie: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Genetech: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen: Consultancy, Honoraria, Other: travel support, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: travel support, Research Funding; MorphoSys: Consultancy, Honoraria, Other: travel support, Research Funding; Roche: Consultancy, Honoraria, Other: travel support, Research Funding.

scholarly journals Impact of Interim FDG-PET (iPET) in the Outcomes of Patients with Aggressive B-Cell Lymphomas Receiving DA-EPOCH-R

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2898-2898
Author(s):  
Vania Phuoc ◽  
Leidy Isenalumhe ◽  
Hayder Saeed ◽  
Celeste Bello ◽  
Bijal Shah ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Fdg Pet ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
End Of Treatment

Introduction: 2-[18F] fluoro-2-deoxy-D-glucose positron emission tomography (FDG-PET) remains the standard of care for baseline and end of treatment scans for aggressive non-Hodgkin lymphomas (NHLs). However, the role of interim FDG-PET remains not as well defined across aggressive NHLs, especially in the era of high-intensity chemoimmunotherapy. Interim FDG-PET (iPET) can serve as an early prognostic tool, and prior studies evaluating the utility of iPET-guided treatment strategies primarily focused on diffuse large B-cell lymphomas (DLBCL) and frontline R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). Classification criteria systems assessing response also differ between studies with no clear consensus between use of Deauville criteria (DC), International Harmonization Project (IHP), and the ΔSUVmax method. Methods: This study evaluates our institutional experience with iPET during treatment with DA-EPOCH ± R (dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin with or without Rituximab) in aggressive NHLs. We retrospectively evaluated 70 patients at Moffitt Cancer Center who started on DA-EPOCH ± R between 1/1/2014 to 12/31/2018 for aggressive NHLs. Response on interim and end-of-treatment (EOT) scans were graded per DC, IHP, and ΔSUVmax methods, and progression free survival (PFS) probability estimates were calculated with chi-square testing and Kaplan Meier method. PFS outcomes were compared between interim negative and positive scans based on each scoring method. Outcomes were also compared between groups based on interim versus EOT positive or negative scans. Results: We identified 70 patients with aggressive NHLs who received DA-EPOCH ± R at our institute. The most common diagnoses were DLBCL (61%) followed by Burkitt's lymphoma (10%), primary mediastinal B-cell lymphoma (9%), plasmablastic lymphoma (7%), gray zone lymphoma (6%), primary cutaneous large B-cell lymphoma (1%), primary effusion lymphoma (1%), and other high-grade NHL not otherwise