Plasma biomarker signature associated with improved survival in advanced non-small cell lung cancer patients on linifanib.

OBJECTIVES: Linifanib, a potent and selective inhibitor of the tyrosine kinase activity of vascular endothelial growth factor and platelet-derived growth factor receptors, has clinical activity in advanced non-small cell lung cancer (NSCLC) both as monotherapy in the relapsed setting or with carboplatin and paclitaxel in the first-line setting. Though benefit was observed in unselected patient populations, identification of predictive biomarkers is critical for further development of this novel agent. MATERIALS AND METHODS: Data from 4 randomized studies in relapsed NSCLC with linifanib (n=116) or other treatments (n=125) were examined in an exploratory analysis to identify a biomarker profile predictive of favorable survival. RESULTS: A signature combining the established tumor markers carcinoembryonic antigen (CEA) and cytokeratin 19 fragments (CYFRA 21-1) was predictive of a favorable outcome. This signature was associated with improved survival in patients receiving linifanib monotherapy (hazard ratio [HR]=0.51 vs signature negative; p=0.002), but not in those receiving other anti-cancer treatments (p=0.716). This signature was validated on baseline plasma samples from patients enrolled in a randomized trial of daily linifanib 7.5 mg, linifanib 12.5 mg, or placebo added to first-line carboplatin and paclitaxel chemotherapy for advanced, nonsquamous NSCLC. Only linifanib-treated signature-positive patients had significant improvement in progression-free survival (PFS). Median PFS with placebo was 5.2 months versus 10.2 months (HR=0.49, p=0.049) for those receiving linifanib 7.5mg, and 8.3 months (HR=0.38, p=0.029) for linifanib 12.5 mg. Overall survival for signature-positive patients was 11.3 months with placebo, 12.5 months with linifanib 7.5mg (HR=1.02, p=0.758), and 17.4 months with linifanib 12.5 mg (HR=0.54, p=0.137). CONCLUSION: This baseline plasma biomarker signature is associated with improved outcome in advanced NSCLC patients receiving linifanib. Utility of the biomarker signature in patient selection for linifanib therapy in NSCLC merits evaluation in larger, prospective trials that are powered to detect a survival benefit.

Randomized phase II study of carboplatin and paclitaxel with either linifanib or placebo for advanced nonsquamous non-small-cell lung cancer.

PURPOSE: Linifanib, a potent, selective inhibitor of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors, has single-agent activity in non-small-cell lung cancer (NSCLC). We evaluated linifanib with carboplatin and paclitaxel as first-line therapy of advanced nonsquamous NSCLC. PATIENTS AND METHODS: Patients with stage IIIB/IV nonsquamous NSCLC were randomly assigned to 3-week cycles of carboplatin (area under the curve 6) and paclitaxel (200 mg/m(2)) with daily placebo (arm A), linifanib 7.5 mg (arm B), or linifanib 12.5 mg (arm C). The primary end point was progression-free survival (PFS); secondary efficacy end points included overall survival (OS) and objective response rate. RESULTS: One hundred thirty-eight patients were randomly assigned (median age, 61 years; 57% men; 84% smokers). Median PFS times were 5.4 months (95% CI, 4.2 to 5.7 months) in arm A (n = 47), 8.3 months (95% CI, 4.2 to 10.8 months) in arm B (n = 44), and 7.3 months (95% CI, 4.6 to 10.8 months) in arm C (n = 47). Hazard ratios (HRs) for PFS were 0.51 for arm B versus A (P = .022) and 0.64 for arm C versus A (P = .118). Median OS times were 11.3, 11.4, and 13.0 months in arms A, B, and C, respectively. HRs for OS were 1.08 for arm B versus A (P = .779) and 0.88 for arm C versus A (P = .650). Both linifanib doses were associated with increased toxicity, including a higher incidence of adverse events known to be associated with VEGF/PDGF inhibition. Baseline plasma carcinoembryonic antigen/cytokeratin 19 fragments biomarker signature was associated with PFS improvement and a trend toward OS improvement with linifanib 12.5 mg. CONCLUSION: Addition of linifanib to chemotherapy significantly improved PFS (arm B), with a modest trend for survival benefit (arm C) and increased toxicity reflective of known VEGF/PDGF inhibitory effects.

