Ibrutinib Research Articles

Tumor Microenvironment (TME) Regulation of Programmed Death-1 (PD1) Receptor and Its Ligands PDL1 and PDL2 in Chronic Lymphocytic Leukemia (CLL)

The PD1 pathway is involved in the inactivation of immune effectors and is potentially reactive against neoplastic cells in the TME. T cells from CLL patients exhibit defective immunity, decreased proliferative capacity, and impaired effector function leading to T cell exhaustion. These functional defects coincide with higher CD244, CD160 and PD1 expression on T cells. In the Eμ-TCL1 transgenic CLL model, PDL1 checkpoint blockade restores immune dysfunction and impairs leukemia growth. PD1/PDL1 ligation also affects BCR signaling, and because PD1 is also expressed on CLL cells, PD1 interference might directly influence tumor growth and proliferation directly.We studied the 1) expression the PD1 receptor and its ligands and their correlation with TFTT; 2) role of the TME in controlling PD1 axis and miRNA expression; 3) role of the BCRi ibrutinib (IB) in the expression of the receptor and ligands.Early-stage CLL patients were prospectively enrolled at diagnosis (n=211, O-CLL1: clinicaltrial.govID:NCT00917540). Gene expression (GE) profiling was performed using the GeneChipVR Gene1.0 STArray (Affymetrix) and validated by qPCR (Thermofisher) and flow-cytometry (FC, BD Biosciences) in an independent subset of patients. CLL lymph node samples underwent in situ immunolocalization analyses. Autologous T-cells (AAT) were obtained by in vitro exposure of patient T-cells with anti CD3/CD28 Dynabeads (Thermofisher) and IL2 in co-culture with CLL cells. Cultures were monitored until substantial clumping was observed and tested for PD1 axis expression. HS5 stromal cells were used in co-culture experiments with B-CLL (ratio 1:100). MiR-424 expression was evaluated by qPCR using validated TaqMan® MicroRNA Assays (Thermofisher). In selected experiments IB was added to cell culture. PD1 GEP of circulating B-CLL cells was significantly higher than of ligands. No differences in GEP of PD1 or its ligands were observed stratifying cases based on CD38, ZAP-70, or FISH markers. Significantly higher PDL1 GE was detected in IGHV-M cases. GEP results were validated by qPCR on 27 independent CLL samples. The impact of GE of the PD1 axis on clinical outcomes in CLL cases (median follow-up 39 months, range 6−82 months) indicated TTFT was significantly shorter in cases with higher levels of PDL2, while no significant differences were detected based on higher or lower PD1 or PDL1 levels. Multivariate analysis indicated higher PDL2 gene expression retained an independent prognostic power (HR=1.9, 95%CI 1.1-3.4, P=.022) in predicting TTFT together with IGHV-UM status, B-lymphocytosis≥5000/mm3 (P=.020), and CD38 expression (P=.021). In situ immunolocalization of CLL tissue infiltrates showed variable expression of PD1 that characterized small lymphoid elements, while PDL1 and PDL2 (PDL2>PDL1) characterized larger medium-sized elements within proliferation centers. Co-localization studies revealed PD1 co-expression of CD20+CLL cells and in scattered CD3+ cells. Both PDL1 and PDL2 were variably expressed in CD20+CLL cells; few T-cells and macrophages showed expression of both ligands within CLL infiltrates. FC analysis of CLL cells after AAT co-culture showed a higher percentage expression of circulating PD1 axis members (n=27, PDL1>>PDL2>PD1) compared to baseline indicating that the changes in PD1 axis expression may be due to events triggered by activation of the TME. In contrast, T cell populations had higher expression of CD3+, CD4+ and CD8+ cells bearing both PD1 and PDL1, while no substantial increment for either CD3+, CD4+ or CD8+/PDL2+ cells following AAT co-culture. GE analysis also showed a similar increase in PDLs (n=15, PDL2>>PDL1>PD1) expression with respect to non-activated B-CLL cells. Interestingly, a 48h co-culture of B-CLL with HS5 stromal cells (n=6) showed a similar increase in PD1 axis GE pattern as AAT cultures. Expression of miR-424 also decreased significantly (<80%) after AAT co-cultures (n=6), indicating its potential involvement in PDL1 regulation. IB reduced PD1/PDL1 gene and protein expression on CLL B-cells; the IB inhibitory effect was also observed only on CD8+ cells.We indicate that PDL2 expression characterizes a subset of high-risk early stage CLL patients. PD1 axis expression is characteristic of the CLL TME whereby modulatory co-stimulatory signals may be counteracted by IB. Further studies will examine the role of miR-424 regulation with this cellular context. DisclosuresNo relevant conflicts of interest to declare.

A Validated Risk Model for Overall Survival in Relapsed/Refractory Chronic Lymphocytic Leukemia Applicable to Patients Treated with Novel Therapies and Standard of Care

