Gayle Marshall

AZD8931, an Equipotent, Reversible Inhibitor of Signaling by Epidermal Growth Factor Receptor, ERBB2 (HER2), and ERBB3: A Unique Agent for Simultaneous ERBB Receptor Blockade in Cancer

Purpose: To test the hypothesis that simultaneous, equipotent inhibition of epidermal growth factor receptor (EGFR; erbB1), erbB2 (human epidermal growth factor receptor 2), and erbB3 receptor signaling, using the novel small-molecule inhibitor AZD8931, will deliver broad antitumor activity in vitro and in vivo. Experimental Design: A range of assays was used to model erbB family receptor signaling in homodimers and heterodimers, including in vitro evaluation of erbB kinase activity, erbB receptor phosphorylation, proliferation in cells, and in vivo testing in a human tumor xenograft panel, with ex vivo evaluation of erbB phosphorylation and downstream biomarkers. Gefitinib and lapatinib were used to compare the pharmacological profile of AZD8931 with other erbB family inhibitors. Results: In vitro, AZD8931 showed equipotent, reversible inhibition of EGFR (IC50, 4 nmol/L), erbB2 (IC50, 3 nmol/L), and erbB3 (IC50, 4 nmol/L) phosphorylation in cells. In proliferation assays, AZD8931 was significantly more potent than gefitinib or lapatinib in specific squamous cell carcinoma of the head and neck and non–small cell lung carcinoma cell lines. In vivo, AZD8931 inhibited xenograft growth in a range of models while significantly affecting EGFR, erbB2, and erbB3 phosphorylation and downstream signaling pathways, apoptosis, and proliferation. Conclusions: AZD8931 has a unique pharmacologic profile providing equipotent inhibition of EGFR, erbB2, and erbB3 signaling and showing greater antitumor activity than agents with a narrower spectrum of erbB receptor inhibition in specific preclinical models. AZD8931 provides the opportunity to investigate whether simultaneous inhibition of erbB receptor signaling could be of utility in the clinic, particularly in the majority of solid tumors that do not overexpress erbB2. Clin Cancer Res; 16(4); 1159–69

Evaluating Robustness and Sensitivity of the NanoString Technologies nCounter Platform to Enable Multiplexed Gene Expression Analysis of Clinical Samples.

Analysis of clinical trial specimens such as formalin-fixed paraffin-embedded (FFPE) tissue for molecular mechanisms of disease progression or drug response is often challenging and limited to a few markers at a time. This has led to the increasing importance of highly multiplexed assays that enable profiling of many biomarkers within a single assay. Methods for gene expression analysis have undergone major advances in biomedical research, but obtaining a robust dataset from low-quality RNA samples, such as those isolated from FFPE tissue, remains a challenge. Here, we provide a detailed evaluation of the NanoString Technologies nCounter platform, which provides a direct digital readout of up to 800 mRNA targets simultaneously. We tested this system by examining a broad set of human clinical tissues for a range of technical variables, including sensitivity and limit of detection to varying RNA quantity and quality, reagent performance over time, variability between instruments, the impact of the number of fields of view sampled, and differences between probe sequence locations and overlapping genes across CodeSets. This study demonstrates that Nanostring offers several key advantages, including sensitivity, reproducibility, technical robustness, and utility for clinical application.

Epidermal growth factor receptor tyrosine kinase inhibitors: similar but different?

Two small-molecule epidermal growth factor receptor tyrosine kinase inhibitors, gefitinib and erlotinib, have been approved for the treatment of non-small-cell lung cancer. Here, we compare the pharmacology and pharmacokinetics of these agents, and reflect on how these properties may affect important clinical questions including the clinical efficacy, optimum dose, and whether there is a relationship between skin rash and clinical outcome for each of these agents. Gefitinib and erlotinib have similar mechanisms of action and pharmacological profiles; however, different molecular structures confer pharmacokinetic differences that may have important clinical implications. Although gefitinib 250 mg/day produces lower mean plasma concentrations and area under the plasma concentration versus time curve compared with erlotinib 150 mg/day, published data suggest that gefitinib significantly accumulates in tumour tissue. This difference may partly explain why it seems possible to achieve maximum clinical efficacy with gefitinib at doses significantly lower than its maximum tolerated dose and, hence, use of an optimal biological dose approach with this agent. We hypothesize that gefitinib is used and is effective at a dose below the maximum tolerated dose as it accumulates in tumour tissue, thus providing the concentration needed at its target to achieve effective epidermal growth factor receptor inhibition in the tumour while causing less skin toxicity than erlotinib; therefore, skin rash is not a useful predictive factor for efficacy with gefitinib.

