Targeting Aberrant Chromatin in Chronic Lymphocytic Leukemia

Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature-appearing malignant lymphocytes (CLL B-cells) in the blood, marrow, lymph nodes, and spleen. Despite improved outcome with the introduction of novel BCR and BCL-2 inhibitors, disease progression is still a therapeutic challenge from either differential responses or acquired drug resistance. Recent studies in CLL reported alterations of the epigenetic landscape as well as mutations of genes encoding key chromatin machineries. These aberrant chromatin structures may provide novel therapeutic targets for CLL. Here, we identify aberrant chromatin features in CLL B-cells as novel therapeutic targets. Methods: Histones were extracted by acid from B cells derived from 10 random selected CLL patients and 10 normal donors and histone modifications were checked by western blot. For ChIP-seq study, published H3K27me3 ChIP-seq data (GSE113336) were downloaded from and analyzed (Control samples, n= 6; CLL samples, n=16). Gene ontology analysis used the Panther Classification System. Cell Survival was determined by CellTiter 96® AQueous Assay (Promega). Results: While most histone modifications do not vary between CLL and controls, H3K27me3 and H3.3S31ph are increased and decreased respectively, albeit variably, in CLL B-cells (Fig1. A and B). Notably, the low level of H3.3S31ph was observed in a subset of samples (7 of 10 CLL samples tested). To further investigate the biology and role of H3K27me3 in CLL, we analyzed its genome-wide distribution by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our analysis showed that the genes with increased H3K27me3 occupancy were mostly enriched in tumor suppression pathways (e.g., negative regulation of PI3K-Akt pathway) or down-regulated genes in CLL such as genes involved in the pro-apoptotic pathway (FAS) (Fig1. C and D). These results suggested that high enrichment of H3K27me3 may regulate the expression of these genes, contributing to CLL survival. H3K27 methylation, an important suppressive histone modification that is associated with transcription repression, is catalyzed by Polycomb Repressive Complex 2 (PRC2). Therefore, inhibition of Enhancer of Zeste Homolog 2 (EZH2), the catalytic subunit of PRC2, could be explored as a therapy approach in CLL. However, feedback activation of H3K27 acetylation (H3K27ac) can promote expression of pro-survival genes that confers EZH2 inhibitor (EZH2i) resistance, which limits its use in human malignancy. Thus, the epigenetic determinants that reliably overcome EZH2i resistance or sensitize cells to EZH2 inhibition have yet to be identified. As we observed that the CLL B-cells in a subset of CLL patients have low levels of H3.3S31ph, and a recent study showed the importance of H3.3S31ph for the enzymatic activity of p300 to acetylate H3 at lysine 27(Martire S et al., Nat Genet. 2019), we assessed the role of H3.3S31ph in the process of EZH2 inhibitor-mediated H3K27ac. Our results showed that inhibition of H3.3S31ph by CHK1 inhibitor MK-8776 abolished the activation of H3K27ac by EZH2i in MEC1 cells, which represents the patients who have CLL cells with relatively high level H3.3S31ph. However, we did not see a major increase of H3K27ac and H3.3S31ph in primary CLL B-cells with EZH2 inhibition (Fig. 1E), consistent with the relatively low expression of CHK1 protein in these cells (Fig. 1F). Because our data shows the requirement of H3.3S31ph in H3K27ac activation by EZH2 inhibition, we next tested if H3.3S31ph inhibition could overcome H3K27ac induced EZH2 inhibition resistance. We found that suppression of H3.3S31ph by CHK1 inhibitor MK-8776 sensitizes the CLL-like line MEC1 to EZH1/2 inhibition (Fig. 1 G). We then showed that an EZH2 inhibitor, Valemetostat, reduce the survival of the primary CLL B-cells (Fig. 1 H). These results suggest that the low level of H3.3S31ph may provide a therapeutic opportunity for CLL treatment with EZH inhibition. Conclusion: In summary, we have elucidated how epigenetic features in leukemic CLL B-cells (H3K27me3 and H3.3S31ph), can provide novel treatment targets for CLL (Fig. 1 I). Moreover, this study may provide a proof of concept to develop new treatment strategies based on epigenetic vulnerabilities in other hematological malignancies. Disclosures Parikh: GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; Genentech: Honoraria; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding; Janssen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Verastem Oncology: Honoraria; Merck: Research Funding. Kenderian:Kite: Research Funding; MorphoSys: Research Funding; Tolero: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; BMS: Research Funding; Gilead: Research Funding; Juno: Research Funding; Lentigen: Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Sunesis: Research Funding. Ding:Beigene: Membership on an entity's Board of Directors or advisory committees; 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; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Research Funding; DTRM: Research Funding; Abbvie: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Oncotracker: Membership on an entity's Board of Directors or advisory committees; 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; 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; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Abbvie: Research Funding; Pharmacyclics: 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; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding.