Introduction:Several prognostic models for overall survival (OS) in chronic lymphocytic leukemia (CLL) have been developed, but none were derived in a relapsed/refractory (R/R) population or in the context of novel therapies. We therefore used a comprehensive model-building approach to derive and validate a novel risk model for OS in a population of previously treated patients (pts) with CLL who received therapy in randomized phase 3 trials of ibrutinib (IBR) vs standard of care (SOC) and idelalisib (IDELA) vs SOC.Methods:In collaboration with Janssen Pharmaceuticals, Gilead Sciences, and Pharmacyclics, the analysis included pts from 5 multicenter randomized phase 3 trials (n=1866): IBR plus bendamustine-rituximab (BR) vs placebo plus BR (HELIOS: NCT01611090), IBR vs ofatumumab (RESONATE: NCT01578707), IDELA plus BR vs placebo plus BR (Study 115: NCT01569295); IDELA plus rituximab vs placebo plus rituximab (Study 116: NCT01539512); and IDELA plus ofatumumab vs ofatumumab (Study 119: NCT01659021). All pts had a diagnosis of CLL, were previously treated, and required treatment.The model-building dataset(n=969) included pts from HELIOS and RESONATE, and was randomly assigned to the training dataset (n=727) and internal-validation dataset (n=242). The external-validation dataset was an independent pooled cohort from studies 115, 116, and 119 (n=897).We applied univariate and multivariate analyses (MVA) to 28 candidate clinical, laboratory and molecular/genetic prognostic factors to derive the risk model in the training dataset. The primary endpoint was OS. We assessed the need for separate models for novel therapies by interactions with treatment in univariate analyses.We evaluated different cutoffs for dichotomized covariates and for definition of risk groups after the final factors were selected. We fit a Cox regression on the training, internal-validation ,and external-validation datasets using the risk categories as the covariate to test the difference among the groups, and calculated the C-statistic as a measure of discrimination.Results:We proceeded with the development of a single risk score for all pts with R/R CLL, as only one potential prognostic factor (deletion 13q, interaction p value <0.10) demonstrated a differential effect necessitating separate models. Six factors were independently prognostic in the multivariate analysis of OS (p<0.05): β2-microglobubulin (B2M), lactose dehydrogenase (LDH), hemoglobin (HGB), immunoglobulin heavy chain variable region (IGHV) mutational status, number of prior therapies, and time from initiation of last therapy.We excluded number of prior therapies from the final multivariate model, reasoning that its optimal cutoff might change with advances in CLL therapies. IGHV did not remain significant (p=0.0592) in MVA of 5 remaining prognostic factors. Notably, deletion 17p was not independently prognostic for OS.The final risk model consisted of 4 prognostic factors: B2M ≥5 mg/L, LDH elevated, HGB <110 mg/L for women or <120 mg/L for men, and time from initiation of last therapy <24 months. Because hazard ratios of these factors were similar (range, 0.55-0.68) with overlapping confidence intervals, 1 point was assigned to each factor. The resulting score separated pts into low (score 0-1), intermediate (score 2-3), and high risk (score 4) groups.Our risk model was prognostic for OS in the training dataset (log-rank p<0.0001; C-statistic 0.74 [95% CI 0.60-0.85]) and was confirmed in the internal-validation dataset (log-rank p<0.0001; C-statistic 0.79 [95% CI 0.56-0.97]), as well as in the external-validation dataset (log-rank p<0.0001; C-statistic 0.71 [95% CI 0.59-0.81]).Conclusions:We present the first validated risk model for OS in relapsed/refractory CLL in the era of targeted therapies. Our model consists of 4 readily available factors (B2M, LDH, HGB, time from initiation of last therapy) and identifies 3 prognostic groups with significantly different OS. This model reliably identifies high risk pts. Investigators can address this unmet need by designing prospective trials targeting these pts. [Display omitted] DisclosuresDarif:Janssen: Employment, Equity Ownership. Londhe:Janssen: Employment, Equity Ownership. Xing:Gilead: Employment, Equity Ownership. Huang:Gilead: Employment. Lin:Gilead: Employment. Byrd:Acerta Pharma: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics: Research Funding; Janssen: Research Funding. Furman:Pharmacyclics: Consultancy, Honoraria; Genentech: Consultancy; Gilead: Consultancy; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sunesis: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy. Hallek:Abbvie: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau. Hillmen:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Research Funding. Jones:Sunesis: Other: Institutional research funding; Abbvie, Pharmacyclics, Genentech, Gilead, Janssen, Merck, and Acerta: Other: Institutional research funding; Genentech, Abbvie, Pharmacyclics, Gilead, Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sharman:Gilead Sciences, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Research Funding; Genentech: Research Funding. Ferrante:Janssen: Employment, Equity Ownership. Reddy:Pharmacyclics/Abbvie: Employment, Equity Ownership. Dreiling:Gilead Sciences, Inc.: Employment. Adewoye:Gilead: Employment. Dubowy:Gilead: Employment. Howes:Janssen R&D: Employment, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Equity Ownership. James:Pharmacyclics LLC, an AbbVie Company: Employment; AbbVie: Equity Ownership. Zelenetz:Celgene: Consultancy; Amgen: Consultancy.