specified (3%). Of the 43 patients with DLBCL, 21/43 (49%) had double hit lymphoma (DHL) while 7/43 (16%) had triple hit lymphoma (THL), and 3/43 (7%) had MYC-rearranged DLBCL while 2/43 (5%) had double expressor DLBCL. Thirty nine out of 70 (56%) were female, and median age at diagnosis was 58.39 years (range 22.99 - 86.86 years). Most patients had stage IV disease (49/70, 70%), and 43/70 (61%) had more than one extranodal site while 45/70 (64%) had IPI score ≥ 3. Forty-six out of 70 (66%) received central nervous system prophylaxis, most with intrathecal chemotherapy (44/70, 63%). Fifty-five out of 70 (79%) had iPET available while 6/70 (9%) had interim computerized tomography (CT) scans. Fifty-six out of 70 (80%) had EOT PET, and 4/70 (6%) had EOT CT scans. Sustained complete remission occurred in 46/70 (66%) after frontline DA-EPOCH ± R (CR1), and 12/70 (17%) were primary refractory while 5/70 (7%) had relapse after CR1. Four of 70 (6%) died before cycle 3, and 3/70 (4%) did not have long-term follow-up due to transition of care elsewhere. Median follow-up was 15.29 months (range 0.85 - 60.09 months). There was significantly better PFS observed if iPET showed DC 1-3 compared to DC 4-5 (Χ2=5.707, p=0.0169), and PFS was better if iPET was negative by IHP criteria (Χ2=4.254, p=0.0392) or ΔSUVmax method (Χ2=6.411, p=0.0113). Comparing iPET to EOT PET, there was significantly better PFS if iPET was negative with EOT PET negative (iPET-/EOT-) compared to iPET positive with EOT negative (iPET+/EOT-), and iPET+/EOT+ and iPET-/EOT+ had worse PFS after iPET-/EOT- and iPET+/EOT- respectively. This pattern in iPET/EOT PFS probability remained consistent when comparing DC (Χ2=30.041, p<0.0001), IHP (Χ2=49.078, p<0.0001), and ΔSUVmax method (Χ2=9.126, p=0.0104). These findings fit clinical expectations with positive EOT scans indicating primary refractory disease. There was no significant difference in PFS when comparing DLBCL versus non-DLBCL (Χ2=3.461, p=0.0628) or DHL/THL versus non-DHL/THL diagnoses (Χ2=2.850, p=0.0914). Conclusion: Our findings indicate a prognostic role of iPET during treatment with DA-EPOCH ± R for aggressive NHLs. Significant differences in PFS were seen when graded by DC, IHP, and ΔSUVmax methods used in prior studies and when comparing interim versus EOT response. Larger studies are needed to confirm these findings. Disclosures Bello: Celgene: Speakers Bureau. Shah:Novartis: Honoraria; AstraZeneca: Honoraria; Spectrum/Astrotech: Honoraria; Adaptive Biotechnologies: Honoraria; Pharmacyclics: Honoraria; Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; Kite/Gilead: Honoraria; Celgene/Juno: Honoraria. Sokol:EUSA: Consultancy. Chavez:Janssen Pharmaceuticals, Inc.: Speakers Bureau; Genentech: Speakers Bureau; Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