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PD-1 Overexpression in Richter's Transformation (RT) and Aggressive Chronic Lymphocytic Leukemia (CLL) after Progression on Ibrutinib Increases Bcl-2 Expression Via Akt/mTOR Pathway

Blood ◽

10.1182/blood-2018-99-112575 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 586-586 ◽

Cited By ~ 1

Author(s):

Sutapa Sinha ◽

Tammy Price-Troska ◽

Shulan Tian ◽

Charla R Secreto ◽

Xiaosheng Wu ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Rna Sequencing ◽

Research Funding ◽

Gene Signature ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Lymphoma Line

Abstract Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p < 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line 'Karpas299'. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response. Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. Disclosures Parikh: Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: 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; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: 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. Ding:Merck: Research Funding.

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Increased Activity of Aldehyde Dehydrogenase, a Stem/Progenitor Cell Marker, in Chronic Lymphocytic Leukemia,

Blood ◽

10.1182/blood.v118.21.3867.3867 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3867-3867

Author(s):

Raymond P. Wu ◽

Christina C.N. Wu ◽

Tomoko Hayashi ◽

Laura Z. Rassenti ◽

Thomas J. Kipps ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Research Funding ◽

Progenitor Cell ◽

Lymphocytic Leukemia ◽

Cell Marker ◽

Aldh Activity ◽

Advisory Committees ◽

Aldefluor Assay

Abstract Abstract 3867 Introduction: Despite their mature appearance, the B cells from chronic lymphocytic leukemia (CLL) possess immature characteristics both functionally and biochemically. CLL B cells display known biochemical markers characteristic of cells early in the blood lineage, including ROR1, Wnt16, and LEF1. In addition, CLL B cells have higher levels of Reactive Oxygen Species (ROS) and of the oxidant-induced transcription factor Nrf2 [NFE2L2], compared to normal peripheral blood mononuclear cells (PBMC). Intracellular ROS status has been suggested to be a marker of cancer stem/progenitor cells possibly due to their high expression of oncogenes. Downstream targets of Nrf2 include the Aldehyde dehydrogenase [ALDH] enzymes, which are believed to play a crucial role in stem cell biology because they protect the cells against oxidative stress caused by accumulation of aldehydes. Here, we use ALDH activity to visualize populations of CLL B cells that may have stem/progenitor properties. Materials and Methods: Isolated PBMC from normal donors and CLL patients with aggressive and indolent disease were stained for ALDH activity with an Aldefluor assay kit (StemCell Technologies). The ALDH inhibitor, diethylaminobenzaldehyde (DEAB), was used to confirm that the fluorescent activity was due to ALDH activity. At the end of the Aldefluor assay, the cells were stained for cell surface markers, CD19, CD5, CD38 and CD34. 50,000 total events were collected for FACS analysis. Normalized Mean Fluorescence Intensity (MFI) values were calculated by dividing each MFI value to average MFI value of normal CD19+ cells for each experiment. Data analyses were performed by FlowJo software and Prizm. P-values were calculated by One-Way ANOVA analysis with Post-Bonferroni's multiple comparison test. Results: We examine the level of ALDH expression and activity in CD19+ cells of healthy donors (n = 9), CLL samples that expressed unmutated IgVH and that were ZAP-70 positive (defined as “aggressive”, n = 14) or samples that expressed mutated IgVH and were ZAP-70 negative (defined as “indolent”, n=12). CLL B cells from patients with aggressive disease had significantly higher ALDH activities compared to normal B cells (p < 0.001) and indolent CLL B cells (p < 0.05) (Figure1). Indolent CLL B cells also have higher level of ALDH activities compared to normal B cells (p < 0.01) (Figure1). Treatment with the ALDH inhibitor, DEAB, suppressed the increased fluorescence observed in CLL B cells. In addition, ALDH high CLL B cells are CD34 negative. These data show that CLL B cells express a marker known to be associated with stem/progenitor cells, but these populations are different from CD34 positive hematopoietic stem cells. In addition, our data show that a stem/progenitor cell marker is associated with the pathogenesis of CLL. Disclosures: Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbot Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

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Bone Marrow Hematopoietic Dysfunction in Untreated Chronic Lymphocytic Leukemia Is Partially Mediated By Exposure to Constituents of the Leukemic Microenvironment

Blood ◽

10.1182/blood-2018-99-118916 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3132-3132

Author(s):