FoxM1: A New Therapeutic Target for Mantle Cell Lymphoma

IntroductionMantle cell lymphoma (MCL) is an aggressive B-cell lymphoma subtype with elevated B-cell receptor activity. Ibrutinib (IBN), the Bruton's tyrosine kinase (BTK) inhibitor, has been shown to have an overall response rate of 68% in relapsed or refractory MCL patients. However, with the emergence of IBN resistance, novel therapies to thwart resistance are urgently needed. FOXM1 (Forkhead box M1) is a proliferation-associated transcription factor that stimulates cell proliferation and exhibits a proliferation-specific expression pattern. FOXM1 has recently been classified as a human proto-oncogene and we previously found this gene to be associated with IBN resistance in our gene expression analysis; therefore, the prognostic significance of FOXM1 and its potential as a new MCL therapeutic target was investigated.MethodsBone marrow aspirates and peripheral blood clinical specimens were obtained from MCL patients. FOXM1 gene expression was assessed in 50 primary MCL patient samples using Quantitative real-time PCR (Q-PCR). To conduct dose-dependent cell viability assays, MCL cell lines (ATCC) were cultured in RPMI1640 (Invitrogen) supplemented with 10% fetal bovine serum (Millipore). The drug screening was performed in a 96-well format in which MCL cells were seeded at 10,000 cells per well and were treated with the FOXM1 inhibitor thiostrepton or ibrutinib at the following concentrations: 0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 uM. Cell viability was tested using the CellTiter-Glo luminescent cell viability assay (Promega) after a 72-hour incubation period. To determine the effects of inhibiting FOXM1 on apoptosis, MCL cells (200,000 cells) were incubated with IBN or thiostrepton at varying doses (1, 3, 5 uM) for 24 hours, and apoptosis was detected using the Annexin V-binding assay. Furthermore, caspase-3/7 activity was assessed in MCL cells with the Caspase-Glo-3/7 Assay (Promega) after 24 hours of treatment with thiostrepton or IBN.ResultsTo evaluate the possibility that FOXM1 is important for MCL, we conducted an analysis of FOXM1 expression in MCL patient samples at the time of diagnosis using Q-PCR. FOXM1 expression was detected in 50 MCL patient samples, and FOXM1 expression levels were significantly higher (82.3%, fold change=2) than the levels observed in peripheral blood mononuclear cells (PBMCs). The association between FOXM1 expression in MCL and clinical pathology was also analyzed by examining the relationship between FOXM1 and Ki-67 expression (a cell-proliferation marker) in 40 diagnosed MCL patient samples, and a significant correlation was noted between the two markers (p<0.05).The effects of inhibiting FOXM1 on MCL cell lines (Jeko-1, Jeko-1-BTK-KO-#1, Jeko-1-BTK-KO-#2, Mino, Maver-1, Z-138, JVM-2, and JVM-13) in vitro were examined by transfection of siRNA targeting FOXM1 or treatment with the FOXM1 inhibitor thiostrepton. Thiostrepton reduced the cell viability of the MCL cell lines in a dose-dependent manner compared with the DMSO control. Likewise, siFOXM1-transfected MCL cell lines showed reduced cell viability compared with the control siRNA-transfected cells (P < 0.01). Annexin V/PI dual staining showed that thiostrepton induced apoptosis in the MCL cell lines in a dose-dependent manner. Furthermore, activated-caspase3-7 expression was increased in a dose-dependent manner in MCL cell lines after treatment with thiostrepton.ConclusionWe have shown that FOXM1 inhibition may be a potential candidate treatment for MCL based on the results of our clinicopathological assessment and in vitro studies. Clinical studies are warranted to further validate FOXM1 as a potential therapeutic target in high-risk MCL. DisclosuresWang:Celgene: Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Oncternal: Research Funding; BeiGene: Research Funding; Juno Therapeutic: Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Consultancy, Honoraria, Research Funding; Oncoceutics: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Asana: Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Karus: Research Funding.

External Stimuli Modulate EZH2 Expression in Chronic Lymphocytic Leukemia. Rationale and Evidence for Synergistic Anti-Tumor Effects of B Cell Signaling and EZH2 Inhibitors

The histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). We have previously reported that EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival and proliferation. Moreover, we demonstrated that EZH2 pharmacological inhibition induced down-regulation of H3K27me3 levels leading to increased cell apoptosis. Here we sought to investigate if EZH2 expression could be affected by microenvironmental triggering, a key driver of CLL development and evolution.The study group included 10 CLL cases (9 with unmutated and 1 with mutated IGHV genes) that had previously been screened and found responsive to cross-linking of the B cell receptor (BcR) with anti-IgM. CD19+ cells were negatively selected from peripheral blood and stimulated or not through the BcR for 14 hours. Using RQ-PCR we found that stimulation with anti-IgM showed opposing effects on EZH2 mRNA levels, since 5 cases upregulated while the remaining 5 downregulated EZH2. In contrast, stimulation with CpG or CpG/CD40L for 14 hours caused pronounced and consistent upregulation of EZH2 mRNA levels compared to unstimulated cells (FD=8.7, p=0.03 and FD=11.3, p=0.03, respectively; n=6). This effect was also obvious at the protein levels in cells treated with CpG, CD40L and CpG/CD40L for 24 hours, where EZH2 protein was significantly upregulated as revealed by Western blot analysis (FD=3.9, p=0.002; FD=1.7, p=0.005; and FD=2.9, p=0.01, respectively; n=6). In CpG-stimulated cultures, the induction of EZH2 was accompanied by a significant increase in cell viability (FD=2, p=0.002; n=6), supporting the conclusion that EZH2 expression likely contributes to the anti-apoptosis phenotype induced in CLL cells by microenvironmental signals. Prompted by these observations, we then investigated whether B cell signaling inhibition might also modulate EZH2 expression and H3K27me3 levels in vivo by longitudinal profiling of another group of 9 CLL patients that were under ibrutinib treatment. In patients at +1 month under ibrutinib, when, as shown by us and others, CLL cells become anergized through the BcR, we observed a significant decrease in EZH2 protein (FD=3, p=0.0007; n=9) as well a trend for reduced H3K27me3 levels (FD=1.4, p=0.07; n=6) compared to the pre-treatment samples, further highlighting the links between BcR signaling and EZH2 expression and functionality. Next, we assessed ex vivo the effects of ibrutinib on CLL cell viability in cases with differential EZH2 expression [EZH2high (n=7) vs EZH2low (n=6)] and found decreased viability in treated cells compared to control cells (DMSO-exposed) in EZH2high cases (FD=1.9, p=0.0025), with no difference in EZH2low cases.Finally, we examined potential synergistic effects of B cell signaling inhibitors [ibrutinib (IB), idelalisib (IDE)] and EZH2 inhibitors (GSK343, GSK126) in primary CLL cells. To this end, CD19+ cells from 6 CLL cases were first pre-stimulated with CpG for 24 hours and then exposed to either single inhibitors (IB, IDE, GSK343, GSK126) or combinations of B cell signaling and EZH2 inhibitors (IB/GSK343, IDE/GSK343, IB/GSK126). Flow cytometry analysis after 3 days in culture revealed significantly lower CLL cell viability and H3K27me3 levels in CLL cells exposed to combined versus single treatment (FD≥ 1.5, p<0.05).In conclusion, we show that EZH2 expression is affected by signals emanating from CLL microenvironment. Moreover, we provide for the first time evidence that the combination of BcR signaling inhibitors widely used in CLL therapy and EZH2 inhibitors currently tested in phase I/II clinical trials in lymphoid malignancies has synergistic effects, suggesting that targeting both processes might represent a rational and promising therapeutic strategy. DisclosuresStavroyianni:Gilead Sciences: Honoraria; Takeda: Honoraria; Janssen Pharmaceuticals: Honoraria. Ghia:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria; Janssen Pharmaceuticals: Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Papakonstantinou:Gilead: Research Funding; Janssen Pharmaceuticals: Research Funding. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding; Novartis SA: Research Funding.

