Daniel J. Freeman

Wild-Type KRAS Is Required for Panitumumab Efficacy in Patients With Metastatic Colorectal Cancer

PURPOSE Panitumumab, a fully human antibody against the epidermal growth factor receptor (EGFR), has activity in a subset of patients with metastatic colorectal cancer (mCRC). Although activating mutations in KRAS, a small G-protein downstream of EGFR, correlate with poor response to anti-EGFR antibodies in mCRC, their role as a selection marker has not been established in randomized trials. PATIENTS AND METHODS KRAS mutations were detected using polymerase chain reaction on DNA from tumor sections collected in a phase III mCRC trial comparing panitumumab monotherapy to best supportive care (BSC). We tested whether the effect of panitumumab on progression-free survival (PFS) differed by KRAS status. RESULTS KRAS status was ascertained in 427 (92%) of 463 patients (208 panitumumab, 219 BSC). KRAS mutations were found in 43% of patients. The treatment effect on PFS in the wild-type (WT) KRAS group (hazard ratio [HR], 0.45; 95% CI: 0.34 to 0.59) was significantly greater (P < .0001) than in the mutant group (HR, 0.99; 95% CI, 0.73 to 1.36). Median PFS in the WT KRAS group was 12.3 weeks for panitumumab and 7.3 weeks for BSC. Response rates to panitumumab were 17% and 0%, for the WT and mutant groups, respectively. WT KRAS patients had longer overall survival (HR, 0.67; 95% CI, 0.55 to 0.82; treatment arms combined). Consistent with longer exposure, more grade III treatment-related toxicities occurred in the WT KRAS group. No significant differences in toxicity were observed between the WT KRAS group and the overall population. CONCLUSION Panitumumab monotherapy efficacy in mCRC is confined to patients with WT KRAS tumors. KRAS status should be considered in selecting patients with mCRC as candidates for panitumumab monotherapy.

Association of K-ras Mutational Status and Clinical Outcomes in Patients with Metastatic Colorectal Cancer Receiving Panitumumab Alone

BACKGROUND Identifying predictive biomarkers is important to optimally treat patients. This analysis evaluated the association of K-ras, BRAF, and PIK3CA gene mutations with tumor resistance to panitumumab alone. PATIENTS AND METHODS From 3 phase II panitumumab metastatic colorectal cancer (mCRC) studies, 62 of 533 patient samples were available. Mutations were identified from genomic DNA by sequencing. RESULTS Of the 62 samples, 24 (38.7%) harbored a K-ras mutation, and 38 (61.3%) were wild type. In the wild-type K-ras group, 11% of patients had a partial response (PR), 53% had stable disease (SD), and 37% had progressive disease (PD). In the mutant K-ras group, 21% of patients had SD, and 79% of patients had PD; there were no responses. The absence of a K-ras mutation was associated with response to panitumumab (PR vs. SD vs. PD; P = .0028). The hazard ratio for wild-type versus mutant K-ras was 0.4 (95% CI, 0.2-0.7) for progression-free survival and 0.5 (95% CI, 0.3-0.9) for overall survival. Four patients had a V600E BRAF mutation, and 2 patients had a PIK3CA mutation. CONCLUSION These data suggest that patients with mCRC with activating K-ras mutations are less likely to respond to panitumumab alone. The small sample size limits us from defining a predictive role of PIK3CA and BRAF mutations for panitumumab treatment.

Discovery and Optimization of a Series of Benzothiazole Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitors

Phosphoinositide 3-kinase α (PI3Kα) is a lipid kinase that plays a key regulatory role in several cellular processes. The mutation or amplification of this kinase in humans has been implicated in the growth of multiple tumor types. Consequently, PI3Kα has become a target of intense research for drug discovery. Our studies began with the identification of benzothiazole compound 1 from a high throughput screen. Extensive SAR studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potent dual inhibitor of PI3K and mTOR. This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82 demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.