Bryce Manso ◽

Kimberly Gwin ◽

Charla R Secreto ◽

Henan Zhang ◽

Wei Ding ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Healthy Controls ◽

Advisory Committees ◽

The Impact

Abstract Peripheral immune dysfunction in B-Chronic Lymphocytic Leukemia (CLL) is well-studied and likely relates to the incidence of serious recurrent infections and second malignancies that plague CLL patients. However, the current paradigms of known immune abnormalities are not able to consistently explain these complications and it is not easy to correct CLL patient immune status. Here, we expand on our preliminary reports that demonstrate bone marrow (BM) hematopoietic dysfunction in early and late stage untreated CLL patients. We found reduced short-term functional capacity of hematopoietic progenitors in BM using colony forming unit assays (Figure 1A-C) and flow cytometry revealed significant reductions in frequencies of hematopoietic stem and progenitor cell (HSPC) populations (exemplified by Lin-CD34+ HSPCs, Figure 1D). We further report that protein levels of the transcriptional regulators HIF-1α, GATA-1, PU.1, and GATA-2 are overexpressed in distinct HSPC subsets from CLL patient BM, providing molecular insight into the basis of HSPC dysfunction. Interestingly, sustained myelopoiesis, evaluated by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays maintained for five weeks, revealed no difference between healthy controls and CLL patients. These new data indicate that when HSPCs are removed from the leukemic microenvironment for ample in vitro culture time, they recover the ability to sustain myelopoiesis. To further assess the impact of the CLL microenvironment on HSPC biology, isolated HSPCs (CD34+ BM cells) from healthy controls were exposed in vitro to known leukemic microenvironment constituents. Exposure to TNFα, a cytokine constitutively produced by CLL B cells, resulted in rapid increases in PU.1 and GATA-2 proteins (Figure 2A-D). Similarly, addition of TNFα to the LTC-IC assay resulted in a striking ablation of myelopoiesis, even at the highest input cell concentration. Further, overexpression of PU.1 and GATA-2 were observed in HSPCs following co-culture with CLL B cells, a result that was not recapitulated when cells were exposed to IL-10, another cytokine constitutively produced by CLL B cells. These findings indicate specific components of the leukemic microenvironment are involved in HSPC modulation. Together, these findings expand on our previous observations of BM hematopoietic dysfunction in untreated CLL patients and offer new molecular insights into the contribution of the leukemic microenvironment on immunodeficiency in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria; AstraZeneca: Honoraria, Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Impact of Idelalisib Treatment Interruption with or without Dose Reduction on Outcomes in Relapsed / Refractory Indolent Non-Hodgkin's Lymphoma and Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-124541 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5468-5468

Author(s):

Shuo Ma ◽

Rebecca J Chan ◽

Lin Gu ◽

Guan Xing ◽

Nishan Rajakumaraswamy ◽

...

Keyword(s):