Systematic Literature Review of the Clinical Efficacy and Safety of Treatments in the Relapsed/Refractory Setting for Patients with Follicular Lymphoma or Marginal Zone Lymphoma

IntroductionThe objective of this study was to assess the efficacy and safety of current and emerging treatments in patients with follicular lymphoma (FL) or marginal zone lymphoma (MZL) who experienced relapse or refractory disease after ≥ 1 treatment.MethodsWe performed a systematic literature review to identify all publicly available randomized controlled trials (RCTs) and nonrandomized (NR) prospective interventional studies in the relapsed/refractory (R/R) setting for FL or MZL patients in EMBASE®, MEDLINE®, and the Cochrane Central Register of Controlled Clinical Trials on March 3, 2017. Additionally, four conference proceedings from 2015 and 2016 (the American Society of Hematology, European Hematology Association, European Society for Medical Oncology, and American Society of Clinical Oncology) were searched to present an up-to-date overview. For safety outcomes we extracted grade 3/4 adverse events (AE). Relevant interventions included rituximab (R), idelalisib, lenalidomide (LEN) ± R, ofatumumab (OFA), obinutuzumab (O), bortezomib (V) ± R, bendamustine (B) ± R, VBR, ibrutinib (IBRU) ± chemoimmunotherapy, brentuximab vedotin, fludarabine (F) + R, O ± B, CHOP ± R, and autologous stem cell transplantation (ASCT). Other interventions which were searched for but not identified are seen in Table 1.ResultsWe screened 3255 abstracts and 126 full papers. Ten RCTs and 23 NR prospective interventional studies were selected for inclusion. In total, 29 studies reported results on chemo-based interventions (nine RCTs and 20 NR prospective interventional studies), and limited ASCT data were reported (one RCT and 3 NR prospective interventional studies). The majority of included studies reported largely FL populations with only two studies being conducted specifically in MZL. There was a considerable amount of heterogeneity across the included studies; factors contributing to this included study design, stage of disease, refractory versus relapsed population, and the median number of prior treatments. In the majority of studies patients had stage III/IV disease. Several studies reported the median number of prior treatments with large variation among the ranges presented. Population age varied, with the median age ranging from 46 to 68.5 years. The most common intervention received was R, either as monotherapy or in combination with chemotherapy.The highest objective response rate was reported for FL/MZL patients treated with B (100%) while the lowest was reported for FL/MZL patients treated with OFA (10%). Median PFS for included studies is summarized in Table 1. Four RCTs provided significant PFS hazard ratios (HRs). Two ASCT studies reported median PFS ranging from 2.4 years (ASCT + interferon [INF]) to 83 months (ASCT + R + INF). In total, 12 studies presented duration of response (DOR) data with one study resulting in a significant DOR HR.Overall survival (OS) data were poorly reported, with only eight studies reporting any OS data. In three of eight studies that intended to report OS, median OS had not been reached.Safety and health-related quality of life (HRQoL) data were not consistently reported in the included studies. The most commonly reported grade 3/4 AE was neutropenia (12 studies) with an incidence ranging from 1.4% (R) to 54.7% (R-CHOP). Fatigue was also reported in nine studies with an incidence ranging from 1% (V + R) to 13% (LEN + R). Other AEs reported were anemia, diarrhea, infection, infusion-related reaction, nausea, pneumonia, sepsis, thrombocytopenia, and vomiting. Only one study presented HRQoL data (EORTC QLQ-C30).ConclusionThis literature review provides a robust overview of response and PFS for treatments of R/R FL/MZL; however, heterogeneity in study design and patient population, as well as a general lack of reporting of OS data, make it challenging to identify an optimal treatment regimen in this setting. HRQoL data were not commonly reported despite the important role patient QoL plays in treatment selection. Additional randomized clinical trials, a more consistent approach to the reporting of clinical end points, and continued monitoring of OS data in this population may address some of these data gaps and unmet needs for patients with R/R FL/MZL. [Display omitted] DisclosuresMonga:Janssen: Other: Janssen employee. Garside:Janssen Pharmaceutica NV: Other: employee of Janssen Pharmaceutica NV. Parisi:Janssen: Other: Janssen employee. Tapprich:Janssen: Other: Janssen employee. Nastoupil:Genentech: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; TG Therapeutics: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Karus Therapeutics: Research Funding; Gilead: Honoraria.