scholarly journals DYRK1A Is Regulated By Oncogenic KMT2A and Required for Survival of KMT2A-Rearranged Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2742-2742
Author(s):  
Christian Hurtz ◽  
Gerald Wertheim ◽  
Rahul S. Bhansali ◽  
Anne Lehman ◽  
Grace Jeschke ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Negative Regulator ◽  
Advisory Committees ◽  
Normal Tissues ◽  
Pharmacologic Inhibition

Background: Research efforts have focused upon uncovering critical leukemia-associated genetic alterations that may be amenable to therapeutic targeting with new drugs. Targeting the oncogenic BCR-ABL1 fusion protein in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (B-ALL) with tyrosine kinase inhibitors to shut down constitutive signaling activation and induce leukemia cell cytotoxicity has remarkably improved patients' survival and has established a precision medicine paradigm for kinase-driven leukemias. However, multiple subtypes of B-ALL are driven through non-tyrosine fusion proteins, including the high-risk KMT2A-rearranged (KMT2A-R) subtype common in infants with B-ALL, leaving many patients with insufficient treatment options. Objectives: KMT2A-R B-ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in older children/adults with B-ALL. Current intensive multiagent chemotherapy regimens induce significant side effects yet fail to cure the majority of patients, demonstrating continued need for novel therapeutic approaches. The goals of our study were to i) identify signaling molecules required for KMT2A-R B-ALL cell survival, ii) select ALL-associated targets that are not essential in normal tissues, and iii) develop new treatment strategies that may benefit patients with KMT2A-R ALL. Results: We performed a genome-wide kinome CRISPR screen using the pediatric KMT2A-R cell line SEM and identified DYRK1A among other signaling molecules as required for leukemia cell survival. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncogene in a murine Down syndrome (DS) model of megakaryoblastic leukemia. In normal hematopoiesis, DYRK1A controls the transition from proliferation to quiescence during lymphoid development. Deletion of DYRK1A results in increased numbers of B cells in S-G2-M phase, yet also significantly reduces cell proliferation. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines (SEM and RS4;11) and a human KMT2A-Aff1-FLAG-transduced ALL model demonstrates that both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-Aff1 fusion directly bind to the DYRK1A promoter. Gene expression and RT-PCR analyses of SEM cells treated with inhibitors against two important KMT2A fusion complex proteins, DOT1L (histone methyltransferase) and menin (tumor suppressor), demonstrate that only menin inhibition induced DYRK1A downregulation. Interestingly, deletion of germline KMT2A in murine B-cells did not decrease DYRK1A expression. Taken together, these results suggest direct transcriptional regulation through the KMT2A fusion complex. Surprisingly, RNA and protein expression of DYRK1A was reduced in KMT2A-R ALL compared to other B-ALL subtypes. We then identified MYC as a potential negative regulator of DYRK1A that could explain the lower RNA and protein expression levels observed. A gain-of-function experiment showed marked downregulation of DYRK1A when MYC was ectopically expressed in murine B-cells, while loss of MYC resulted in DYRK1A upregulation. Parallel analysis of publicly available gene expression data from children with high-risk B-ALL (NCI TARGET database) showed significantly higher MYC RNA expression levels in KMT2A-R ALL as compared to other ALL subtypes, further validating our findings that MYC acts as a negative regulator of DYRK1A. Finally, to assess pharmacologic inhibition, we treated multiple KMT2A-rearranged ALL cell lines with the novel DYRK1A inhibitor EHT 1610 and identified sensitivity to DYRK1A inhibition. We then queried the Achilles database and identified that DYRK1A is not a common essential gene in normal tissues, suggesting minimal potential for on-target/off-tumor effects of DYRK1A inhibition. Conclusions: We identified a novel mechanism in KMT2A-R ALL in which DYRK1A is positively regulated by the KMT2A fusion protein and negatively regulated by MYC. Genetic deletion and pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells. While further studies are needed, we predict that combining DYRK1A inhibitors with chemotherapy could decrease relapse risk and improve long-term survival of patients with KMT2A-R B-ALL. Disclosures Crispino: MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy; Scholar Rock: Research Funding; Forma Therapeutics: Research Funding. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy.