Targeting DNA repair with combination veliparib (ABT-888) and temozolomide in patients with metastatic castration-resistant prostate cancer.

Androgen receptor-mediated transcription is directly coupled with the induction of DNA damage, and castration-resistant tumor cells exhibit increased activity of poly (ADP-ribose) polymerase (PARP)-1, a DNA repair enzyme. This study assessed the efficacy and safety of low dose oral PARP inhibitor veliparib (ABT-888) and temozolomide (TMZ) in docetaxel-pretreated patients with metastatic castration-resistant prostate cancer (mCRPC) in a single-arm, open-label, pilot study. Patients with mCRPC progressing on at least one docetaxel-based therapy and prostate specific antigen (PSA) ≥ 2 ng/mL were treated with veliparib 40 mg twice daily on days 1-7 and TMZ once daily (150 mg/m(2)/day cycle 1; if well tolerated then 200 mg/m(2)/day cycle 2 onwards) on days 1-5 q28 days. Patients received 2 (median) treatment cycles (range, 1-9). The primary endpoint was confirmed PSA response rate (decline ≥ 30 %). Twenty-six eligible patients were enrolled, 25 evaluable for PSA response. Median baseline PSA was 170 ng/mL. Two patients had a confirmed PSA response (8.0 %; 95 % CI: 1.0-26.0), 13 stable PSA, and 10 PSA progression. The median progression-free survival was 9 weeks (95 % CI: 7.9-17) and median overall survival 39.6 weeks (95 % CI: 26.6-not estimable). The most frequent treatment-emergent adverse events (AEs) were thrombocytopenia (77 %), anemia (69 %), fatigue (50 %), neutropenia (42 %), nausea (38 %), and constipation (23 %). Grade 3/4 AEs occurring in > 10 % of patients were thrombocytopenia (23 %) and anemia (15 %). Veliparib and TMZ combination was well tolerated but with modest activity. Biomarker analysis supported the proof of concept that this combination has some antitumor activity in mCRPC.

Phase 2 trial of linifanib (ABT-869) in patients with unresectable or metastatic hepatocellular carcinoma.

BACKGROUND: The efficacy and safety of linifanib (ABT-869), a selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases, were assessed in this phase 2, single-arm, open-label, multicenter trial. METHODS: Eligible patients had unresectable or metastatic hepatocellular carcinoma and had received ≤ 1 prior systemic therapy. Patients received oral linifanib at a fasting dose of 0.25 mg/kg,. The primary endpoint was the progression-free rate at 16 weeks. Tumor response was assessed every 8 weeks. Secondary endpoints included the time to disease progression, overall survival, and objective response rate. Safety was also assessed. RESULTS: Of the 44 patients enrolled, the majority were Asian (89%), had received no prior systemic therapy (82%), had Child-Pugh class A hepatic function (86%), and had hepatitis B virus infection (61%). The estimated progression-free rate at 16 weeks was 31.8% (34.2% for patients with Child-Pugh class A hepatic function), the estimated objective response rate was 9.1% (10.5% for patients with Child-Pugh class A hepatic function), the median time to disease progression was 3.7 months (3.7 months for patients with Child-Pugh class A hepatic function), and the median overall survival was 9.7 months (10.4 months for patients with Child-Pugh class A hepatic function). The most common linifanib-related adverse events were diarrhea (55%) and fatigue (52%). The most common linifanib-related grade 3/4 adverse events were hypertension (25%) and fatigue (14%). Serum levels of biomarkers cancer antigen (CA) 125, cytokeratin fragment (CYFRA)21.1, and protein induced by vitamin K absence or antagonist II (PIVKA) demonstrated potential as prognostic indicators of patient response or outcome. CONCLUSIONS: Single-agent linifanib was found to be clinically active in patients with advanced hepatocellular carcinoma, with an acceptable safety profile.

Phase II study of single-agent navitoclax (ABT-263) and biomarker correlates in patients with relapsed small cell lung cancer.