The Influence of Sodium Glycocholate and Other Additives on the in vivo Transfection of Plasmid DNA in the Lungs

AbstractPurpose. A plasmid containing the luciferase ‘marker’ cDNA was constructed to test non viral gene delivery formulations in vivo. Methods. A scale up procedure was devised to produce up to gram quantities of plasmid. Sufficient quantities were generated to process and test the DNA with various additives and to generate a spray-dried powder formulation of the plasmid. Male Sprague-Dawley rats (250 g) were intratracheally instilled with 200–250 µl of solution containing 200 µg plasmid ± lipid [DC Chol:DOPE 1:1 molar (2mg/kg)] growth factors [KGF (10 mg/kg), EGF (5 mg/kg)], permeation enhancers [sodium glycocholate (0.01 to 10% w/v)), sodium deoxycholate (1% w/v), beta-cyclodextrin (1% w/v)], surfactant [Tween 80 (1% w/v)], a mucolytic [N-acetylcysteine (10% w/v)] and positively charged synthetic polymers [PVAVAM 6 and 14%]. Animals were sacrificed 24 hr post-dose and the lungs were assayed for luciferase using a chemiluminescent assay. Results. The relative ability of the materials to promote luciferase production in the lungs was permeation enhancer >> DNA alone ≥ lipid, mucolytic, surfactant, growth factor > polymer. Protein production in the lungs ranged from 10 times below the DNA control (≈16 pg) using the polymers (≈1.5 pg) to ≈125 times greater than the control using the permeation enhancer (≈2050 pg). The transfection capabilities of the majority of additives was low. The enhancing effects of sodium glycocholate were dose-dependent and perhaps associated with the critical micelle concentration. Although the bile salt was the most successful of the tested compounds, it resulted in significant mortality when used at concentrations greater than 1 % w/v. Conclusions. The results suggest that transfection can be significantly enhanced by additives such as NaGC but some toxicity may be unavoidable.

A comparability study of 5 commercial KRAS tests

BackgroundActivating mutations in the KRAS gene occur frequently in human tumors, including colorectal carcinomas; most mutations occur in codons 12 and 13. Mutations in KRAS have been associated with poor response to anti-epidermal growth factor receptor antibodies. Therefore, an accurate and readily available analysis of KRAS mutational status is needed. The aim of this study was to evaluate concordance between KRAS assays performed by 6 different laboratories.MethodsForty formalin-fixed paraffin-embedded colorectal cancer tumor samples were obtained. Sample sections were submitted for KRAS mutation analysis to 5 independent commercial laboratories (Agencourt, Gentris, Genzyme, HistoGeneX, and Invitek) and to the Amgen DNA Sequencing Laboratory for direct polymerase chain reaction sequencing. The assay used by Invitek is no longer commercially available and has been replaced by an alternative technique. Results from the commercial services were compared with those from Amgen direct sequencing by κ statistics.ResultsKRAS mutations were observed in codon 12 and/or 13 in 20 of 40 (50%) samples in Amgen direct sequencing assays. Results from HistoGeneX (κ = 0.95), Genzyme (κ = 0.94), and Agencourt (κ = 0.94) were in almost perfect agreement with these results, and the results from Gentris were in substantial agreement with the results from Amgen (κ = 0.75). The Invitek allele-specific assay demonstrated slight agreement (κ = 0.13).ConclusionsThis study provides data on the comparability of KRAS mutational analyses. The results suggest that most (but not all) commercial services provide analysis that is accurate and comparable with direct sequencing.

Structure-activity relationships of phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitors: investigations of various 6,5-heterocycles to improve metabolic stability.

N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-yl)benzo[d]thiazol-2-yl)acetamide (1) is a potent and efficacious inhibitor of PI3Kα and mTOR in vitro and in vivo. However, in hepatocyte and in vivo metabolism studies, 1 was found to undergo deacetylation on the 2-amino substituent of the benzothiazole. As an approach to reduce or eliminate this metabolic deacetylation, a variety of 6,5-heterocyclic analogues were examined as an alternative to the benzothiazole ring. Imidazopyridazine 10 was found to have similar in vitro potency and in vivo efficacy relative to 1, while only minimal amounts of the corresponding deacetylated metabolite of 10 were observed in hepatocytes.

Augmentation of Radiation Response by Panitumumab in Models of Upper Aerodigestive Tract Cancer