PHASE I STUDY OF BENDAMUSTINE, RITUXIMAB, IBRUTINIB, AND VENETOCLAX IN RELAPSED, REFRACTORY MANTLE CELL LYMPHOMA

Introduction: Bendamustine (B) and rituximab (R) with ibrutinib (IBR) was well-tolerated and efficacious (Maddocks Blood 2015). Ibrutinib and venetoclax (VEN) were synergistic and active in relapsed, refractory (R/R) MCL (Tam NEJM 2018). VEN increases apoptotic priming and can act as a chemosensitizing agent. The aim of this phase I study was to define the safety and tolerability of VEN with BR-IBR in R/R MCL (Clinical trial: NCT03295240). Methods: Patients (pts) with R/R MCL received six 28-day cycles of BR-IBR-VEN in a 3+3 dose escalation design. Dose level 1 (DL1) included B 90 mg/m2 day (d) 1 and 2, R 375 mg/m2 d1, and IBR 560mg daily. For cycle 1, VEN dose ramp-up was 20, 50, 100, and 200mg daily weekly. During cycles 2-6, VEN 400mg daily was given d1-5. Dosing cohorts with longer duration of VEN (7d and 10d) were planned. The study was amended to include dose level-1 (DL-1) with B at 70mg/m2, d1 and 2. Results: As of March 2021, 7 pts were treated with BR-IBR-VEN at DL1 and 3 pts at DL-1. Baseline characteristics were median age 68 years (range 60-81);90% male;80% with Ki67 ≥30%;10% blastic MCL;40% TP53 mutant, and 100% one prior line of treatment (tx). One dose limiting toxicity (DLT) was observed at DL1: grade 3 thrombocytopenia lasting >14 days. At DL1, 2 pts had only 5 cycles of BR-IBR-VEN due to cytopenias (grade 3 neutropenia and grade 2 thrombocytopenia) despite B dose reduction. The frequent tx-related adverse events (AEs) were thrombocytopenia (n = 7), constipation (n = 6), fatigue (n = 4), neutropenia (n = 3), and nausea (n = 4). The grade 3-4 AEs were neutropenia (n = 1) thrombocytopenia (n = 3), and rash (n = 1). There was one tx-related serious AE: an elderly pt was briefly hospitalized during VEN ramp-up for volume overload and diuretic tx. Most pts had count recovery post-tx, but one had persistent neutropenia. Given cytopenias observed at DL1, the study was amended to include DL-1. At DL-1 (n = 3), there were no DLTs, dose reductions or delays, significant cytopenias, or ≥grade 3 txrelated AEs. The overall response rate (ORR) was 80% (8/10), all complete responses (CRs). For TP53 mutant MCL, the ORR was 50% (2/4), all CRs. There were 5 deaths (Fig 1). Two TP53 mutant MCL pts with primary refractory disease died of disease rapidly. A 17p deleted MCL pt achieved a CR and underwent allogeneic stem cell transplant and died of pneumonia (PNA) 10 mos after. A pt in CR with post-tx neutropenia died of PNA. The TP53 mutant MCL pt with a DLT after 2 cycles of BR-IBR-VEN received single-agent BTKi for 27 mos until progression and then died of COVID. Conclusions: BR-IBR-VEN showed acceptable safety and tolerability and preliminary efficacy at DL-1. During the COVID pandemic, enrollment for this immunosuppressive 4-drug regimen with intravenous tx was challenging. The study was closed after enrolling 3 of 6 pts at DL-1 and a definitive maximum tolerated dose was not determined. Future study is needed to determine the role of BR-IBR-VEN in MCL.

Improving physicochemical properties of Ibrutinib with cocrystal strategy based on structures and natures of the carboxylic acid co-formers

Ibrutinib(IBR) is an irreversible Bruton's tyrosine kinase inhibitor and has been classified as the Biopharmaceutical Classification System (BCS) class Ⅱ drug. The objective of the current study was to improve the physicochemical properties of IBR by co-crystallization. Based on ΔpKa analysis and structure analysis, we reported seven IBR pharmaceutical cocrystals with different structure types of co-formers such as hydroxy-2-naphthoic acids, mono-hydroxybenzoic acids, and aliphatic acids via the slurry method and ultrasonic method. The seven prepared cocrystals were characterized by powder X-ray diffraction(PXRD), differential scanning calorimetry(DSC), thermogravimetric analysis(TGA) and Fourier transform infrared spectroscopy(FT-IR). The solubility and accumulative dissolution of IBR cocrystals were explored. It was found that the solubility and dissolution were optimally increased up to 6.64 times and 1.85 times than that of IBR Form A, respectively. Moreover, all the IBR cocrystals maintained a stable cocrystal state after 90-d under 40 °C/75% RH condition and after 30-d under 60 °C/0% RH condition. According to the results analysis, there may be hydrogen bond(H-bond) and π-π stacking interactions in the IBR cocrystal system. The current study demonstrated that the co-crystallization of IBR with specific co-formers proposed a prospective technology and optional strategy to modify the physicochemical nature of IBR.

PRE-EXISTING CARDIOVASCULAR DISEASE INCREASES RISK OF ATRIAL ARRHYTHMIA IN CANCER PATIENTS TREATED WITH IBRUTINIB

PRE-EXISTING CARDIOVASCULAR DISEASE INCREASES RISK OF ATRIAL ARRHYTHMIA IN CANCER PATIENTS TREATED WITH IBRUTINIB

Comparison of ibrutinib and idelalisib plus rituximab in real-life relapsed/resistant chronic lymphocytic leukemia cases.

To compare the capacity of ibrutinib (IB) and idelalisib-rituximab (IDELA-R) of prolonging overall survival (OS) as in CLL patients, previously treated with chemotherapy only. A real-life cohort of 675 cases has been identified and investigated in the database of the groups participating in the study. At an unadjusted univariate analysis, a significant death risk reduction was observed favoring IB (IDELA-R vs IB HR=0.5, 95% CI=0.36-0.71) although with some limitations due to the non-randomized and retrospective nature of the study and to the lower number of patients in the IDELA-R group (112 cases) related to the current prescribing practice. To overcome the potential problem of confounding by indication, we adjusted the association between the type of therapy and mortality for all variables significantly associated with OS at Cox univariate analysis. Furthermore, those variables, differently distributed between the two study groups, were introduced into the multivariate Cox model to improve the effectiveness of the analysis. By introducing all these variables into the multiple Cox regression model, we confirmed the protective effect of IB vs IDELA-R (HR=0.67, 95% CI=0.45-0.98, P=.04) independent of potential confounders. Although our analysis presents some constraints, that is, the unavailability of additional potential confounders, and the retrospective nature of the study, this observation may be of help for the daily clinical practice, particularly in the absence of randomized trials comparing the two schedules.

Photodynamic/ photothermal therapy enhances neutrophil-mediated ibrutinib tumor delivery for potent tumor immunotherapy: More than one plus one?