PURPOSE: Bcl-2 is a critical regulator of apoptosis that is overexpressed in the majority of small cell lung cancers (SCLC). Nativoclax (ABT-263) is a potent and selective inhibitor of Bcl-2 and Bcl-x(L). The primary objectives of this phase IIa study included safety at the recommended phase II dose and preliminary, exploratory efficacy assessment in patients with recurrent and progressive SCLC after at least one prior therapy. EXPERIMENTAL DESIGN: Thirty-nine patients received navitoclax 325 mg daily, following an initial lead-in of 150 mg daily for 7 days. Study endpoints included safety and toxicity assessment, response rate, progression-free and overall survival (PFS and OS), as well as exploratory pharmacodynamic correlates. RESULTS: The most common toxicity associated with navitoclax was thrombocytopenia, which reached grade III-IV in 41% of patients. Partial response was observed in one (2.6%) patient and stable disease in 9 (23%) patients. Median PFS was 1.5 months and median OS was 3.2 months. A strong association between plasma pro-gastrin-releasing peptide (pro-GRP) level and tumor Bcl-2 copy number (R = 0.93) was confirmed. Exploratory analyses revealed baseline levels of cytokeratin 19 fragment antigen 21-1, neuron-specific enolase, pro-GRP, and circulating tumor cell number as correlates of clinical benefit. CONCLUSION: Bcl-2 targeting by navitoclax shows limited single-agent activity against advanced and recurrent SCLC. Correlative analyses suggest several putative biomarkers of clinical benefit. Preclinical models support that navitoclax may enhance sensitivity of SCLC and other solid tumors to standard cytotoxics. Future studies will focus on combination therapies.

An open-label, phase 1 study evaluating safety, tolerability, and pharmacokinetics of linifanib (ABT-869) in Japanese patients with solid tumors.

PURPOSE: This phase 1 study assessed the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of linifanib in Japanese patients with advanced solid tumors. METHODS: Patients were assigned to one of four sequential cohorts (0.05, 0.10, 0.20, or 0.25 mg/kg) of oral, once-daily linifanib on a 21-day cycle. Adverse events (AEs) were assessed per common terminology criteria for adverse events v3.0; tumor responses were assessed by response evaluation criteria in solid tumors. RESULTS: Eighteen patients were enrolled. Eleven (61%) received ≥3 prior therapies. Dose-limiting toxicities were Grade 3 ALT increase (0.10 mg/kg linifanib) and Grade 1 T-wave inversion (0.25 mg/kg linifanib) requiring dose interruption for >7 days and discontinuation on day 29. The most common linifanib-related AE was hypertension. Other significant treatment-related AEs included proteinuria, fatigue, and palmar-plantar erythrodysaesthesia. Linifanib pharmacokinetics were dose-proportional across 0.10-0.25 mg/kg. Two patients (11.1%) had confirmed partial responses, 12 had a best response of stable disease (11 had stable disease for ≥12 weeks), and four patients were not evaluable due to incomplete data. Four patients (lung cancer, breast cancer, thymic cancer, sarcoma) have continued linifanib for ≥48 weeks (range, 48-96+ weeks). CONCLUSION: Linifanib was well tolerated with promising preliminary clinical activity in Japanese patients. Later-phase global studies examining linifanib efficacy will include Japanese patients.

A novel multi-targeted tyrosine kinase inhibitor, linifanib (ABT-869), produces functional and structural changes in tumor vasculature in an orthotopic rat glioma model.