scholarly journals Waldenström's Macroglobulinemia (WM) Is Preceded By Clonal Lymphopoiesis Including MYD88 L265P in Progenitor B Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1527-1527
Author(s):  
Sara Rodríguez ◽  
Cirino Botta ◽  
Jon Celay ◽  
Ibai Goicoechea ◽  
Maria J Garcia-Barchino ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Somatic Mutations ◽  
Advisory Committees ◽  
Normal Cells ◽  
Cell Clones ◽  
B Cell Precursors ◽  
Myd88 L265p

Background: Although MYD88 L265P is highly frequent in WM, by itself is insufficient to explain disease progression since most cases with IgM MGUS also have mutated MYD88. In fact, the percentage of MYD88 L265P in CD19+ cells isolated from WM patients is typically &lt;100%, which questions if this mutation initiates the formation of B-cell clones. Furthermore, a few WM patients have detectable MYD88 L265P in total bone marrow (BM) cells and not in CD19+ selected B cells, raising the possibility that other hematopoietic cells carry the MYD88 mutation. However, no one has investigated if the pathogenesis of WM is related to somatic mutations occurring at the hematopoietic stem cell level, similarly to what has been shown in CLL or hairy cell leukemia. Aim: Define the cellular origin of WM by comparing the genetic landscape of WM cells to that of CD34 progenitors, B cell precursors and residual normal B cells. Methods: We used multidimensional FACSorting to isolate a total of 43 cell subsets from BM aspirates of 8 WM patients: CD34+ progenitors, B cell precursors, residual normal B cells (if detectable), WM B cells, plasma cells (PCs) and T cells (germline control). Whole-exome sequencing (WES, mean depth 74x) was performed with the 10XGenomics Exome Solution for low DNA-input due to very low numbers of some cell types. We also performed single-cell RNA and B-cell receptor sequencing (scRNA/BCRseq) in total BM B cells and PCs (n=32,720) from 3 IgM MGUS and 2 WM patients. Accordingly, the clonotypic BCR detected in WM cells was unbiasedly investigated in all B cell maturation stages defined according to their molecular phenotype. In parallel, MYD88p.L252P (orthologous position of the human L265P mutation) transgenic mice were crossed with conditional Sca1Cre, Mb1Cre, and Cγ1Cre mice to selectively induce in vivo expression of MYD88 mutation in CD34 progenitors, B cell precursors and germinal center B cells, respectively. Upon immunization, mice from each cohort were necropsied at 5, 10 and 15 months of age and screened for the presence of hematological disease. Results: All 8 WM patients showed MYD88 L265P and 3 had mutated CXCR4. Notably, we found MYD88 L265P in B cell precursors from 1/8 cases and in residual normal B cells from 3/8 patients, which were confirmed by ASO-PCR. In addition, CXCR4 was simultaneously mutated in B cell precursors and WM B cells from one patient. Overall, CD34+ progenitors, B-cell precursors and residual normal B cells shared a median of 1 (range, 0-4; mean VAF, 0.16), 2 (range, 1-5; mean VAF, 0.14), and 4 (range, 1-13; mean VAF, 0.26) non-synonymous mutations with WM B cells. Some mutations were found all the way from CD34+ progenitors to WM B cells and PCs. Interestingly, concordance between the mutational landscape of WM B cells and PCs was &lt;100% (median of 85%, range: 25%-100%), suggesting that not all WB B cells differentiate into PCs. A median of 7 (range, 2-19; mean VAF, 0.39) mutations were unique to WM B cells. Accordingly, many clonal mutations in WM B cells were undetectable in normal cells. Thus, the few somatic mutations observed in patients' lymphopoiesis could not result from contamination during FACSorting since in such cases, all clonal mutations would be detectable in normal cells. Of note, while somatic mutations were systematically detected in normal cells from all patients, no copy number alterations (CNA) present in WM cells were detectable in normal cells. scRNA/BCRseq unveiled that clonotypic cells were confined mostly within mature B cell and PC clusters in IgM MGUS, whereas a fraction of clonotypic cells from WM patients showed a transcriptional profile overlapping with that of B cell precursors. In mice, induced expression of mutated MYD88 led to a moderate increase in the number of B220+CD138+ plasmablasts and B220-CD138+ PCs in lymphoid tissues and BM, but no signs of clonality or hematological disease. Interestingly, such increment was more evident in mice with activation of mutated MYD88 in CD34+ progenitors and B-cell precursors vs mice with MYD88 L252P induced in germinal center B cells. Conclusions: We show for the first time that WM patients have somatic mutations, including MYD88 L265P and in CXCR4, at the B cell progenitor level. Taken together, this study suggests that in some patients, WM could develop from B cell clones carrying MYD88 L265P rather than it being the initiating event, and that other mutations or CNA are required for the expansion of B cells and PCs with the WM phenotype. Disclosures Roccaro: Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; European Hematology Association: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. San-Miguel:Amgen, Bristol-Myers Squibb, Celgene, Janssen, MSD, Novartis, Roche, Sanofi, and Takeda: Consultancy, Honoraria. Paiva:Amgen, Bristol-Myers Squibb, Celgene, Janssen, Merck, Novartis, Roche, and Sanofi; unrestricted grants from Celgene, EngMab, Sanofi, and Takeda; and consultancy for Celgene, Janssen, and Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau.

scholarly journals 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 ◽  
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.

IKZF3 Overexpression Phenocopies Gain-of-Function Mutation in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Shanye Yin ◽  
Gregory Lazarian ◽  
Elisa Ten Hacken ◽  
Tomasz Sewastianik ◽  
Satyen Gohil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Dna Binding ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Bcr Signaling ◽  
Experimental Group