Overall Survival ◽

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Dose Reduction ◽

Clinical Outcomes ◽

Research Funding ◽

Treatment Interruption ◽

Lymphocytic Leukemia ◽

Hodgkin's Lymphoma ◽

Advisory Committees

Introduction: Idelalisib (IDELA) is the first-in-class PI3Kδ inhibitor and is approved as a monotherapy for relapsed or refractory (R/R) follicular lymphoma and in combination with rituximab for R/R chronic lymphocytic leukemia (CLL). We previously evaluated IDELA treatment interruption as a mechanism to mitigate treatment-emergent adverse events (TEAEs) and found that limited interruption with clinically appropriate re-challenging resulted in superior clinical outcomes. These findings did not comprehensively address the potential confound of interruptions inherently being associated with longer duration of therapy (DoT). Furthermore, the compound effect of IDELA dose reduction together with treatment interruption on IDELA efficacy was not assessed. Objectives: 1) To evaluate whether the benefit of IDELA interruption is retained in patients on therapy >180 days, a duration previously found to be associated with longer overall survival among patients who discontinued IDELA due to an AE; and 2) To compare clinical outcomes of patients who reduced IDELA dosing in addition to interrupting IDELA with those of patients who interrupted IDELA without additional dose reduction. Methods: Using data from Gilead-sponsored trials of patients with R/R indolent non-Hodgkin's lymphoma (iNHL) treated with IDELA monotherapy (N=125, Gopal et al., N. Engl. J. Med., 2014) or with R/R CLL treated with IDELA + anti-CD20 (N=110, Furman et al., N. Engl. J. Med., 2014; and N=173, Jones et al., Lancet Haematol., 2017), DoT, progression-free survival (PFS), and overall survival (OS) were compared between patients on IDELA therapy >180 days with vs. without interruption and between patients who experienced Interruption and Dose Reduction (IDR) vs. patients who experienced Interruption but NoDose Reduction (INoDR) at any point during IDELA treatment. Interruption was defined as missing at least one IDELA treatment day due to an AE and dose reduction could have occurred before or after the first interruption. PFS and OS were estimated using the Kaplan-Meier method and were compared using a log-rank test. Results: Sixty-nine of 125 patients with R/R iNHL (55.2%) and 222 of 283 patients with R/R CLL (78.4%) remained on IDELA therapy >180 days with 29 (42.0%) and 103 (46.4%) of them, respectively, experiencing interruption on or after day 180 (Table 1). The proportions of patients with interruption before day 180 were similar within each of these populations. Among patients on therapy >180 days, those with treatment interruption on or after 180 days had a longer median (m) DOT than patients without interruption (Table 1). Both PFS and OS were longer in CLL patients who interrupted compared to those who did not interrupt (mPFS=28.9 mos. vs. 17.3 mos. and mOS=not reached [NR] vs. 40.4 mos. for with interruption vs. without interruption, respectively, Table 1 and Figure 1). In patients with iNHL, no difference was observed in PFS or OS between patients who interrupted vs. those who did not (Table 1). Of patients who experienced at least one AE-induced interruption at any point during IDELA therapy (n=63 iNHL and n=157 CLL), 47 iNHL patients (74.6%) and 84 CLL patients (53.5%) also had dose reduction. Two iNHL patients (1.6%) and 5 CLL patients (1.8%) had IDELA dose reduction but no interruption. Both iNHL and CLL patients with IDR experienced a similar PFS compared to patients with INoDR (mPFS=16.5 mos. vs. 14.2 mos. for iNHL and 21.8 mos. vs. 22.1 mos. for CLL with IDR vs. INoDR, respectively, Table 2). However, OS was longer in both iNHL and CLL patients with IDR compared to INoDR (mOS=61.2 mos. vs. 35.3 mos. for iNHL and NR vs. 42.4 mos. for CLL, respectively, Table 2; CLL patients shown in Figure 2). Discussion: IDELA treatment interruption is not associated with rapid clinical deterioration, as observed with some B-cell receptor signaling pathway inhibitors. No clear relationship between IDELA DoT and frequency of interruption was observed. When normalized for DoT >180 days, IDELA treatment interruption retained its clinical benefit in the CLL population. When utilized together with IDELA interruption, dose reduction did not lead to inferior clinical outcomes but instead extended OS in both iNHL and CLL populations. Adherence to treatment interruption and dose reduction guidance as outlined in the IDELA USPI may optimize IDELA tolerability and efficacy for patients with iNHL and CLL. Disclosures Ma: Janssen: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Gilead: Research Funding; Abbvie: Research Funding; Juno: Research Funding; Incyte: Research Funding; Xeme: Research Funding; Beigene: Research Funding; Novartis: Research Funding; Astra Zeneca: Consultancy, Research Funding, Speakers Bureau; Kite: Consultancy; Acerta: Research Funding; Bioverativ: Consultancy; Genentech: Consultancy. Chan:Gilead Sciences, Inc.: Employment, Equity Ownership. Gu:Gilead Sciences, Inc.: Employment. Xing:Gilead Sciences, Inc.: Employment. Rajakumaraswamy:Gilead Sciences, Inc.: Employment. Ruzicka:Gilead Sciences, Inc.: Employment. Wagner-Johnston:Gilead: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Jannsen: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees.

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

Blood ◽

10.1182/blood-2020-137085 ◽

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<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 (<median) (p=0.0015 and p<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<0.0001), whereas no such change was observed in matched T cells (n ranging between 62 to 652 per experimental group, p>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<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.

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IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽

10.1182/blood.v116.21.2431.2431 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2431-2431

Author(s):

Marta Coscia ◽

Francesca Pantaleoni ◽

Chiara Riganti ◽

Candida Vitale ◽

Micol Rigoni ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Board Of Directors ◽

Stromal Cells ◽

Peripheral Blood ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Spontaneous Apoptosis ◽

Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

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The Influence Of KIR Haplotype In ITP Incidence, Treatment Response and Bleeding Symptoms

Blood ◽

10.1182/blood.v122.21.2316.2316 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2316-2316

Author(s):

Bethan Psaila ◽

Nayla Boulad ◽

Emily Leven ◽

Naznin Haq ◽

Christina Soo Lee ◽

...

Keyword(s):