Tyrosine kinase inhibitors represent a class of targeted therapy that has proven to be successful for cancer treatment. Linifanib is a novel, orally active multi-targeted receptor tyrosine kinase (RTK) inhibitor that exhibits potent antitumor and antiangiogenic activities against a broad spectrum of experimental tumors and malignancies in patients. The compound is currently being evaluated in phase 2 and 3 clinical trials. To investigate the effectiveness of linifinib against gliomas and the mechanism of drug action, we characterized treatment-induced antitumor and antiangiogenic responses to linifanib in an orthotopic rat glioma model. The effect of linifanib treatment on tumor growth was determined by tumor volume assessment using anatomical magnetic resonance imaging (MRI). Changes in tumor microvessel function were evaluated with dynamic contrast-enhanced MRI (DCE-MRI). Immunohistochemistry (IHC) was applied to excised tumor samples to examine underlying changes in vascular structures and target receptor expression. Linifanib (10 mg/kg) given twice daily inhibited tumor growth following treatment for 7 days with tumor volumes being 149 ± 30 and 66 ± 7 mm(3) for vehicle-and linifanib-treated groups, respectively. A significant reduction of 37 ± 13% in tumor perfusion and microvessel permeability (measured by K (trans)) was observed as early as 2 h after administration compared with vehicle treatment. Continuous linifanib administration further reduced K (trans) at later time points until the end of the study (7 days post-treatment). At day 7, K (trans) was reduced by 75 ± 32% for linifanib treatment compared with vehicle treatment. Significant reduction in total blood vessel density and improved vessel wall integrity were observed, and staining for target receptor expression confirmed inhibition of phospho VEGFR-2 and PDGFR-ß by linifanib treatment. These results demonstrate significant antitumor and antiangiogenic activity against gliomas by linifanib, a property that may result from the inhibition of VEGFR-2 and PDGFR-ß-mediated vascular changes. DCE-MRI measured K (trans) changes at early treatment stages may be a useful pharmacodynamic marker for linifanib activity in clinical trials, and basal K (trans) may provide predictive value for tumor progression.

Substantial susceptibility of chronic lymphocytic leukemia to BCL2 inhibition: results of a phase I study of navitoclax in patients with relapsed or refractory disease.

PURPOSE: BCL2 overexpression is a hallmark of chronic lymphocytic leukemia (CLL). The novel BH3 mimetic navitoclax (ABT-263) specifically inhibits BCL2 and related proteins BCL-x(l) and BCL-w, potently inducing apoptosis of CLL cells in vitro. A phase I trial in patients with CLL was conducted to evaluate the safety, pharmacokinetics, and biologic activity of oral navitoclax. PATIENTS AND METHODS: Twenty-nine patients with relapsed or refractory CLL received daily navitoclax for 14 days (10, 110, 200, or 250 mg/d; n = 15) or 21 days (125, 200, 250, or 300 mg/d; n = 14) of each 21-day cycle. Dose escalation decisions were informed by continual reassessment methodology. RESULTS: Lymphocytosis was reduced by more than 50% in 19 of 21 patients with baseline lymphocytosis. Among 26 patients treated with navitoclax ≥ 110 mg/d, nine (35%) achieved a partial response and seven maintained stable disease for more than 6 months. Median treatment duration was 7 months (range, 1 to ≥ 29 months). Median progression-free survival was 25 months. Activity was observed in patients with fludarabine-refractory disease, bulky adenopathy, and del(17p) CLL. Thrombocytopenia due to BCL-x(l) inhibition was the major dose-limiting toxicity and was dose-related. Low MCL1 expression and high BIM:MCL1 or BIM:BCL2 ratios in leukemic cells correlated with response. We determined that the navitoclax dose of 250 mg/d in a continuous dosing schedule was optimal for phase II studies. CONCLUSION: BCL2 is a valid therapeutic target in CLL, and its inhibition by navitoclax warrants further evaluation as monotherapy and in combination in this disease.

ABT-869, a multitargeted receptor tyrosine kinase inhibitor, reduces tumor microvascularity and improves vascular wall integrity in preclinical tumor models.

N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N1-(2-fluoro-5-methyl phenyl)-urea (ABT-869) is a novel multitargeted receptor tyrosine kinase inhibitor that demonstrates single-agent activity in preclinical studies and has undergone phase I and II clinical trials. We characterized the mechanism of action of ABT-869 by examining vascular changes after treatment (25 mg/kg per day) in HT1080 fibrosarcoma and SW620 colon carcinoma cells, using immunohistochemistry, dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI), and hypoxic protein detection. We observed the inhibition of vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor ß phosphorylation in both tumors and changes in tumor vasculature. Reductions in microvessel density and diameter were observed. Vascular-wall integrity was assessed by colocalization of pericytes and basement membrane. Although both microvessel density and total number of pericytes decreased with treatment, the percentage of pericyte coverage on remaining vessels significantly increased. These data suggest the selective ablation of microvessels lacking pericyte coverage. Functional vascular measures DCE-MRI and hypoxia formation were also tested. After 2 days of treatment on the HT1080 model, vascular permeability, K(trans), was reduced by >60% and hypoxic tumor fraction was significantly decreased, which was also seen in the SW620 tumors after 4 days of treatment. Taken together, decreases in vascular permeability and changes in vascular integrity observed in these studies define the mode of action of ABT-869 and may aid in optimizing the timing of therapeutic window for combination therapies.