A hotspot mutation within the DNA-binding domain of IKZF3 (IKZF3-L162R) has been identified as a putative driver in chronic lymphocytic leukemia (CLL); however, its functional effects are unknown. We recently confirmed its role as a CLL driver in a B cell-restricted conditional knock-in model. IKZF3 mutation altered mature B cell development and signaling capacity, and induced CLL-like disease in elderly mice (~40% penetrance). Moreover, we found IKZF3-L162R acts as a gain-of-function mutation, altering DNA binding specificity and target selection of IKZF3, and resulting in overexpression of multiple B-cell receptor (BCR) genes. Consistent with the murine data, RNA-sequencing analysis showed that human CLL cells with mut-IKZF3 [n=4] have an enhanced signature of BCR-signaling gene expression compared to WT-IKZF3 [n=6, all IGHV unmutated] (p&lt;0.001), and also exhibited general upregulation of key BCR-signaling regulators. These results confirm the role of IKZF3 as a master regulator of BCR-signaling gene expression, with the mutation contributing to overexpression of these genes. While mutation in IKZF3 has a clear functional impact on a cardinal CLL-associated pathway, such as BCR signaling, we note that this driver occurs only at low frequency in patients (~3%). Because somatic mutation represents but one mechanism by which a driver can alter a cellular pathway, we examined whether aberrant expression of IKZF3 could also yield differences in BCR-signaling gene expression. We have observed expression of the IKZF3 gene to be variably dysregulated amongst CLL patients through re-analysis of transcriptomic data from two independent cohorts of human CLL (DFCI, Landau et al., 2014; ICGC, Ferreira et al., 2014). We thus examined IKZF3 expression and BCR-signaling gene expression, or the 'BCR score' (calculated as the mean expression of 75 BCR signaling-associate genes) in those cohorts (DFCI cohort, n=107; ICGC cohort, n=274). Strikingly, CLL cells with higher IKZF3 expression (defined as greater than median expression) had higher BCR scores than those with lower IKZF3 expression (&lt;median) (p=0.0015 and p&lt;0.0001, respectively). These findings were consistent with the notion that IKZF3 may act as a broad regulator of BCR signaling genes, and that IKZF3 overexpression, like IKZF3 mutation, may provide fitness advantage. In support of this notion, our re-analysis of a gene expression dataset of 107 CLL samples (Herold Leukemia 2011) revealed that higher IKZF3 expression associated with poorer prognosis and worse overall survival (P=0.035). We previously reported that CLL cells with IKZF3 mutation appeared to increase in cancer cell fraction (CCF) with resistance to fludarabine-based chemotherapy (Landau Nature 2015). Instances of increase in mut-IKZF3 CCF upon treatment with the BCR-signaling inhibitor ibrutinib have been reported (Ahn ASH 2019). These studies together suggest an association of IKZF3 mutation with increased cellular survival following either chemotherapy or targeted treatment. To examine whether higher expression of IKZF3 was associated with altered sensitivity to ibrutinib, we performed scRNA-seq analysis (10x Genomics) of two previously treatment-naïve patients undergoing ibrutinib therapy (paired samples, baseline vs. Day 220). We analyzed an average of 11,080 cells per patient (2000 genes/cell). Of note, following ibrutinib treatment, remaining CLL cells expressed higher levels of IKZF3 transcript compared to pretreatment baseline (both p&lt;0.0001), whereas no such change was observed in matched T cells (n ranging between 62 to 652 per experimental group, p&gt;0.05), suggesting that cells with high expression of IKZF3 were selected by ibrutinib treatment. Moreover, we showed that ibrutinib treatment resulted in consistent upregulation of BCR-signaling genes (e.g., CD79B, LYN, GRB2, FOS, RAC1, PRKCB and NFKBIA) (n ranging between 362 to 1374 per experimental group, all p&lt;0.0001), which were likewise activated by mutant IKZF3. Altogether, these data imply that IKZF3 mutation or overexpression may influence upregulation of BCR-signaling genes and enhance cellular fitness even during treatment with BCR-signaling inhibitors. We highlight our observation that IKZF3 mutation appears to be phenocopied by elevated IKZF3 expression, and suggest that alterations in mRNA or protein level that mimic genetic mutations could be widespread in human cancers. Disclosures Kipps: Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

Poor Outcomes Among Patients Failing Dose-Adjusted EPOCH in Aggressive Lymphoma: A Collaborative, Retrospective Study

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1518-1518 ◽  
Author(s):  
Jackie Vandermeer ◽  
Allison M Winter ◽  
Ajay K. Gopal ◽  
Ryan D. Cassaday ◽  
Brian T. Hill ◽  
...  
Keyword(s):  
B Cell ◽  
Board Of Directors ◽  
Salvage Therapy ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
First Line ◽  
Advisory Committees ◽  
First Line Therapy