Neutrophil-mediated drug-delivery systems have gained widespread attention owing to their superior efficacy in cancer therapy. Neutrophils, the most abundant white cells in peripheral blood, are known to migrate to inflamed tumors. Here, we elaborate on a novel strategy to enhance tumor infiltration of neutrophils by photodynamic/photothermal therapy (PDT/PTT) to deliver ibrutinib (IBR) nanocomplexes for cancer immunotherapy. DiR-loading liposomes (DiR-lipos) were administered to induce acute inflammation, and sialic acid (SA) derivative-coated IBR-loading nanocomplexes (SA-2@NCs) were fabricated for targeting activated peripheral blood neutrophils (PBNs). This in vitro and in vivo attempt, therefore, proved the hypothesis that inducing acute inflammation via PDT/PTT could facilitate the migration of PBNs, which could deliver SA-2@NCs into the tumor. The enhanced tumor delivery of SA-2@NCs was accompanied by enhanced antitumor T-cell immune responses in a mouse orthotopic breast cancer model. Our findings indicate that the combination of IBR-mediated immunotherapy with DiR-mediated PDT/PTT bring together two leading novel strategies, taking advantage of their synergistic mechanisms of action for a potent anti-tumor efficacy for breast cancer therapy.

Ibrutinib and Obinutuzumab in CLL: MRD Responses Sustained for Several Years with Deepest MRD Depletion in Patients with >1 Year Prior Ibrutinib Exposure

Ibrutinib and Obinutuzumab in CLL: MRD Responses Sustained for Several Years with Deepest MRD Depletion in Patients with >1 Year Prior Ibrutinib Exposure

Clinical Outcomes Among Real-World Patients with Chronic Lymphocytic Leukemia (CLL) Initiating First-Line Ibrutinib or Chemoimmunotherapy (CIT) Stratified By Risk Status: Results from a US Retrospective Chart Review Study

Clinical Outcomes Among Real-World Patients with Chronic Lymphocytic Leukemia (CLL) Initiating First-Line Ibrutinib or Chemoimmunotherapy (CIT) Stratified By Risk Status: Results from a US Retrospective Chart Review Study

Ibrutinib Off-Target Inhibition Inhibits Antibody-Dependent Cellular Phagocytosis but Not Efferocytosis of CLL Cells

Background Combinations of different targeted therapies, including Bruton tyrosine kinase (BTK) inhibitors and anti-CD20 monoclonal antibodies (mAbs) could improve treatment for CLL. Unexpectedly, the combination of ibrutinib (IBR) with rituximab did not show additional clinical benefit. However, IBR inhibits many off-target molecules that may limit therapeutic mAb clinical effectiveness and a more selective BTK inhibitor, such as acalabrutinib (ACALA), could be more effective in combination with mAb therapy. Initial data from the ELEVATE TN trial support this possibility. IBR off-target effects on antibody-dependent cellular phagocytosis (ADCP), the major mechanism of therapeutic mAb activity could explain this difference. Additionally, IBR induces a higher and longer duration increase in circulating lymphocytes than ACALA. IBR off-target effects on efferocytosis, another phagocytic process involved in apoptotic cell removal, might explain this difference. Methods Using state-of-the-art direct kinetic measurements of phagocytosis by time-lapse video, (Chu et al. J Cell Sci 2020;133:jcs237883) we investigated the effects of IBR and ACALA on phagocytosis (ADCP or efferocytosis) by human monocyte-derived macrophages (hMDM) in vitro. Live cell time-lapse video of 10 μg/ml rituximab (Genentech) mediated ADCP of CLL cells by CellTracker Deep Red (CTDR, Thermofisher) labeled hMDM (20:1 CLL:hMDM cell ratio) either untreated or treated with IBR or ACALA (3-fold serial dilutions from 100 to 0.41 μM) was imaged in a stage-top environmental chamber (37°C and 5% CO2) mounted onto a Nikon Ti-Eclipse inverted microscope with an ELWD 20x/0.45NA S Plan Fluor Ph1 objective and an Andor Zyla 5.5 sCMOS camera. Images were captured sequentially every 4 min over 2.8 h. For each experiment (n = 18), duplicate or triplicate wells for each drug concentration were imaged. For efferocytosis, live cell time-lapse video imaging of phagocytosis of pHrodo iFL Red STP ester (pHrodo Red, Thermo Fisher Scientific) labeled apoptotic CLL cells by CTDR-labeled hMDM (20:1 CLL:hMDM cell ratio) either untreated or treated with IBR or ACALA (2-fold serial dilutions from 10 to 1.25 μM) was collected every 4 min over 2.8 h. For each experiment (n = 7), duplicate or triplicate wells for each drug concentration was imaged and analyzed. Finally, for efferocytosis, the intensity of pHrodo Red dye, a pH-sensitive dye that increases in intensity with acidic pH, as found in the endolysosomes, was measured in the pHrodo Red color channel and analyzed. Results IBR significantly inhibited ADCP at all measured drug concentrations (0.41 μM, p &amp;lt; 0.05; 1.2 μM, p &amp;lt; 0.01; 3.7 - 100 μM, p &amp;lt; 0.001). The mean peak free drug concentration (Cmax) achieved clinically by standard doses for IBR is ~0.5 μM. ACALA only significantly inhibited ADCP at the highest concentration (100 μM, p &amp;lt; 0.001). The Cmax achieved clinically by standard doses for ACALA is ~1.2 μM. ACALA did not inhibit efferocytosis or subsequent transition to endolysosomal compartment at all tested concentrations (p &amp;gt; 0.05). IBR did not inhibit efferocytosis (p &amp;gt; 0.05) and only inhibited transition to endolysosomal compartment at highest concentration tested (10 μM, p &amp;lt; 0.01) Conclusion Our study shows that BTK inhibition does not block ADCP and a more selective BTK inhibitor may prove effective in combination with therapeutic anti-CD20 mAbs. IBR off-target inhibition specifically blocks ADCP and not efferocytosis. Thus, IBR off-target inhibition of ADCP should be via proximal signaling by antibody Fc receptors and not subsequent downstream phagocytic mechanisms in common with efferocytosis. These results also imply the lack of BTK and IBR off-target molecules involvement in efferocytosis. Finally, the increased lymphocytosis seen with IBR compared to ACALA treatment in CLL cannot be explained by IBR off-target effects on efferocytosis. These findings provide a critical understanding of macrophage phagocytosis reduction by BTK inhibitor selectivity that will have important consequences for the development of combination targeted therapies with mAbs. Disclosures Chu: Acerta Pharma/AstraZeneca: Research Funding; Pfizer: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; TG Therapeutics: Research Funding. Izumi:AstraZeneca: Current equity holder in publicly-traded company; Acerta Pharma: Current equity holder in private company, Ended employment in the past 24 months, Patents &amp; Royalties: Acalabrutinib patents (no royalties). Munugalavadla:Gilead Sciences: Current equity holder in publicly-traded company; AstraZeneca: Current equity holder in publicly-traded company; Acerta Pharma: Current Employment. Barr:Gilead: Consultancy; Morphosys: Consultancy; TG therapeutics: Consultancy, Research Funding; Seattle Genetics: Consultancy; Celgene: Consultancy; AstraZeneca: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Genentech: Consultancy; Abbvie/Pharmacyclics: Consultancy, Research Funding; Verastem: Consultancy. VanDerMeid:Acerta Pharma / AstraZeneca: Research Funding. Elliott:Acerta Pharma / AstraZeneca: Research Funding. Zent:Mentrik Biotech: Research Funding; TG Therapeutics, Inc: Research Funding; Acerta / Astra Zeneca: Research Funding.