Phase I study of Navitoclax (ABT-263), a novel Bcl-2 family inhibitor, in patients with small-cell lung cancer and other solid tumors.

PURPOSE: Resistance to chemotherapy-induced apoptosis represents a major obstacle to cancer control. Overexpression of Bcl-2 is seen in multiple tumor types and targeting Bcl-2 may provide therapeutic benefit. A phase I study of navitoclax, a novel inhibitor of Bcl-2 family proteins, was conducted to evaluate safety, pharmacokinetics, and preliminary efficacy in patients with solid tumors. PATIENTS AND METHODS: Patients enrolled to intermittent dosing cohorts received navitoclax on day -3, followed by dosing on days 1 to 14 of a 21-day cycle. Patients on continuous dosing received a 1-week lead-in dose of 150 mg followed by continuous daily administration. Blood samples were collected for pharmacokinetic analyses, biomarker analyses, and platelet monitoring. RESULTS: Forty-seven patients, including 29 with small-cell lung cancer (SCLC) or pulmonary carcinoid, were enrolled between 2007 and 2008, 35 on intermittent and 12 on continuous dosing cohorts. Primary toxicities included diarrhea (40%), nausea (34%), vomiting (36%), and fatigue (34%); most were grade 1 or 2. Dose- and schedule-dependent thrombocytopenia was seen in all patients. One patient with SCLC had a confirmed partial response lasting longer than 2 years, and eight patients with SCLC or carcinoid had stable disease (one remained on study for 13 months). Pro-gastrin releasing peptide (pro-GRP) was identified as a surrogate marker of Bcl-2 amplification and changes correlated with changes in tumor volume. CONCLUSION: Navitoclax is safe and well tolerated, with dose-dependent thrombocytopenia as the major adverse effect. Preliminary efficacy data are encouraging in SCLC. Efficacy in SCLC and the utility of pro-GRP as a marker of treatment response will be further evaluated in phase II studies.

Reducing agent-mediated precipitation of high-abundance plasma proteins.

Depletion of high-abundance proteins is regarded as a critical sample preparation step for most plasma proteomic analyses and profiling strategies. This report describes a process that rapidly and reproducibly precipitates high-abundance disulfide-rich proteins, including albumin and transferrin, from serum and plasma. A low volume of concentrated reducing agent, viz. dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), was added directly to plasma followed by a brief incubation at ambient temperature. Removal of the precipitate via centrifugation and identification of the protein content revealed an albumin-enriched pellet. Direct analysis of the supernatant by MALDI-TOF-MS afforded peptidome and small protein profiles with enhanced features and minimal ionization of full-length albumin. The reproducible and quantitative nature of the method has been demonstrated by monitoring the plasma levels of an antiangiogenic protein biologic, rKringle5 (rK5). The 10.5-kDa analyte was only reliably detected in plasma after treatment with reducing agent, ionizing linearly from 150 to 1200 fmol (on-target) with a mean CV of 7%. This method distinguishes itself from immunoaffinity resin-based approaches since it can be scaled to large milliliter quantities and it is compatible with plasma from all species tested.

Identification and characterization of novel antagonists of the CCR3 receptor.

Eotaxin, an inducer of eosinophil migration and activation, exerts its activity by binding to CCR3, the C-C chemokine receptor 3. An inhibitor of the eotaxin-CCR3 binding interaction may have potential as an anti-inflammatory drug for treatment of asthma, parasitic infections, and allergic disorders. A radioligand binding assay was developed using HEK cells transfected with CCR3, with (125)I eotaxin as the ligand. Whole cells grown on polylysine-coated plates were used as the receptor source for the screen. Screening of more than 200,000 compounds with this assay yielded a number of screening hits, and of these, 2 active novel antagonists were identified. These compounds showed inhibitory effects on eosinophil chemotaxis in both in vitro and in vivo assays.