Abstract Introduction Among patients with aggressive B-NHL who fail RCHOP, about half respond to standard salvage regimens and may proceed to curative-intent, transplant-based therapy. However, whether pts failing more intensive regimens such as dose-adjusted, infusional EPOCH benefit from standard salvage regimens is unclear. We hypothesized that such patients comprise a higher-risk cohort, facing inferior response rates and outcomes using standard salvage regimens. We undertook a collaborative study to assess response rates and survival among pts failing EPOCH for aggressive B-NHL, to inform patient management and design of clinical trials in this setting. Methods Pharmacy records and institutional databases were queried, identifying pts receiving EPOCH over the last 10 years at the University of Washington/SCCA and the Cleveland Clinic Foundation, for combined analysis. Under IRB approval, patient characteristics, histology, outcome with EPOCH, time to EPOCH failure, response to salvage, and overall survival were analyzed. Diffuse large B cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma, B-cell-lymphoma unclassifiable, HIV-associated B cell lymphoma, and transformed B cell non-Hodgkin lymphoma were included. Pts receiving <2 cycles EPOCH, or who had inadequate follow-up (<3 months), were excluded. Failure of EPOCH was defined as failure to respond or progression during therapy, need for initiation of salvage therapy, or death during therapy of any cause. Adverse events or treatment change due to toxicity were not included in the definition of failure. JMP 11 was used to generate kaplan-meier survival estimates. Results 124 pts with aggressive B-NHL receiving EPOCH were identified. 54 had not relapsed, and among 70 remaining da-EPOCH failures, 37 met the above inclusion criteria. Median age was 55. 27% were female, and 23 received EPOCH as first-line therapy. All but 3 received rituximab with EPOCH. Histologies were primarily DLBCL in 22/37 (60%) and BCL-U in 12/37 (32%) carrying a MYC translocation; most of these harbored additional translocations in BCL2 and/or BCL6 (10/12). However, data regarding MYC rearrangement was not available for all pts. 2 had HIV-associated B-NHL and 3 had PMBCL. With 18 months follow up, the median time to EPOCH failure was 5 months. Only 3 EPOCH failures occurred late (>12 months). Median OS from the date of EPOCH failure was 10 months (Figure 1). Those receiving EPOCH as first-line therapy (23) had a median OS of 14 months from EPOCH failure, as opposed to 4 months for those receiving EPOCH as salvage therapy (log-rank p=.01). Salvage chemotherapy regimens after EPOCH were diverse, and generally ineffective; 6/28 (21%) regimens produced a response (Table 1). Among patients failing EPOCH within a year, platinum-containing salvage (RICE/RDHAP) was effective in only 2/13 patients (15%). 9 patients did not receive any salvage, most of whom died or proceeded to palliative measures and/or hospice care. Conclusions A relatively low overall response rate (21%) was observed in this retrospective analysis of patients failing EPOCH. Analogous to early RCHOP failure in the CORAL study, those failing EPOCH within a year may face inferior outcomes with platinum-based salvage therapy. While combined from two institutions, our data represent a modest sample size and require confirmation. If verified, examination of mechanisms of resistance to EPOCH, and selecting EPOCH failures for clinical trials of novel targeted therapies and transplant-based approaches, may prove critical. Table 1. Salvage Therapy for REPOCH failures Regimen: response/total number treated Notes Response to any salvage: 6/28 (21%) Some patients received more than 1 chemo salvage; responses were tabulated per regimen. RICE: 4/12 2/3 alive post transplant(1 auto 1 allo; 1 declined transplant and survived; 1 died) RDHAP: 1/6 Gemcitabine-based: 0/5 HyperCVAD (Part A and/or B): 1/5 Survivor had CNS only relapse, received regimen B and transplant 9- received no systemic treatmen; most died or proceeded to palliative measures and/or hospice Figure 1. Figure 1. Disclosures Gopal: Gilead: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Emergent/Abbott: Research Funding; Sanofi-Aventis: Honoraria; Seattle Genetics: Consultancy, Honoraria; BioMarin: Research Funding; Piramal: Research Funding; Janssen: Consultancy; Millenium: Honoraria, Research Funding; BMS: Research Funding; Merck: Research Funding. Hill:Seattle Genetics: 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; Pfizer: Consultancy, 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. Till:Roche/Genentech: Research Funding; Pfizer: Research Funding.