Platelet Count ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Kir Genes ◽

Number Of Patients

Abstract The pathogenesis of immune thrombocytopenia (ITP) is multifactorial, with both cellular and humoural immune dysfunction. The role of NK cells has not been well defined in ITP but in other diseases NK cells have a role in rejecting “foreign” eg transplanted organ or tumor, and also acting against self as occurs in autoimmunity. NK cell activity is orchestrated by the balance of activating vs. inhibitory signalling, in particular via the killer cell immunoglobulin-like receptor (KIR) family of receptors. Significant variation exists in KIR allelic subtype and copy number for the KIR between individuals, and associations have been made with certain haplotypes and a number of autoimmune disorders including rheumatoid arthritis, scleroderma and diabetes. Previous reports have demonstrated a reduction in natural killer (NK) cell number and function in ITP and expression of inhibitory KIR genes is increased in patients in remission vs. active ITP. Methods To explore whether a particular KIR haplotype might predispose to ITP, and also affect response to ITP treatment, we performed KIR genotyping using the Invitrogen SSP kit on 92 patients attending a haematology centre in New York and compared the results to data from 213 controls taken from the USA Eastern Database. Genomic DNA was typed for the inhibitory KIR genes KIR2DL1, KIR2DL2, KIR2DL5A (alleles 001 and 002), KIR2DL5B (alleles 002-004, 06, and 007), KIR3DL1, KIR3DL3; the activating KIR genes KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1; the framework genes KIR2DL3, KIR2DL4, KIR3DL2, KIR3DP1; and the pseudogene KIR2DP1. The patients with ITP had been or were receiving treatment with IVIG (n=64), corticosteroids (72) and rituximab (37). Bleeding symptoms were recorded. Response to treatment was defined as complete - platelet count increase to > 100 x 109/mL; partial - platelet count increase to > 50 x 109/mL; or no response. For the purpose of analysis, PRs and CRs were combined. A comprehensive database allowed a logistic regression, assessing both responses to treatments, platelet counts, neutrophil counts, CRP, lymphocyte subsets and bleeding symptoms. Results The expression of two inhibitory KIR genes, 2DL1 and 3DL1, was significantly lower in the patients with ITP as compared to controls (87% 2DL1 and 87% 3DL1 compared to 99% in controls - P < 0.02). Response to rituximab was strongly related to KIR haplotype expression. 2DL1 expression was higher among nonresponders to Rituximab (100% of non responders compared to 82% of responders), whereas 2DL3 expression was significantly lower (79% compared to 90%) (P < 0.05, Figure 1B). Separately, patients with the 2DS3 allele, an activatory KIR, were 5.5 times more likely to have experienced significant bleeding. Conclusions Although these findings are preliminary and require further investigation, these data suggest that increased cytotoxic autoimmunity due to reduced KIR inhibition may be associated with the development of ITP and possibly contribute importantly to the pathogenesis. Anti-CD20 targeting therapy directed at B cells was strongly influenced by 2 different KIRs (1 upregulated and one down-regulated) emphasizing the potential role of NK cells in elimination of tissue-based (nodal) B cells. Finally a more pronounced clinical phenotype with a markedly higher incidence of severe bleeding associated with an increased activatory KIR expression demonstrates the role of NK cells in bleeding presumably via their effects on either endothelial cells or platelet function. These exciting findings will be pursued for confirmation in a larger number of patients. Disclosures: Bussel: Amgen: Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; GlaxoSmithKline: Family owns stock, Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity’s Board of Directors or advisory committees.

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Head-To-Head Comparison Of Obinutuzumab (GA101) Plus Chlorambucil (Clb) Versus Rituximab Plus Clb In Patients With Chronic Lymphocytic Leukemia (CLL) and Co-Existing Medical Conditions (Comorbidities): Final Stage 2 Results Of The CLL11 Trial

Blood ◽

10.1182/blood.v122.21.6.6 ◽

2013 ◽

Vol 122 (21) ◽

pp. 6-6 ◽

Cited By ~ 10

Author(s):

Valentin Goede ◽

Kirsten Fischer ◽

Raymonde Busch ◽

Anja Engelke ◽

Barbara Eichhorst ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Final Stage ◽