Combined Ibrutinib and Venetoclax for First-Line Treatment for Patients with Chronic Lymphocytic Leukemia (CLL): Focus on MRD Results

Combined Ibrutinib and Venetoclax for First-Line Treatment for Patients with Chronic Lymphocytic Leukemia (CLL): Focus on MRD Results

Final Results of a Phase II Study of Fc Engineered, CD19 Antibody Tafasitamab in Combination with Lenalidomide or Ibrutinib in Patients with Chronic Lymphocytic Leukemia (CLL)

Final Results of a Phase II Study of Fc Engineered, CD19 Antibody Tafasitamab in Combination with Lenalidomide or Ibrutinib in Patients with Chronic Lymphocytic Leukemia (CLL)

Minimal Residual Disease-Driven Treatment Intensification By Sequential Addition of Ibrutinib to Venetoclax in Relapsed/Refractory Chronic Lymphocytic Leukemia: Results of the Monotherapy and Combination Phases of the Improve Study

Minimal Residual Disease-Driven Treatment Intensification By Sequential Addition of Ibrutinib to Venetoclax in Relapsed/Refractory Chronic Lymphocytic Leukemia: Results of the Monotherapy and Combination Phases of the Improve Study

High Clonal Complexity of Resistance Mechanisms Occurring at Progression after Single-Agent Targeted Therapy Strategies in Chronic Lymphocytic Leukemia

High Clonal Complexity of Resistance Mechanisms Occurring at Progression after Single-Agent Targeted Therapy Strategies in Chronic Lymphocytic Leukemia

Comparative Efficacy of First-Line Chemotherapy-Free Combinations in Chronic Lymphocytic Leukemia (CLL): A Network Meta-Analysis

Introduction: Chronic Lymphocytic Leukemia (CLL) is an incurable B-cell malignancy which disproportionately affects the elderly. Although first-line chemoimmunotherapy (CIT) improved CLL clinical outcomes, recent randomized trials revealed superior outcomes with novel chemotherapy-free combinations (CFC) incorporating anti-CD20 monoclonal antibodies and inhibitors of BTK or Bcl-2. So far, these CFC have not been compared head-to-head. We conducted this network meta-analysis to evaluate their relative efficacy to each other. Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language; diagnosis of CLL; trials that explored the efficacy of first-line CFC with Obinutuzumab (O), Rituximab (R), Ibrutinib (IB), Acalabrutinib (ACAL), Venetoclax (VEN) compared to standard CIT that included Chlorambucil (CHLOR) with either R or O, Bendamustine+Rituximab, or Fludarabine+ Cyclophosphamide+R; and phase 3 randomized studies reporting responses, progression, death, and adverse (AE) events. A frequentists network meta-analysis was conducted using netmeta package and random-effects model. Results: Five studies comprising a total of 2,272 participants were included. When O-based CFC data was analyzed, only ACAL-O had a significant lower relative risk (RR) of progression and death (P&amp;D). There were no significant differences with respect to overall response rates (ORR), complete remission (CR), minimal residual disease (MRD), or grade &amp;gt;3 adverse events (Grd3+) among O-based CFC. When R-based CFC data was analyzed, IB and IB-R were not different with respect to RR of P&amp;D, ORR, CR, MRD, or Grd3+. When the data was analyzed as CFC versus combined CIT, only ACAL-O was found to be significantly superior to other O- and R-based CFC with respect to RR of P&amp;D. ORR and Grad3+ rates of O- and R-based CFC were not significantly different. While ACAL-O, IB-O, and VEN-O had superior CR and MRD rates compared to other CFC, there were no significant differences among each other. Conclusions: This network meta-analysis is the first to compare and rank first-line CFC therapies in CLL. It indicates that ACAL-O has a superior profile having the lowest RR of P&amp;D without significant difference in Grd3+ among CFC. Disclosures No relevant conflicts of interest to declare.