Phase I/Ib Study of the Novel Immunotoxin MT-3724 in Relapsed/Refractory Non-Hodgkin's B-Cell Lymphoma (NHL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5112-5112
Author(s):  
Paul A Hamlin ◽  
Catherine S. Diefenbach ◽  
David J. Valacer ◽  
Jack Higgins ◽  
Michelle A. Fanale
Keyword(s):  
B Cells ◽  
Phase I ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
B Cell Lymphoma ◽  
Advisory Committees ◽  
Dose Cohort ◽  
Mixed Response

Abstract Background CD20 is selectively expressed on the surface of early pre-B-cells, remains throughout B-cell development, and is then lost from plasma cells. Because CD20 is present on the majority of B-cell lymphomas, anti-CD20 monoclonal antibody (MAb) therapy is widely employed in the treatment of NHL. However a majority of NHL patients eventually become refractory to CD20 MAb(s). Resistance mechanisms may include increased MAb catabolism, initial or post treatment selection of low CD20 expressing tumor cells, trogocytosis of surface CD20, failure of MAb effector mechanisms and/or impaired patient immune cell function. MT-3724 is a recombinant fusion protein consisting of a CD20 binding variable fragment (scFv) fused to the enzymatically active Shiga-like toxin-I A1 subunit (SLT-I A1). SLT-I A1 is an N-glycosidase that catalytically inactivates 60S ribosomal subunits causing inhibition of protein synthesis. Upon its scFv binding to cell surface CD20 in vitro, SLT-I A1 forces MT-3724 internalization which then routes in a predictable fashion to the cytosol and irreversibly inactivates the cell ribosomes triggering cell death. MT-3724 has been shown to specifically bind and kill CD20+ malignant human B-cells in vitro and non-human primate (NHP) B-cells in vivo. MT-3724 was tested for safety in healthy NHPs: 6 intravenous (IV) doses of MT-3724 were given over 12 days at doses of 50, 150, and 450 mcg/kg. There were no deaths or effects on serum chemistries in the NHP studies. The major observed toxicity (inappetence) resolved within 48 hours of last dose. There was a significant, dose-dependent NHP B-cell depletion by Day 3 at all doses. Given the preclinical activity and mechanism of action, a Phase I/Ib study of MT-3724 was initiated in NHL. Methods MT-3724 is being tested for safety and tolerability in a first-in-human, open label, ascending dose study (3 + 3 design) in sequential cohorts of 5, 10, 20 and 50 mcg/kg/dose. Eligible subjects who previously responded to a CD20 MAb containing therapy followed by relapse/recurrence of NHL receive 6 doses by 2 hour IV infusions over the first 12 days of a 28 day cycle (first cycle). With continued safety, tolerability and lack of tumor progression, subjects may receive up to 4 additional 6-dose cycles (21 days) with tumor assessments after cycles 2, 4 and 5. Dose escalation is based on < 33% dose limiting toxicities (DLTs) observed during the first 28 day cycle. Results Three NHL subjects (2 transformed DLBCL, 1 FL) have completed at least one cycle in the 5 mcg/kg/dose cohort with no protocol DLTs or infusion related reactions and are evaluable for safety. Non-DLTs included grade (Gr) 2-3 transient hyperglycemic episodes related to pre-infusion corticosteroid therapy (n=1); transient Gr 4 neutropenia, possibly related to MT-3724 during cycle 1, week 4 (n=1); Gr 4 hypercalcemia and acute kidney injury with Gr 3 hypophosphatemia during cycle 1, week 4 due to leukemic disease progression (n=1). Subject 1 completed 5 cycles of therapy, with a partial response achieved post cycle 2 sustained through cycle 5; Subject 3 had a mixed response (both subjects had transformed DLBCL). Three subjects have now initiated treatment in the 10 mcg/kg/dose cohort with updated data to be presented at the meeting. Conclusions MT-3724 at 5 mcg/kg/dose has been safely administered for up to 5 cycles in this first-in-human study in relapsed/refractory NHL subjects. Treatment with the 10 mcg/kg cohort has commenced with continuing dose ascension planned. There is early evidence of clinical activity. Disclosures Diefenbach: Gilead: Equity Ownership, Research Funding, Speakers Bureau; Jannsen Oncology: Consultancy; Idera: Consultancy; Immunogen: Consultancy; Incyte: Research Funding; Genentech: Research Funding; Celgene: Consultancy; Molecular Templates: Research Funding; Seattle Genetics: Consultancy, Honoraria, Research Funding. Valacer:Molecular Templates: Employment. Higgins:Molecular Templates: Employment. Fanale:Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Research Funding; Infinity: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Research Funding; Genentech: Research Funding; Medimmune: Research Funding; Novartis: Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Molecular Templates: Research Funding; ADC Therapeutics: Research Funding; Onyx: Research Funding; Gilead: Research Funding.

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