Benefits of mTOR kinase targeting in oncology: pre-clinical evidence with AZD8055

AZD8055 is a small-molecule inhibitor of mTOR (mammalian target of rapamycin) kinase activity. The present review highlights molecular and phenotypic differences between AZD8055 and allosteric inhibitors of mTOR such as rapamycin. Biomarkers, some of which are applicable to clinical studies, as well as biological effects such as autophagy, growth inhibition and cell death are compared between AZD8055 and rapamycin. Potential ways to develop rational combinations with mTOR kinase inhibitors are also discussed. Overall, AZD8055 may provide a better therapeutic strategy than rapamycin and analogues.

Identification of biomarkers in human head and neck tumor cell lines that predict for in vitro sensitivity to gefitinib.

Potential biomarkers were identified for in vitro sensitivity to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in head and neck cancer. Gefitinib sensitivity was determined in cell lines, followed by transcript profiling coupled with a novel pathway analysis approach. Eleven cell lines were highly sensitive to gefitinib (inhibitor concentration required to give 50% growth inhibition [GI50] < 1 μM), three had intermediate sensitivity (GI50 1–7 μM), and six were resistant (GI50 > 7 μM); an exploratory principal component analysis revealed a separation between the genomic profiles of sensitive and resistant cell lines. Subsequently, a hypothesis‐driven analysis of Affymetrix data (Affymetrix, Inc., Santa Clara, CA, USA) revealed higher mRNA levels for E‐cadherin (CDH1); transforming growth factor, alpha (TGF‐α); amphiregulin (AREG); FLJ22662; EGFR; p21‐activated kinase 6 (PAK6); glutathione S‐transferase Pi (GSTP1); and ATP‐binding cassette, subfamily C, member 5 (ABCC5) in sensitive versus resistant cell lines. A hypothesis‐free analysis identified 46 gene transcripts that were strongly differentiated, seven of which had a known association with EGFR and head and neck cancer (human EGF receptor 3 [HER3], TGF‐α, CDH1, EGFR, keratin 16 [KRT16], fibroblast growth factor 2 [FGF2], and cortactin [CTTN]). Polymerase chain reaction (PCR) and enzyme‐linked immunoabsorbant assay analysis confirmed Affymetrix data, and EGFR gene mutation, amplification, and genomic gain correlated strongly with gefitinib sensitivity. We identified biomarkers that predict for in vitro responsiveness to gefitinib, seven of which have known association with EGFR and head and neck cancer. These in vitro predictive biomarkers may have potential utility in the clinic and warrant further investigation.

Vandetanib inhibits both VEGFR-2 and EGFR signalling at clinically relevant drug levels in preclinical models of human cancer

Vandetanib is a multi-targeted receptor tyrosine kinase inhibitor that is in clinical development for the treatment of solid tumours. This preclinical study examined the inhibition of two key signalling pathways (VEGFR-2, EGFR) at drug concentrations similar to those achieved in the clinic, and their contribution to direct and indirect antitumour effects of vandetanib. For in vitro studies, receptor phosphorylation was assessed by Western blotting and ELISA, cell proliferation was assessed using a cell viability endpoint, and effects on cell cycle determined using flow cytometry. For in vivo studies, Western blotting, ELISA and immunohistochemistry (IHC) were used to assess receptor phosphorylation. Cell culture experiments demonstrated that anti-proliferative effects of vandetanib resulted from inhibition of either EGFR or VEGFR-2 signalling in endothelial cells, but were associated with inhibition of EGFR signalling in tumour cells. Vandetanib inhibited both EGFR and VEGFR-2 signalling in normal lung tissue and in tumour xenografts. In a lung cancer model expressing an activating EGFR mutation, the activity of vandetanib was similar to that of a highly selective EGFR inhibitor (gefitinib), and markedly greater than that of a highly selective VEGFR inhibitor (vatalanib). These data suggest that at the plasma exposures achieved in the clinic, vandetanib will significantly inhibit both VEGFR-2 and EGFR signalling, and that both inhibition of angiogenesis and direct inhibition of tumour cell growth can contribute to treatment response.

Abstract 3029: Profiling HER3/ErbB3 activation in formalin-fixed, paraffin-embedded (FFPE) breast tumor samples that express high and low HER2/ErbB2 levels using proximity-based immunoassays.