PURPOSE To examine the interaction between panitumumab, a fully human anti-epidermal growth factor receptor monoclonal antibody, and radiation in head-and-neck squamous cell carcinoma and non-small-cell lung cancer cell lines and xenografts. METHODS AND MATERIALS The head-and-neck squamous cell carcinoma lines UM-SCC1 and SCC-1483, as well as the non-small-cell lung cancer line H226, were studied. Tumor xenografts in athymic nude mice were used to assess the in vivo activity of panitumumab alone and combined with radiation. In vitro assays were performed to assess the effect of panitumumab on radiation-induced cell signaling, apoptosis, and DNA damage. RESULTS Panitumumab increased the radiosensitivity as measured by the clonogenic survival assay. Radiation-induced epidermal growth factor receptor phosphorylation and downstream signaling through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) was inhibited by panitumumab. Panitumumab augmented radiation-induced DNA damage by 1.2-1.6-fold in each of the cell lines studied as assessed by residual gamma-H(2)AX foci after radiation. Radiation-induced apoptosis was increased 1.4-1.9-fold by panitumumab, as evidenced by Annexin V-fluorescein isothiocyanate staining and flow cytometry. In vivo, the combination therapy of panitumumab and radiation was superior to panitumumab or radiation alone in the H226 xenografts (p = 0.01) and showed a similar trend in the SCC-1483 xenografts (p = 0.08). In vivo, immunohistochemistry demonstrated the ability of panitumumab to augment the antiproliferative and antiangiogenic effects of radiation. CONCLUSION These studies have identified a favorable interaction in the combination of radiation and panitumumab in upper aerodigestive tract tumor models, both in vitro and in vivo. These data suggest that clinical investigations examining the combination of radiation and panitumumab in the treatment of epithelial tumors warrant additional pursuit.

Structure-based design of a novel series of potent, selective inhibitors of the class I phosphatidylinositol 3-kinases.

A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

Selective Class I Phosphoinositide 3-Kinase Inhibitors: Optimization of a Series of Pyridyltriazines Leading to the Identification of a Clinical Candidate, AMG 511

The phosphoinositide 3-kinase family catalyzes the phosphorylation of phosphatidylinositol-4,5-diphosphate to phosphatidylinositol-3,4,5-triphosphate, a secondary messenger which plays a critical role in important cellular functions such as metabolism, cell growth, and cell survival. Our efforts to identify potent, efficacious, and orally available phosphatidylinositol 3-kinase (PI3K) inhibitors as potential cancer therapeutics have resulted in the discovery of 4-(2-((6-methoxypyridin-3-yl)amino)-5-((4-(methylsulfonyl)piperazin-1-yl)methyl)pyridin-3-yl)-6-methyl-1,3,5-triazin-2-amine (1). In this paper, we describe the optimization of compound 1, which led to the design and synthesis of pyridyltriazine 31, a potent pan inhibitor of class I PI3Ks with a superior pharmacokinetic profile. Compound 31 was shown to potently block the targeted PI3K pathway in a mouse liver pharmacodynamic model and inhibit tumor growth in a U87 malignant glioma glioblastoma xenograft model. On the basis of its excellent in vivo efficacy and pharmacokinetic profile, compound 31 was selected for further evaluation as a clinical candidate and was designated AMG 511.

Activity of panitumumab alone or with chemotherapy in non-small cell lung carcinoma cell lines expressing mutant epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) kinase domain mutations cause hyperresponsiveness to ligand and hypersensitivity to small-molecule tyrosine kinase inhibitors. However, little is known about how these mutations respond to antibodies against EGFR. We investigated the activity of panitumumab, a fully human anti-EGFR monoclonal antibody, in vitro in mutant EGFR-expressing non-small cell lung carcinoma (NSCLC) cells and in vivo with chemotherapy in xenograft models. Mutant EGFR-expressing NSCLC cells (NCI-H1975 [L858R+T790M] and NCI-H1650 [Δ746-750]) and CHO cells were treated with panitumumab before EGF stimulation to assess the inhibition of EGFR autophosphorylation. Established tumors were treated with panitumumab (25, 100, or 500 μg/mouse twice a week) alone or with docetaxel (10 or 20 mg/kg once a week) or cisplatin (7.5 mg/kg once a week). Antitumor activity and levels of proliferation markers were analyzed. Treatment of mutant EGFR-expressing CHO and NSCLC cells with panitumumab inhibited ligand-dependent autophosphorylation. In NCI-H1975 and NCI-H1650 xenografts, treatment with panitumumab alone or with cisplatin inhibited tumor growth compared with control (P < 0.0003). With panitumumab plus docetaxel, enhanced antitumor activity was seen in both xenografts versus panitumumab alone. Panitumumab treatment alone decreased Ki-67 and phospho- mitogen-activated protein kinase (pMAPK) staining in both xenografts compared with control. Docetaxel enhanced panitumumab activity in NCI-H1650 xenografts (decreased Ki-67 and pMAPK staining by >60%) when compared with either agent alone. Panitumumab inhibits ligand-induced EGFR phosphorylation, tumor growth, and markers of proliferation alone or with docetaxel in NSCLC cell lines that express clinically observed EGFR kinase domain mutations, including the small-molecule tyrosine kinase inhibitor-resistant T790M mutation. [Mol Cancer Ther 2009;8(6):1536–46]