Targeting Glutamine Metabolism Overcomes Resistance to Targeted Therapies in Refractory Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin's lymphoma characterized by high refractory occurrence following drug treatment. Despite the encouraging initial MCL tumor response to ibrutinib (IBN), relapse occurs only after few months of treatment due to multiple resistance mechanisms. Thus, the novel therapeutic strategies targeting resistant mechanisms are crucial. Our group has recently shown that among the highly proliferative MCL population, a subpopulation of IBN-R cells exhibits increased OXPHOS activity that is fueled by increased glutaminolysis and rely more on mitochondrial respiration for their grow and survival. The aim of this work was to uncover potential targets responsible for the upregulation of OXPHOS pathway in the refractory/relapsed (R/R) MCL by using multiple biochemical and biological strategies. We focused the present study on glutaminase (GLS), the enzyme that converts glutamine to glutamate, a precursor of α-ketoglutarate (α-KG) that links glutamate to the TCA cycle. Incorporation of α-KG into the TCA cycle is a major anaplerotic step in proliferating cells and is critical for the maintenance of TCA cycle function. To further demonstrate the reliance of OXPHOS on glutamine anaplerosis, we have further tested the combinatory effects of targeting GLS and OXPHOS using their respective inhibitors, CB-839 and IACS-010759, on tumor killing activity in R/R MCL. Methods:Primary MCL cells from patient leukapheresis or whole blood specimens, as well as established MCL cell lines were used as experimental models of MCL. Metabolomic profiling was used to determine intracellular metabolite fluxes and levels. Cell Titer Glo assay was used to measure cell proliferation/viability after treatment with inhibitors. Annexin V and propidium iodide were used to measure cell apoptosis and cell cycle arrestviaflow cytometry analysis. Magnetic microbeads-based B-cell isolation method were used for the purification of malignant B cells from patient samples. Western blot analysis was used to evaluate protein level expression. Patient-derived Xenograft (PDX) mouse model created from patients with MCL was used to evaluate the in vivo anti-tumor activity and potential clinical value of GLS and OXPHOS inhibitors. Results:Our recent metabolomic profiling studies have demonstrated that glutaminolysis and OXPHOS are upregulated in IBN-R MCL, manifested by increased glutamine uptake in the ibrutinib-resistant MCL cell lines (p=0.03).Inhibition of glutamine metabolism with the allosteric GLS1-selective inhibitor BPTES resulted in inhibition of cell viability (0.2381uM-9.98uM), indicating that MCL cells are dependent on glutamine metabolism for their proliferation. To corroborate with the above finding, we also presented evidence that GLS1 is highly increased in IBN-R and CART-R MCL patient samples and cell lines confirmed by immunoblotting. Inhibiting of GLS would lead to significant reduction in OXPHOS, mitochondria membrane potential and ATP production, as either single drug or in combination with other targeting agents. To identify a clinical actionable GLS inhibitor for the treatment of MCL, we chose a GLS1 specific inhibitor CB-839 (Selleckchem), currently under several phase II and III clinical trials investigation on solid tumors. Inhibiting GLS1 with CB-839 leads to the decreased cell viability in MCL (0.5626nM-308.4nM). Of note, the treatment with CB-839 to MCL cell lines induces S phase reduction in both Jeko-1 (17.23%) and Z-138 (14.01%), as well as induces significant apoptosis (p=0.013 and p=0.002 in Jeko-1 and Z-138 cells). GLS inhibition will be further explored in the context of mitochondria defect or hypoxia, where OXPHOS maybe deficient. Importantly, while CB-839 is continuing its validation in several solid tumor models, this is the first study providing data on its efficacy in preclinical models of MCL. Conclusion:In conclusion, we report that glutaminolysis and OXPHOS are upregulated in IBN-R MCL that could be partially due to high expression of GLS1. Our preliminary results revealed that the new GLS inhibitor, GCB-839, may present a clinical potential for a new indication and warrants more in-depth investigation. Deciphering the mechanisms involved in MCL metabolic heterogeneity and adaptability during drug resistance development would be crucial to identify key actors enabling MCL cells to escape from therapy. Disclosures Wang: Acerta Pharma:Research Funding;Molecular Templates:Research Funding;InnoCare:Consultancy;Oncternal:Consultancy, Research Funding;Celgene:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Targeted Oncology:Honoraria;MoreHealth:Consultancy;Kite Pharma:Consultancy, Other: Travel, accommodation, expenses, Research Funding;Lu Daopei Medical Group:Honoraria;OMI:Honoraria, Other: Travel, accommodation, expenses;Verastem:Research Funding;Nobel Insights:Consultancy;BioInvent:Research Funding;Guidepoint Global:Consultancy;AstraZeneca:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Pharmacyclics:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Janssen:Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding;Juno:Consultancy, Research Funding;Dava Oncology:Honoraria;Loxo Oncology:Consultancy, Research Funding;Pulse Biosciences:Consultancy;OncLive:Honoraria;Beijing Medical Award Foundation:Honoraria;VelosBio:Research Funding.

Ibrutinib and carboxylic acid coamorphous system with increased solubility and dissolution: A potential interaction mechanism

The aim of this study was to improve the solubility and dissolution of Ibrutinib (IBR), a biopharmaceutical classification system (BCS) class II drug, by coamorphization with four kind of carboxylic acids, tartaric acid (TA), citric acid (CA), malic acid (MA) and succinic acid (SA) at molar ratios of 1:1 and 2:1, and investigate the interaction mechanism between IBR and acids responsible for enhancement. Compared with crystalline IBR Form A, eight IBR-Acid coamorphous systems (CASs) showed prominent improvement in solubility and dissolution. The solubility and dissolution of IBR-TA (2:1) CAS were increased up to 3.5–5.5 times and 4.3 times than IBR Form A, which showed optimal properties. Moreover, IBR-TA (2:1) CAS kept stable amorphous state after 180 days under 40 °C/75 %RH condition and kept stable amorphous state for 30 days under 60 °C/0 %RH condition. From Fourier Transform-Infrared spectra and glass transition temperature analysis, the possible molecular interaction model in IBT-TA (2:1) CAS was proposed to explain the improvement of solubility and dissolution. The above investigation indicated that coamorphization of IBR and acid was an effective strategy to improve solubility and dissolution of poor soluble IBR.