Background: HER3 is widely accepted as a potent heterodimerization partner for HER2 tyrosine kinase. HER3 activation may promote intrinsic and/or acquired resistance to HER2-targeted therapies. Therapeutic strategies that target HER3 directly or in combination with HER2 are being evaluated both preclinically and clinically. Historically, such strategies have focused exclusively on tumors that exhibit HER2 gene-amplification and/or over-expression (HER2 high/positive). Recent studies suggest distinct or additional utility in non-amplified, low HER2 tumor settings (HER2 low/negative). Attempts to identify appropriate HER2-positive patient populations that are likely to benefit from HER3-targeted therapies have revealed an important need for more quantitative, sensitive methods of measuring HER2 and HER3 protein expression other than traditional IHC, and also for quantitative, directed measurements of HER3 activation. Methods: Dual-antibody proximity-binding assays (VeraTag) were used to measure HER2 total (HERmark®), HER3 total, HER2-HER3 heterodimer, phospho-HER3 and HER3-PI3 kinase (p85) protein expression across a broad dynamic range in a group of HER2-(low) and HER2+ (high) FFPE breast tumors (HER2 status determined by IHC and verified by HERmark). Dual-antibody proximity-binding assays developed for use in high HER2 FFPE tumor specimens (PloS ONE: 28 Jan 2011 10.1371/journal.pone.0016443) were optimized for use in low HER2 samples. Results: HER2 expression spanned nearly a 2-log10 dynamic range in the HER2 breast tumor set. HER3 expression spanned a 1.5-log10 dynamic range in these tumors and trended with HER2 expression levels. HER2-HER3 heterodimerization correlated more closely with HER2 expression than HER3 expression in both low and high HER2 breast tumors, suggesting that HER2 may be the primary driver of heterodimerization. Correlations between phospho-HER3, HER3-PI3K, and HER2-HER3 heterodimer measurements were observed, with each analyte spanning a ∼1-log10 dynamic range for the corresponding measurement. HER3 signaling patterns in low and high HER2 breast tumors were compared to assess the level of HER3 activation relative to HER2 expression levels. Conclusions: We have developed multiple assays for quantitative measurements of HER3 activation in FFPE breast tumors samples that express low and high levels of the HER2 receptor. These assays are presently being investigated preclinically to define mechanism of drug action, and in clinical trials to predict response to targeted therapies. Citation Format: Jerry Wallweber, Ahmed Chenna, Roy Ravanera, Weidong Huang, David Stathas, Gayle Marshall, Chris Womack, Alison Pritchard, Ken Thress, Carl Barrett, Christos Petropoulos, Lisa DeFazio Eli, John Winslow. Profiling HER3/ErbB3 activation in formalin-fixed, paraffin-embedded (FFPE) breast tumor samples that express high and low HER2/ErbB2 levels using proximity-based immunoassays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3029. doi:10.1158/1538-7445.AM2013-3029

Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

BackgroundAdvanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE).MethodsHere we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated.ResultsOut of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment.ConclusionsCareful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material.

Abstract 912: Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease.

Effective therapies for women with low-HER2 expressing breast cancer remain a significant unmet clinical need. We have detected significant phosphorylated-HER and HER2:HER3 dimer expression in clinical breast cancer samples without HER2 amplification suggesting that HER signaling may play a role in these tumors. It is our hypothesis that ligand-driven signaling is the major route of HER family activation in the absence of HER2 amplification. AZD8931 is an orally bioavailable and highly selective small molecule inhibitor of EGFR, HER2 and HER3 signaling, which shows greatest potency when HER signaling is ligand-driven(1). In a range of low HER2 breast cancer cell lines, AZD8931 inhibited heregulin-driven proliferation (GI50 range 0.05 to 0.38μM) HER2:HER3 signaling, HER3:PI3K interaction and downstream signaling. In vivo, twice-daily oral dosing of AZD8931 showed significant monotherapy anti-tumor efficacy in a xenograft model of low HER2 breast cancer (MDA-MB-175VII) at well-tolerated doses (90% TGI at 12.5 mg/kg/bid). We further evaluated the activity of AZD8931 in combination with paclitaxel, a commonly used standard of care chemotherapy for patients with advanced breast cancer expressing low levels of HER2. Pre-clinically, the combination of AZD8931 with paclitaxel showed at least additive activity in vitro in a range of breast cell lines and additive efficacy in vivo in a BT474 (high HER2) xenograft model at well tolerated doses (AZD8931 12.5 mg/kg/bid TGI 63%; paclitaxel 7.5mg/kg/qw TGI 41%; combination TGI 95%). Combination work in the low HER2 xenograft model is ongoing. These data demonstrate that AZD8931 inhibits ligand-driven HER family receptor activation, receptor dimerization, PI3K interaction and downstream signaling leading to anti-tumor activity in vivo. These data also support the potential clinical utility of AZD8931 for the therapeutic treatment of low HER2 expressing breast cancers in combination with paclitaxel. 1 Hickinson et al. Clin.Cancer Res (2010) 16:1159-69. Citation Format: Gayle Marshall, Susan Ashton, Georgina Speake, Celina D9Cruz, Michael Grondine, Cath Trigwell, Graham Bigley, Garry Beran, Katy Lynaugh, Teresa C. Klinowska. Targeting HER family signaling in low HER2-expressing breast cancer: activity of the selective and equipotent EGFR, HER2 and HER3 signaling inhibitor, AZD8931, in models of low HER2-expressing disease. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 912. doi:10.1158/1538-7445.AM2013-912