Research Funding ◽

Observation Time ◽

Lymphocytic Leukemia ◽

Type Ii ◽

Efficacy And Safety ◽

Advisory Committees ◽

Stage 1

Abstract Introduction CLL11 is a large randomized phase 3 trial investigating first-line chemoimmunotherapy in CLL patients with comorbidities, i.e. patients typically treated in daily practice. Here, we present: (i) The final stage 2 analysis with efficacy and safety results of the head-to-head comparison between GA101 plus Clb (GClb) and rituximab plus Clb (RClb); at the pre-planned interim analysis, the primary endpoint was met early and the results were released by the independent data monitoring board. (ii) An update on the stage I analysis (GClb vs. Clb and RClb vs. Clb comparisons) with longer observation time; the final stage 1 analysis recently showed that GClb or RClb has superior efficacy to chemotherapy with Clb alone. Methods Treatment-naïve CLL patients with a Cumulative Illness Rating Scale (CIRS) total score >6 and/or an estimated creatinine clearance (CrCl) <70 mL/min were eligible. Patients received Clb alone (0.5 mg/kg po d1, d15 q28 days, 6 cycles), GClb (100 mg iv d1, 900 mg d2, 1000 mg d8, d15 of cycle 1, 1000 mg d1 cycles 2-6), or RClb (375 mg/m2 iv d1 cycle 1, 500 mg/m2 d1 cycles 2-6). Primary endpoint was investigator-assessed progression-free survival (PFS). Response rates, minimal residual disease (MRD), and overall survival (OS) were key secondary efficacy endpoints. Results Final results of the stage 2 analysis: Median observation time was 19 months. The GClb and RClb treatment arms were well balanced for baseline characteristics. Median age, CIRS score, and CrCl at baseline were 73 years, 8, and 63 mL/min respectively. Key efficacy and safety results are shown in the table. The PFS benefit of GClb over RClb was supported by all pre-planned subgroup analyses (including the cytogenetic subgroups 17p-, 11q-, 12+, 13q-). The number of patients with MRD negative blood samples at end-of-treatment was more than 10-fold higher with GClb compared with RClb (63/214 [29.4%] vs. 6/243 [2.5%]). Grade 3-4 infusion-related reactions with GClb occurred at first infusion only. Updated results of the stage 1 analysis: Median observation time was 23 months. Confirming the primary stage 1 results, GClb or RClb compared with Clb alone was associated with statistically significant and clinically meaningful improvement in PFS (GClb vs. Clb: HR 0.18, CI 0.13-0.24, p<.0001, RClb vs. Clb: HR 0.44, CI 0.34-0.57, p<.0001). The updated median PFS in GClb, RClb and Clb were 26.7, 16.3 and 11.1 months, respectively. Updated OS analysis demonstrated a benefit of GClb over Clb (HR 0.41, CI 0.23-0.74, p=0.002). OS analysis for RClb over Clb showed HR 0.66, CI 0.39-1.11, p=0.113. At the data cut-off, 9%, 15%, and 20% of the patients in the GClb, RClb, and Clb arms, respectively, had died. OS medians were not reached. Conclusions GA101, a novel, glycoengineered, type II CD20 antibody, in combination with Clb (GClb regimen) demonstrated statistically significant and clinically meaningful prolongation of PFS, and higher complete response rate and MRD negativity rate compared with RClb in previously untreated CLL patients with comorbidities. Infusion-related reactions and neutropenia were more common with GClb without an increase in infections. Furthermore, GClb vs. Clb alone demonstrated a prolongation of OS. Overall, GClb is superior to RClb and a highly active treatment in this typical CLL patient population. Disclosures: Goede: Mundipharma: Honoraria; F. Hoffmann-La Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Off Label Use: GA101 is a novel, glycoengineered, type II anti-CD20 monoclonal antibody that is designed to enhance direct cell death and antibody-dependent cellular cytotoxicity. It is being investigated in chronic lymphocytic leukemia, Non-Hodgkin’s Lymphoma and other hematologic indications. Fischer:Mundipharma: Travel grants, Travel grants Other; F. Hoffmann-La Roche: Travel grants Other. Engelke:F. Hoffmann-La Roche: Travel grants Other. Eichhorst:Mundipharma: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Consultancy; F. Hoffman-La Roche: Honoraria, Research Funding. Wendtner:F. Hoffmann-La Roche: Consultancy, Research Funding. Dilhuydy:F. Hoffmann-La Roche: Consultancy. Opat:F. Hoffmann-La Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Alexion Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Novartis Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Owen:F. Hoffmann-La Roche: Honoraria. Kreuzer:F. Hoffmann-La Roche: Consultancy, Honoraria. Langerak:F. Hoffmann-La Roche: Research Funding. Ritgen:F. Hoffmann-La Roche: Research Funding. Stilgenbauer:F. Hoffmann-La Roche: Consultancy, Honoraria, Research Funding. Asikanius:F. Hoffmann-La Roche: Employment. Humphrey:F. Hoffmann-La Roche: Employment. Wenger:F. Hoffmann-La Roche: Employment, Ownership interests (including stock options) in a start-up company, the stock of which is not publicly traded Other. Hallek:F. Hoffmann-La Roche: Consultancy, Honoraria, Research Funding.

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Preliminary Safety Results from the Phase IIIb GREEN Study of Obinutuzumab (GA101) Alone or in Combination with Chemotherapy for Previously Untreated or Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v124.21.3345.3345 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3345-3345 ◽

Cited By ~ 7

Author(s):

Francesc Bosch ◽

Thomas Illmer ◽

Mehmet Turgut ◽

Agostino Cortelezzi ◽

Susan F. Lasserre ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Safety Profile ◽

Research Funding ◽

Safety Data ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Phase Iiib ◽

Unfit Patients ◽

Grade 3

Abstract Background: The novel, glycoengineered type II anti-CD20 monoclonal antibody, obinutuzumab (GA101) has demonstrated superior efficacy to chlorambucil (Clb) monotherapy and to Clb in combination with rituximab (R-Clb) with an acceptable safety profile in CLL. However, an increased rate of infusion-related reactions (IRRs) has been observed with the obinutuzumab(G)-Clb combination compared with R-Clb during the first cycle of treatment. The GREEN study (NCT01905943) is an ongoing phase IIIb, multicenter, open-label trial investigating the safety and efficacy of obinutuzumab alone or in combination with chemotherapy in patients with previously untreated or relapsed/refractory CLL. We report safety data from cohort 1, which aimed to reduce IRRs on the first day of obinutuzumab administration in previously untreated patients using a lower dose and slower infusion rate than in previous studies. Methods: Subjects aged ≥18 years withdocumented CLL, an Eastern Cooperative Oncology Group (ECOG) performance status of 0–2 and adequate hematologic function are enrolled. Treatment includes obinutuzumab (1000mg) administered intravenously on days (D) 1 (25mg) and 2 (975mg), D8, and D15 of cycle (C) 1, and on D1 of C2–6, alone (any patient: n=18) or in combination with 28-day cycles of chemotherapy: fludarabine plus cyclophosphamide (FC; n=46) for fit patients (cumulative illness rating scale [CIRS] ≤6 and creatinine clearance [CrCl] ≥70mL/min), Clb (n=8) for unfit patients (CIRS >6 and/or CrCl <70mL/min) or bendamustine (B; n=86) for fit/unfit patients. The primary outcome is safety, including the frequency, type and severity of adverse events (AEs). The present analysis focuses on IRRs, defined as treatment-related AEs occurring during or within 24 hours of infusion. Results were assessed to determine if a low obinutuzumab dose (25mg) and slow infusion rate (12.5mg/hour) on D1 (the current recommended C1D1 regimen is 100mg at 25mg/hour) could reduce IRRs. Analysis was based on a data cut-off of 28 April 2014, planned for when the first 150 previously untreated patients had completed cohort 1. Results: Of 158 subjects eligible for the IRR analysis (Table), median age was 65.0 (34.0–83.0) years and the majority were males (65.2%) with Binet stage B (52.5%) or C (31.0%) CLL. Median observation time was 2.09 (0.2–6.0) months and median exposure time was 1.0 (0.0–4.8) month. IRRs occurring in ≥10% of patients were chills (14.6%) and pyrexia (15.2%). Serious IRRs in ≥1% of patients were tumor lysis syndrome (TLS; 3.8%) and pyrexia (1.3%). Grade ≥3 IRRs experienced by ≥1% of patients were TLS (5.7%), hypertension (1.3%) and hypotension (1.3%). IRRs were most frequent in C1D1 (Fig). In the overall safety population (n=172; previously untreated patients) the most frequently reported serious AEs of special interest included IRR (8.1%) and neutropenia (11.0%). AEs of particular interest, thrombocytopenia, cardiac, and hemorrhagic events, were experienced by 16.3%, 3.5% and 3.5% of patients, respectively. Table. Table. Conclusions: Preliminary safety data from the GREEN study, assessing the use of obinutuzumab alone or in combination with chemotherapy (B, FC or Clb) in subjects with untreated CLL, are in line with the known safety profile of obinutuzumab in similar populations. Although there is limited exposure time available for subjects in GREEN, IRRs seemed to be more manageable and a lower proportion of subjects with IRRs grade ≥3 was observed compared with previous studies. No new safety signals were reported. However, since the number of discontinuations during C1 was comparable with previous obinutuzumab studies, the decision was taken to further improve IRR rates by assessing additional dexamethasone premedication in cohort 2. Final safety data from the study will be presented at a later timepoint. Figure 1 Figure 1. Disclosures Bosch: Roche: Consultancy, Research Funding, Speakers Bureau. Off Label Use: GAZYVA (obinutuzumab) is a CD20-directed cytolytic antibody and is indicated, in combination with chlorambucil, for the treatment of patients with previously untreated chronic lymphocytic leukemia (CLL). This abstract reports on obinutuzumab alone or in combination with chemotherapy for previously untreated or relapsed/refractory CLL.. Lasserre:F. Hoffmann–La Roche: Employment. Truppel-Hartmann:F. Hoffmann–La Roche: Employment. Leblond:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Foà:Roche-Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Stilgenbauer:Roche: Consultancy, Honoraria, Research Funding.