Evaluation of AZD8931, an Equipotent Inhibitor of erbB1, erbB2, and erbB3 Receptor Signaling, on Ligand Stimulated Breast Cancer Cell Lines with Differing Levels of erbB2 Expression.

Background: Further treatment options for patients whose breast cancers do not overexpress erbB2 are required. AZD8931, an equipotent, reversible inhibitor of erbB1 (HER1, EGFR), erbB2 (HER2), and erbB3 (HER3) receptor signaling may be useful in this setting and may lead to inhibition of tumor cell proliferation, invasion, metastasis, angiogenesis and tumor cell survival. Objectives: To compare the activity of AZD8931 with other erbB inhibitors (gefitinib [G] and lapatinib [L]) in breast cancer cell lines stimulated with erbB ligands. Methods: A panel of 9 breast cancer cell lines with differing erbB2 expression levels were used: erbB2+/ER+ (BT474c; MDA-MB-361); erbB2+/ER- (MDA-MB-453; SKBR-3); erbB2-/ER+ (MCF7; T47D; ZR75-1); or erbB2-/ER- (MDA-MB-231; MDA-MB-468). Following overnight serum starvation, cells were incubated with AZD8931, G or L (0-10 µM) for 90 min and then stimulated with erbB ligands (50 ng/ml; EGF, TGFα, amphiregulin, epiregulin, betacellulin, neuregulin1, or HBEGF) for 5 min before lysis. Levels of phosphorylated erbB1, erbB2, and erbB3 were analyzed by ELISA. For IC 50 determination, mean basal phosphorylation was subtracted. Geometric mean IC 50 s were calculated from triplicate assays and 2-sided unequal variance t-tests compared logIC 50 s . Results: AZD8931 demonstrated potent inhibitory activity (IC 50 s ≤10610 nM) when phosphorylation of erbB1, erbB2 or erbB3 receptors was driven by any erbB ligand. G demonstrated potent inhibitory activity (IC 50 s ≤20 nM) when the phosphorylation of erbB1 and erbB2 was driven by any erbB ligand. L more strongly inhibited the phosphorylation of erbB2 (IC 50 s ≤10 nM) than erbB1 (IC 50 s 50 s were lower for AZD8931 (1-5 nM) than for G (1-120 nM) or L (20-80 nM) in the majority of the cell lines tested. Inhibition of erbB1 phosphorylation driven by EGF (IC 50 s ≤ vs >20 nM), TGFα (≤5 vs >20 nM), HB-EGF (≤ vs >25 nM), or betacellulin (≤6 vs 10-118 nM) also indicated more potent inhibitory activity for AZD8931 over L in cell lines that respond to ligand stimulation. No phosphorylation response to amphiregulin was seen in any of the cell lines. Conclusion: This study demonstrates that in a range of breast cell lines with varying levels of erbB2 expression, AZD8931 is a potent and balanced inhibitor of erbB1, erbB2, and erbB3 signaling. The pharmacological profile of AZD8931 is thus distinct from G and L and suggests that AZD8931 offers an agent to test the hypothesis that combined, balanced inhibition of erbB signaling could provide clinical benefit. AZD8931 may be particularly useful in the treatment of solid tumors that do not overexpress erbB2 including trastuzumab-ineligible breast cancer, an area of unmet medical need. AZD8931 is being evaluated in a Phase I clinical trial. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5059.