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Five-Year Experience with Single-Agent Ibrutinib in Patients with Previously Untreated and Relapsed/Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.233.233 ◽

2016 ◽

Vol 128 (22) ◽

pp. 233-233 ◽

Cited By ~ 53

Author(s):

Susan M. O'Brien ◽

Richard R. Furman ◽

Steven E. Coutre ◽

Ian W. Flinn ◽

Jan Burger ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Board Of Directors ◽

Research Funding ◽

Single Agent ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Equity Ownership ◽

Grade 3 ◽

Over Time

Abstract Background: Ibrutinib (ibr), a first-in-class, once-daily Bruton's tyrosine kinase inhibitor, is approved by the US FDA for treatment of patients (pts) with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) including pts with del17p. The phase 1b/2 PCYC-1102 trial showed single-agent efficacy and tolerability in treatment-naïve (TN; O'Brien, Lancet Oncol 2014) and relapsed/refractory (R/R) CLL/SLL (Byrd, N Engl J Med 2013). We report efficacy and safety results of the longest follow-up to date for ibr-treated pts. Methods: Pts received 420 or 840 mg ibr QD until disease progression (PD) or unacceptable toxicity. Overall response rate (ORR) including partial response (PR) with lymphocytosis (PR-L) was assessed using updated iwCLL criteria. Responses were assessed by risk groups: unmutated IGVH, complex karyotype (CK; ≥3 unrelated chromosomal abnormalities by stimulated cytogenetics assessed by a reference lab), and in hierarchical order for del17p, then del11q. In the long-term extension study PCYC-1103, grade ≥3 adverse events (AEs), serious AEs, and AEs requiring dose reduction or discontinuation were collected. Results: Median age of the 132 pts with CLL/SLL (31 TN, 101 R/R) was 68 y (range, 37-84) with 43% ≥70 y. Baseline CK was observed in 41/112 (37%) of pts. Among R/R pts, 34 (34%) had del17p, 35 (35%) del11q, and 79 (78%) unmutated IGVH. R/R pts had a median of 4 prior therapies (range, 1-12). Median time on study was 46 m (range, 0-67) for all-treated pts, 60 m (range, 0-67.4) for TN pts, and 39 m (range, 0-67) for R/R pts. The ORR (per investigator) was 86% (complete response [CR], 14%) for all-treated pts (TN: 84% [CR, 29%], R/R: 86% [CR, 10%]). Median progression-free survival (PFS) was not reached (NR) for TN and 52 m for R/R pts with 60 m estimated PFS rates of 92% and 43%, respectively (Figure 1). In R/R pts, median PFS was 55 m (95% confidence intervals [CI], 31-not estimable [NE]) for pts with del11q, 26 m (95% CI,18-37) for pts with del17p, and NR (95% CI, 40-NE) for pts without del17p, del11q, trisomy 12, or del13q. Median PFS was 33 m (95% CI, 22-NE) and NR for pts with and without CK, and 43 m (95% CI, 32-NE) and 63 m (95% CI, 7-NE) for pts with unmutated and mutated IGVH, respectively(Figure 2). Among R/R pts, median PFS was 63 m (95% CI, 37-NE) for pts with 1-2 prior regimens (n=27, 3 pts with 1 prior therapy) and 59 m (95% CI, 22-NE) and 39 m (95% CI, 26-NE) for pts with 3 and ≥4 prior regimens, respectively. Median duration of response was NR for TN pts and 45 m for R/R pts. Pts estimated to be alive at 60 m were: TN, 92%; all R/R, 57%; R/R del17p, 32%; R/R del 11q, 61%; R/R unmutated IGVH, 55%. Among all treated pts, onset of grade ≥3 treatment-emergent AEs was highest in the first year and decreased during subsequent years. With about 5 years of follow-up, the most frequent grade ≥3 AEs were hypertension (26%), pneumonia (22%), neutropenia (17%), and atrial fibrillation (9%). Study treatment was discontinued due to AEs in 27 pts (20%) and disease progression in 34 pts (26%). Of all treated pts, 38% remain on ibr treatment on study including 65% of TN pts and 30% of R/R pts. Conclusions: Single-agent ibrutinib continues to show durable responses in pts with TN or R/R CLL/SLL including those with del17p, del11q, or unmutated IGVH. With extended treatment, CRs were observed in 29% of TN and 10% of R/R pts, having evolved over time. Ibrutinib provided better PFS outcomes if administered earlier in therapy than in the third-line or beyond. Those without CK experienced more favorable PFS and OS than those with CK. Ibrutinib was well tolerated with the onset of AEs decreasing over time, allowing for extended dosing for 65% of TN and 30% of R/R pts who continue treatment. Disclosures O'Brien: Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Furman:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Coutre:Janssen: Consultancy, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding. Flinn:Janssen: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership. Burger:Pharmacyclics, LLC, an AbbVie Company: Research Funding; Gilead: Research Funding; Portola: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Roche: Other: Travel, Accommodations, Expenses. Sharman:Gilead: Research Funding; TG Therapeutics: Research Funding; Acerta: Research Funding; Seattle Genetics: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding. Wierda:Abbvie: Research Funding; Genentech: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Gilead: Research Funding. Jones:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Luan:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment, Other: Travel, Accommodations, Expenses. James:AbbVie: Equity Ownership; Pharmacyclics, LLC, an AbbVie Company: Employment. Chu:Pharmacyclics, LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership.

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